Quality control method and application of traditional Chinese medicine compound containing gardenia kernel

Abstract

The invention discloses a construction method of a fingerprint spectrum of a traditional Chinese medicine compound containing gardenia fruit. The construction method comprises the following steps: preparing a traditional Chinese medicine composition test solution; preparing a reference substance solution; and obtaining the fingerprint spectrum of the traditional Chinese medicine composition according to results of the test solution and the reference solution of the traditional Chinese medicine composition through high performance liquid chromatography detection. The detection method is simple, accurate, efficient, good in stability, high in precision, good in reproducibility, convenient and easy to master, energy can be effectively saved, the analysis cost is reduced, and the technical content is not disclosed in the prior art.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a quality control method for a traditional Chinese medicine compound containing gardenia seeds and its application. Background Technology

[0002] In recent years, the integration of research on "Chinese herbal medicine chemistry" in the sense of phytochemistry with research on compound Chinese herbal medicine chemistry and life science research has enabled people to gain a deeper understanding of the in vivo processes of Chinese herbal medicine. Subsequently, the connotation and extension of drug metabolism have been expanded, not only referring to therapeutic drugs, but also including all exogenous chemical substances. The research content also includes not only the biotransformation process of chemical substances, but also the properties and quantitative laws of drug metabolism, involving processes such as absorption, distribution, metabolism and excretion. With the great progress of science and technology, there is also a richer content, such as predicting drug interactions and the impact of environmental factors on drug metabolism, and using these influences to improve therapeutic effects and drug safety. The research on the active and toxic components of Chinese herbal medicine has always occupied a pivotal position in the scientific research on complex systems of Chinese herbal medicine. It is a fundamental and core issue.

[0003] Traditional Chinese medicine (TCM) compound prescriptions, as a whole, require the combined action of all the individual medicinal materials to achieve therapeutic effects. As a primary form of clinical application, TCM compound prescriptions possess the advantages of multiple components, multiple targets, and multiple effects. However, the active substances in TCM compound prescriptions interact after entering the human body, producing a "natural compatibility" effect. Through multi-target binding, they achieve synergistic effects and reduce toxicity. These active substances may originate from the original components of the TCM, compounds generated during the preparation process, or metabolic products of the human body. These active substances do not exert their medicinal effects in a single medicinal material; they only exert their therapeutic effect when combined as a whole. To ensure safety and efficacy, reflect the inherent quality characteristics of this product, and embody its holistic nature, a series of studies have been conducted on the medicinal ingredients and preparation quality of this product, adhering to the principles of practicality, science, and standardization, based on the effects of each medicinal ingredient in the prescription.

[0004] Currently, no literature reports a comprehensive analysis of the chemical composition, content determination, and fingerprint spectral study of the traditional Chinese medicine composition containing gardenia seed claimed in this invention. In the prior art, only the quality analysis of a single component in the traditional Chinese medicine compound is performed. There is no comprehensive and systematic quality control method to reflect the quality status of the main reference sample components in the traditional Chinese medicine compound and finished product of this invention. It is impossible to effectively control its production process and product quality, and cannot better guarantee its clinical efficacy. Therefore, it is necessary to adopt a fingerprint spectral quality control method that can comprehensively control the overall quality of the traditional Chinese medicine compound of this invention to control its key quality. Summary of the Invention

[0005] Based on this, the present invention provides a method for constructing the fingerprint spectrum of a traditional Chinese medicine compound containing gardenia seed, the method comprising the following steps:

[0006] Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the decoction of the traditional Chinese medicine compound containing gardenia seed, concentrate it, add the first solvent and weigh it, extract for a period of time, cool it and weigh it again, use the first solvent to make up for the lost weight, shake it well, filter it, and take the filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes gypsum, gardenia seed, saposhnikovia root, patchouli leaf and licorice;

[0007] Preparation of reference solutions: Weigh appropriate amounts of geniposide, cimicifugain, 5-O-methylvisamiloside, glycyrrhizin, ammonium glycyrrhizate, cimicifugain, and 5-p-hydroxymaltol reference standards, and add methanol solution to prepare reference solutions with concentrations of 5~200 μg / ml for each of geniposide, cimicifugain, 5-O-methylvisamiloside, glycyrrhizin, ammonium glycyrrhizate, cimicifugain, and 5-p-hydroxymaltol.

[0008] The fingerprint spectrum of the traditional Chinese medicine compound was obtained based on the results of high performance liquid chromatography detection of the test solution and the reference solution.

[0009] The chromatographic conditions for this high-performance liquid chromatography (HPLC) detection were as follows: a column packed with octadecylsilane-bonded silica gel was used; mobile phase A was selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B was an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program was as follows: 0–6 min, 97%B→85%B; 6–20 min, 85%B→79%B; 20–23 min, 79%B→76%B; 23–26 min, 76%B→75%B; 26–40 min, 75%B→62%B; 40–55 min, 62%B→32%B; 55–60 min, 32%B→20%B; 60–62 min, 20%B→97%B; 62–75 min, 97%B; the flow rate was 0.92–1.08 ml / min; the column temperature was 27–33 °C; and the detection wavelength was 200–400 nm. nm, injection volume is 1~20 μl.

[0010] Furthermore, the mass ratio of the gypsum, the gardenia kernel, the saposhnikovia root, the patchouli leaf and the licorice is (15~25):(2~10):(130~200):(20~40):(80~160).

[0011] Furthermore, the mass ratio of the gypsum, the gardenia seed, the saposhnikovia root, the patchouli leaf, and the licorice is approximately 20.65: approximately 4.13: approximately 165.2: approximately 28.91: approximately 123.9.

[0012] Furthermore, the mass ratio of the gypsum, the gardenia seed, the saposhnikovia root, the patchouli leaf, and the licorice is (1~10):(0.5~5):(10~70):(2~20):(10~50).

[0013] Furthermore, the mass ratio of the gypsum, the gardenia seed, the saposhnikovia root, the patchouli leaf, and the licorice is approximately 5:1:40:7:30.

[0014] Furthermore, this windproof is a baked windproof.

[0015] Furthermore, the mass / volume (g / ml) ratio of the traditional Chinese medicine decoction containing gardenia kernel to the first solvent is 0.5 to 2, for example, about 1.

[0016] Furthermore, the mass of the traditional Chinese medicine decoction containing gardenia seeds is 20-30 g, for example, about 25 g.

[0017] Furthermore, the preparation method of this traditional Chinese medicine compound decoction includes the following steps: take appropriate amounts of each medicinal herb slices, crush them into coarse particles, weigh out the crushed gypsum, gardenia, patchouli leaves, saposhnikovia root (baked), and licorice root, mix them evenly, add honey wine to moisten, stir-fry, cool, pulverize, and pass through a No. 4 sieve; take one dose of the prescription amount, add solvent, cover, heat over high heat to boiling, then simmer over low heat to obtain a concentrated liquid, filter while hot to obtain the traditional Chinese medicine compound decoction.

[0018] Furthermore, the concentration of this mead is 10-40%.

[0019] Furthermore, the ratio of honey to wine in the mead is approximately 1:1.

[0020] Furthermore, the humidification period is 1 to 4 hours.

[0021] Furthermore, the frying temperature is between 50°C and 80°C.

[0022] Furthermore, the stir-frying time is 30 to 90 minutes.

[0023] Furthermore, the solvent is water.

[0024] Furthermore, the ratio of the mass of the prescription dose to the mass / volume of the solvent is 0.02 to 0.03, in g / mL.

[0025] Furthermore, the simmering time is 30 to 60 minutes.

[0026] Furthermore, the volume ratio of the concentrate to the solvent is 0.4 to 0.6.

[0027] Furthermore, the volume of the first solvent is 20-30 ml, for example, about 25 ml.

[0028] Furthermore, the first solvent is an ethanol solution.

[0029] Furthermore, the volume percentage concentration of the ethanol solution is 30% to 70%, for example, about 50%.

[0030] Furthermore, the extraction method is ultrasonic extraction or reflux extraction, for example, ultrasonic extraction.

[0031] Furthermore, the frequency of the ultrasound extraction is 20-60 kHz, for example, about 40 kHz.

[0032] Furthermore, the power of the ultrasound extraction is 200~600 W, for example, about 500 W.

[0033] Furthermore, the ultrasonic extraction time is 15-60 minutes, for example, about 30 minutes.

[0034] Furthermore, in the preparation of this reference solution, the volume percentage concentration of the methanol solution is 30% to 70%, for example, about 50%.

[0035] Furthermore, the concentration of geniposide in the reference solution was approximately 20 μg / ml.

[0036] Furthermore, the concentration of cimicifugoside in the reference solution was approximately 30 μg / ml.

[0037] Furthermore, the concentration of 5-O-methylvisamidol in the reference solution was approximately 30 μg / ml.

[0038] Furthermore, the concentration of glycyrrhizin in the reference solution was approximately 30 μg / ml.

[0039] Furthermore, the concentration of ammonium glycyrrhizate in the reference solution was approximately 150 μg / ml.

[0040] Furthermore, the concentration of cimicifugain in the reference solution was approximately 10 μg / ml.

[0041] Furthermore, the concentration of 5-p-hydroxymaltol in the reference solution was approximately 10 μg / ml.

[0042] Furthermore, the flow rate for this high-performance liquid chromatography detection is 0.94~1.06 ml / min, for example, about 1.0 ml / min.

[0043] Furthermore, the column temperature is 28~32℃, for example, about 30℃.

[0044] Furthermore, between the 0th and 10th minute, the detection wavelength is approximately 284 nm.

[0045] Furthermore, between the 10th and 27th minutes, the detection wavelength was approximately 238 nm.

[0046] Furthermore, between the 27th and 35th minutes, the detection wavelength was approximately 370 nm.

[0047] Furthermore, between the 35th and 70th minutes, the detection wavelength was approximately 254 nm.

[0048] Furthermore, the injection volume is 5~15 μl, for example, about 10 μl.

[0049] Furthermore, the theoretical plate number of the chromatographic peak corresponding to the 5-O-methylvisamidol glycoside is not less than 10,000.

[0050] Furthermore, the chromatographic column is a YMC-Pack ODS-A column or a ZORBAX SB-C18 column.

[0051] Furthermore, the specifications of the chromatographic column are as follows: column length 250 mm, inner diameter 4.6 mm, particle size 5 μm.

[0052] Furthermore, mobile phase A is acetonitrile.

[0053] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer salt solution are selected from one or more weak acids and their salts, weak bases and their salts of different concentrations.

[0054] Furthermore, the aqueous acid solution, aqueous alkaline solution, and / or aqueous buffer solution are selected from different concentrations of formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.

[0055] Furthermore, the acid aqueous solution is a 0.01% to 1% acid aqueous solution.

[0056] Furthermore, the acidic aqueous solution is a 0.01%~0.2% phosphoric acid aqueous solution.

[0057] Furthermore, the acidic aqueous solution is an aqueous solution of approximately 0.05% phosphoric acid.

[0058] Furthermore, the buffer salt solution is a phosphate solution and / or a phosphate solution.

[0059] Furthermore, the pH value of this buffer salt solution is no greater than 7.0.

[0060] Furthermore, when the detection wavelength is 284 nm between 0 and 10 minutes, 238 nm between 10 and 27 minutes, 370 nm between 27 and 35 minutes, and 254 nm between 35 and 70 minutes, the fingerprint spectrum includes peaks 1-16. The retention times of these peaks are approximately 7.288 min, 8.514 min, 11.058 min, 12.546 min, 13.254 min, 16.129 min, 20.699 min, 21.301 min, 22.030 min, 24.691 min, 30.582 min, 32.140 min, 35.108 min, 41.861 min, 45.170 min, and 46.890 min, respectively. min.

[0061] Furthermore, the detection wavelength is 284 nm between 0 and 10 minutes, 238 nm between 10 and 27 minutes, 370 nm between 27 and 35 minutes, and 254 nm between 35 and 70 minutes. At nm, the fingerprint spectrum includes peaks 1-16, with peak 10 serving as a reference peak. The average relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, and 16 are approximately 0.30, 0.34, 0.45, 0.51, 0.54, 0.65, 0.84, 0.86, 0.89, 1.24, 1.30, 1.42, 1.70, 1.83, and 1.90, respectively.

[0062] Furthermore, when the detection wavelength is 284 nm between 0 and 10 minutes, 238 nm between 10 and 27 minutes, 370 nm between 27 and 35 minutes, and 254 nm between 35 and 70 minutes, peak 1 is 5-p-hydroxymaltol, peak 5 is geniposide, peak 6 is cimicifugoside, peak 7 is glycyrrhizin, peak 8 is cimicifugoside, peak 10 is 5-O-methylvisamidol glycoside, and peak 16 is glycyrrhizic acid.

[0063] Furthermore, the detection wavelength is 284 nm between 0 and 10 minutes, 238 nm between 10 and 27 minutes, 370 nm between 27 and 35 minutes, and 254 nm between 35 and 70 minutes. At nm, peak 1 comes from licorice root, gardenia seed, and saposhnikovia root; peak 2 comes from licorice root, gardenia seed, patchouli leaf, gypsum, and saposhnikovia root; peak 3 comes from gardenia seed; peak 4 comes from licorice root; peak 5 comes from gardenia seed; peak 6 comes from saposhnikovia root; peak 7 comes from licorice root; peak 8 comes from licorice root; peak 9 comes from saposhnikovia root; peak 10 comes from saposhnikovia root; peak 11 comes from licorice root; peak 12 comes from licorice root; peak 13 comes from licorice root; peak 14 comes from licorice root; peak 15 comes from licorice root; and peak 16 comes from licorice root.

[0064] According to another aspect of the present invention, a quality control method for a traditional Chinese medicine compound containing gardenia seeds is provided, the quality control method comprising the following steps:

[0065] (1) Establish standard fingerprint spectra of traditional Chinese medicine compound reference samples based on the above fingerprint spectra construction method;

[0066] (2) Take the test solution of the traditional Chinese medicine compound and perform detection according to the chromatographic conditions in the above fingerprint chromatogram construction method to obtain the fingerprint chromatogram of the traditional Chinese medicine compound sample; and

[0067] (3) Compare the fingerprint spectrum of the Chinese medicine compound sample to be tested obtained in step (2) with the standard fingerprint spectrum of the Chinese medicine compound reference sample obtained in step (1). If it meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

[0068] Furthermore, the compliance requirement includes one or more of the following:

[0069] (1) The fingerprint spectrum of the Chinese herbal compound sample showed 16 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak was within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint spectrum of the Chinese herbal compound reference sample.

[0070] (2) Taking the 5-O-methylvisamidol glycoside peak as the S peak, the relative retention times of each characteristic chromatographic peak in the fingerprint chromatogram of the Chinese herbal compound sample to be tested and the S peak are within ±10% of the relative retention times of each characteristic chromatographic peak in the standard fingerprint chromatogram of the Chinese herbal compound reference sample; and

[0071] (3) According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the fingerprint of the sample to be tested of the traditional Chinese medicine compound and the standard fingerprint of the reference sample of the traditional Chinese medicine compound shall not be less than 0.90.

[0072] According to another aspect of the present invention, the above-described construction method or the above-described quality control method is provided for use in the quality testing and / or quality evaluation and / or quality control of traditional Chinese medicine compound containing gardenia seeds.

[0073] The beneficial effects of this invention are:

[0074] In summary, this invention provides a method for fingerprint analysis and quality control of a reference sample of a traditional Chinese medicine composition containing gardenia seeds. This method identifies 16 common characteristic peaks, is simple to implement, and has a short analysis time. It solves the problems of difficult separation of fingerprint characteristic peaks and interference from impurity peaks, ensuring the chemical composition stability and safety of the reference sample. This provides an important reference for the quality control of subsequent preparations, ensuring product quality stability, guaranteeing the efficacy of the traditional Chinese medicine compound, and enabling the traditional Chinese medicine compound to better serve human health.

[0075] Specifically, compared with the prior art, the beneficial effects of the present invention are as follows: (1) The fingerprint spectrum of the traditional Chinese medicine compound reference sample of the present invention is established, which overcomes the defect that the content determination of a single component is difficult to reflect the overall content. It can control the intrinsic quality of the traditional Chinese medicine compound reference sample of the present invention from an overall and macroscopic perspective, ensuring the efficacy of the drug. It utilizes the main means of modern drug research to realize the inheritance of classics and make the classic prescriptions more formally controlled in terms of quality; (2) The chemical composition of the traditional Chinese medicine compound reference sample of the present invention is complex, and it is difficult to separate its characteristic peaks. In the process of establishing the fingerprint spectrum, the present invention adopts the gradient elution method, which solves the problem of the fingerprint characteristic peaks being difficult to separate and the interference of impurity peaks; (3) In the process of establishing the fingerprint spectrum of the traditional Chinese medicine compound reference sample of the present invention, the present invention has achieved the following benefits: Sixteen common characteristic peaks were identified, and their relative retention time, relative peak area and similarity were studied to ensure the chemical composition stability and safety of the reference sample, providing an important reference for the quality control of subsequent compound preparations; (4) The fingerprint spectrum of each effective component in the traditional Chinese medicine compound reference sample of the present invention is regarded as a whole, and attention is paid to the order and interrelationship of each characteristic peak. This avoids the one-sidedness of judging the overall quality of the traditional Chinese medicine compound reference sample of the present invention by only measuring one or two chemical components, and reduces the possibility of artificial processing to meet the quality standards. This provides a new method and means for the complete and accurate evaluation of the quality of the traditional Chinese medicine compound reference sample of the present invention; (5) The method of the present invention has good stability, high precision, good reproducibility, convenience and easy mastery. Attached Figure Description

[0076] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without exceeding the scope of protection claimed by the present invention.

[0077] Figure 1 The image shows the 3D absorbance diagram of the test sample 1, a traditional Chinese medicine composition containing gardenia seeds.

[0078] Figure 2 This is a schematic diagram of the characteristic chromatogram (16 characteristic peaks) of the reference sample 1 of the traditional Chinese medicine composition containing gardenia kernel.

[0079] Figure 3 This is a schematic diagram showing the characteristic chromatogram results of reference samples 2, 3, and 4 of the traditional Chinese medicine composition containing gardenia seed. In the diagram, A represents reference sample decoction 2; B represents reference sample decoction 3; and C represents reference sample decoction 4. Detailed Implementation

[0080] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0081] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the field of this invention or the field of application of such terms. While any methods, conditions, substances, or materials similar to or equivalent to those disclosed herein may be used in the practice of this invention, preferred methods, conditions, substances, or materials are described herein.

[0082] This invention is intended to cover all options, variations, and equivalents that may be included in the field of prior art as defined in the claims. Those skilled in the art will recognize many similar or equivalent methods and substances described herein that can be applied in the practice of this invention. This invention is by no means limited to the description of methods and substances.

[0083] The singular forms “a,” “an,” and “the” used in the specification and appended claims include plural indicators unless the context clearly specifies otherwise.

[0084] In this invention, the term "comprising" and "including" are synonymous. The terms "comprising," "including," "having," "containing," or any other variations thereof as used herein are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such a composition, step, method, article, or apparatus.

[0085] As described in the background section, no literature reports a comprehensive analysis of the chemical components, content determination, and fingerprinting of the traditional Chinese medicine composition containing gardenia seed claimed in this invention. To address the above problems, this invention provides a method for constructing a fingerprint of a traditional Chinese medicine compound containing gardenia seed, the method comprising the following steps:

[0086] Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the decoction of the traditional Chinese medicine compound containing gardenia seed, concentrate it, add the first solvent and weigh it, extract for a period of time, cool it and weigh it again, use the first solvent to make up for the lost weight, shake it well, filter it, and take the filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes gypsum, gardenia seed, saposhnikovia root, patchouli leaf and licorice;

[0087] Preparation of reference solutions: Weigh appropriate amounts of geniposide, cimicifugain, 5-O-methylvisamiloside, glycyrrhizin, ammonium glycyrrhizate, cimicifugain, and 5-p-hydroxymaltol reference standards, and add methanol solution to prepare reference solutions with concentrations of 5~200 μg / ml for each of geniposide, cimicifugain, 5-O-methylvisamiloside, glycyrrhizin, ammonium glycyrrhizate, cimicifugain, and 5-p-hydroxymaltol.

[0088] The fingerprint spectrum of the traditional Chinese medicine compound was obtained based on the results of high performance liquid chromatography detection of the test solution and the reference solution.

[0089] The chromatographic conditions for this high-performance liquid chromatography (HPLC) detection were as follows: a column packed with octadecylsilane-bonded silica gel was used; mobile phase A was selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B was an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program was as follows: 0–6 min, 97%B→85%B; 6–20 min, 85%B→79%B; 20–23 min, 79%B→76%B; 23–26 min, 76%B→75%B; 26–40 min, 75%B→62%B; 40–55 min, 62%B→32%B; 55–60 min, 32%B→20%B; 60–62 min, 20%B→97%B; 62–75 min, 97%B; the flow rate was 0.92–1.08 ml / min; the column temperature was 27–33 °C; and the detection wavelength was 200–400 nm. nm, injection volume is 1~20 μl.

[0090] In this invention, when time, flow rate, column temperature, wavelength, injection volume, ratio, volume, volume percentage concentration, frequency, power, concentration, or other values ​​or parameters are expressed as ranges, preferred ranges, or a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “200~400” is disclosed, the described range should be interpreted as including ranges “200~400”, “200~350”, “200~300”, “200~250”, “250~400”, “250~350”, “250~300”, “300~400”, “300~350”, “350~400”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0091] In a preferred embodiment, the mass ratio of the gypsum, gardenia kernel, saposhnikovia root, patchouli leaf and licorice is (15~25):(2~10):(130~200):(20~40):(80~160).

[0092] In a preferred embodiment, the mass ratio of the gypsum, gardenia kernel, saposhnikovia root, patchouli leaf, and licorice is about 20.65: about 4.13: about 165.2: about 28.91: about 123.9.

[0093] In a preferred embodiment, the mass ratio of the gypsum, gardenia kernel, saposhnikovia root, patchouli leaf and licorice is (1~10):(0.5~5):(10~70):(2~20):(10~50).

[0094] In a preferred embodiment, the mass ratio of the gypsum, gardenia kernel, saposhnikovia root, patchouli leaf, and licorice is about 5: about 1: about 40: about 7: about 30.

[0095] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20.65" includes ±5% of 20.65, or from 19.6175 to 21.6825; "about 4.13" includes ±5% of 4.13, or from 3.9235 to 4.3365; "about 165.2" includes ±5% of 165.2, or from 156.94 to 173.46; "about 28.91" includes ±5% of 28.91, or from 27.4645 to 30.3555; "about 12" includes ±5% of 28.91, or from 27.4645 to 30.3555; "about 12" includes ±5% of 20.65, or from 19.6175 to 21.6825; "about 4.13" includes ±5% of 4.13, or from 3.9235 to 4.3365; "about 12" includes ±5% of 20.65, or from 19.6175 to 21.6825; "about 4.13" includes ±5% of 20.65, or from 19.6175 to 21.6825; "about 4.13" includes ±5% of 28.91, or from 27.4645 to 30.3555; "about 12" includes ±5% of 20.65, or from 19.6175 to 21.6825; "about 12" includes ±5% of 20.65, or from 19.6175 to 21.6825; "about 12" includes ±5% of 20.65, or from 3.9” includes 123.9 ± 5%, or from 117.705 to 130.095; “about 5” includes 5 ± 5%, or from 4.75 to 5.25; “about 1” includes 1 ± 5%, or from 0.95 to 1.05; “about 40” includes 40 ± 5%, or from 38 to 42; “about 7” includes 7 ± 5%, or from 6.65 to 7.35; “about 30” includes 30 ± 5%, or from 28.5 to 31.5.

[0096] In a preferred embodiment, the windproof is a baked windproof.

[0097] In a preferred embodiment, the mass / volume (g / ml) ratio of the traditional Chinese medicine decoction containing gardenia seeds to the first solvent is 0.5 to 2, for example, about 1.

[0098] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1" includes ±5% of 1, or from 0.95 to 1.05.

[0099] In a preferred embodiment, the mass of the traditional Chinese medicine decoction containing gardenia seeds is 20-30 g, for example, about 25 g.

[0100] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0101] In a preferred embodiment, the preparation method of the traditional Chinese medicine compound decoction includes the following steps: take appropriate amounts of each medicinal herb slices, crush them into coarse particles, weigh out the crushed gypsum, gardenia, patchouli leaves, saposhnikovia root (baked), and licorice root, mix them evenly, add honey wine to moisten, stir-fry, cool, pulverize, and pass through a No. 4 sieve; take one dose of the prescription amount, add solvent, cover, heat over high heat to boiling, then simmer over low heat to obtain a concentrated liquid, filter while hot to obtain the traditional Chinese medicine compound decoction.

[0102] In a preferred embodiment, the concentration of the mead is 10-40%.

[0103] In a preferred embodiment, the ratio of honey to wine in the mead is approximately 1:approximately 1.

[0104] In a preferred embodiment, the soaking time is 1 to 4 hours.

[0105] In a preferred embodiment, the frying temperature is 50°C to 80°C.

[0106] In a preferred embodiment, the frying time is 30 to 90 minutes.

[0107] In a preferred embodiment, the solvent is water.

[0108] In a preferred embodiment, the mass ratio of the prescription dose to the mass / volume ratio of the solvent is 0.02 to 0.03, in g / mL.

[0109] In a preferred embodiment, the simmering time is 30 to 60 minutes.

[0110] In a preferred embodiment, the volume ratio of the concentrate to the solvent is 0.4 to 0.6.

[0111] In a preferred embodiment, the volume of the first solvent is 20-30 ml, for example, about 25 ml.

[0112] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 25" includes ±5% of 25, or from 23.75 to 26.25.

[0113] In a preferred embodiment, the first solvent is an ethanol solution.

[0114] In a preferred embodiment, the volume percentage concentration of the ethanol solution is 30% to 70%, for example, about 50%.

[0115] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50%" includes 50% ± 5%, or from 47.5% to 52.5%.

[0116] In a preferred embodiment, the extraction method is ultrasonic extraction or reflux extraction, for example, ultrasonic extraction.

[0117] In a preferred embodiment, the frequency of the ultrasonic extraction is 20-60 kHz, for example, about 40 kHz.

[0118] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 40" includes ±5% of 40, or from 38 to 42.

[0119] In a preferred embodiment, the power of the ultrasonic extraction is 200-600 W, for example, about 500 W.

[0120] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 500" includes ±5% of 500, or from 475 to 525.

[0121] In a preferred embodiment, the ultrasonic extraction time is 15 to 60 minutes, for example, about 30 minutes.

[0122] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0123] In a preferred embodiment, the methanol solution has a volume percentage concentration of 30% to 70%, for example, about 50%, in the preparation of the reference solution.

[0124] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 50%" includes 50% ± 5%, or from 47.5% to 52.5%.

[0125] In a preferred embodiment, the concentration of geniposide in the reference solution is about 20 μg / ml.

[0126] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 20" includes ±5% of 20, or from 19 to 21.

[0127] In a preferred embodiment, the concentration of cimicifugoside in the reference solution is about 30 μg / ml.

[0128] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0129] In a preferred embodiment, the concentration of 5-O-methylvisamidol in the reference solution is about 30 μg / ml.

[0130] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0131] In a preferred embodiment, the concentration of glycyrrhizin in the reference solution is about 30 μg / ml.

[0132] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0133] In a preferred embodiment, the concentration of ammonium glycyrrhizate in the reference solution is about 150 μg / ml.

[0134] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 150" includes ±5% of 150, or from 142.5 to 157.5.

[0135] In a preferred embodiment, the concentration of cimicifugain in the reference solution is about 10 μg / ml.

[0136] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0137] In a preferred embodiment, the concentration of 5-p-hydroxymaltol in the reference solution is about 10 μg / ml.

[0138] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0139] In a preferred embodiment, the flow rate of the high-performance liquid chromatography (HPLC) detection is 0.94 to 1.06 ml / min, for example, about 1.0 ml / min.

[0140] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 1.0" includes ±5% of 1.0, or from 0.95 to 1.05.

[0141] In a preferred embodiment, the column temperature is 28~32°C, for example, about 30°C.

[0142] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 30" includes 30 ±5%, or from 28.5 to 31.5.

[0143] In a preferred embodiment, the detection wavelength is approximately 284 nm between the 0th and 10th minute.

[0144] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 284" includes ±5% of 284, or from 269.8 to 298.2.

[0145] In a preferred embodiment, the detection wavelength is approximately 238 nm between the 10th and 27th minutes.

[0146] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 238" includes ±5% of 238, or from 226.1 to 249.9.

[0147] In a preferred embodiment, the detection wavelength is approximately 370 nm between the 27th and 35th minutes.

[0148] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 370" includes ±5% of 370, or from 351.5 to 388.5.

[0149] In a preferred embodiment, the detection wavelength is approximately 254 nm between the 35th and 70th minutes.

[0150] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 254" includes ±5% of 254, or from 241.3 to 266.7.

[0151] In a preferred embodiment, the injection volume is 5 to 15 μl, for example, about 10 μl.

[0152] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 10" includes ±5% of 10, or from 9.5 to 10.5.

[0153] In a preferred embodiment, the theoretical plate number of the chromatographic peak corresponding to the 5-O-methylvisamidol glycoside is not less than 10,000.

[0154] In a preferred embodiment, the chromatographic column is a YMC-Pack ODS-A column or a ZORBAX SB-C18 column.

[0155] In a preferred embodiment, the chromatographic column has the following specifications: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

[0156] In a preferred embodiment, the mobile phase A is acetonitrile.

[0157] In a preferred embodiment, the acid aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution are selected from one or more weak acids and their salts, and weak bases and their salts of different concentrations.

[0158] In a preferred embodiment, the acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution is selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid.

[0159] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 1% acidic aqueous solution.

[0160] In a preferred embodiment, the acidic aqueous solution is a 0.01% to 0.2% phosphoric acid aqueous solution.

[0161] In a preferred embodiment, the acidic aqueous solution is an aqueous solution of about 0.05% phosphoric acid.

[0162] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.05%" includes 0.05% ± 5%, or from 0.0475% to 0.0525%.

[0163] In a preferred embodiment, the buffer salt solution is a phosphate solution and / or a phosphate solution.

[0164] In a preferred embodiment, the pH value of the buffer saline solution is not greater than 7.0.

[0165] In a preferred embodiment, when the detection wavelength is 284 nm between 0 and 10 minutes, 238 nm between 10 and 27 minutes, 370 nm between 27 and 35 minutes, and 254 nm between 35 and 70 minutes, the fingerprint spectrum includes peaks 1-16, with retention times corresponding to approximately 7.288 min, 8.514 min, 11.058 min, 12.546 min, 13.254 min, 16.129 min, 20.699 min, 21.301 min, 22.030 min, 24.691 min, 30.582 min, 32.140 min, 35.108 min, 41.861 min, 45.170 min, and 46.890 min, respectively. min.

[0166] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 7.288" includes ±5% of 7.288, or from 6.9236 to 7.6524; "about 8.514" includes ±5% of 8.514, or from 8.0883 to 8.9397; "about 11.058" includes ±5% of 11.058, or from 10.5051 to 11.6109; "about 12.546" includes ±5% of 12.546, or from 11.9187 to 13.1733; "about 13..." "254" includes 13.254 ± 5%, or from 12.5913 to 13.9167; "about 16.129" includes 16.129 ± 5%, or from 15.32255 to 16.93545; "about 20.699" includes 20.699 ± 5%, or from 19.66405 to 21.73395; "about 21.301" includes 21.301 ± 5%, or from 20.23595 to 22.36605; "Approximately 22.030" includes ±5% of 22.030, or from 20.9285 to 23.1315; "Approximately 24.691" includes ±5% of 24.691, or from 23.45645 to 25.92555; "Approximately 30.582" includes ±5% of 30.582, or from 29.0529 to 32.1111; "Approximately 32.140" includes ±5% of 32.140, or from 30.533 to 33.747; "Approximately 35.108" includes 35.108 ± 5%, or from 33.3526 to 36.8634; "Approximately 41.861" includes 41.861 ± 5%, or from 39.76795 to 43.95405; "Approximately 45.170" includes 45.170 ± 5%, or from 42.9115 to 47.4285; "Approximately 46.890" includes 46.890 ± 5%, or from 44.5455 to 49.2345.

[0167] In a preferred embodiment, the detection wavelength is 284 nm between 0 and 10 minutes, 238 nm between 10 and 27 minutes, 370 nm between 27 and 35 minutes, and 254 nm between 35 and 70 minutes. At nm, the fingerprint spectrum includes peaks 1-16, with peak 10 serving as a reference peak. The average relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, and 16 are approximately 0.30, 0.34, 0.45, 0.51, 0.54, 0.65, 0.84, 0.86, 0.89, 1.24, 1.30, 1.42, 1.70, 1.83, and 1.90, respectively.

[0168] In this invention, "about" refers to a value within a range of ±5% of a specific value. For example, "about 0.30" includes ±5% of 0.30, or from 0.285 to 0.315; "about 0.34" includes ±5% of 0.34, or from 0.323 to 0.357; "about 0.45" includes ±5% of 0.45, or from 0.4275 to 0.4725; "about 0.51" includes ±5% of 0.51, or from 0.4845 to 0.5355; "about 0.54" includes ±5% of 0.54, or from 0.513 to 0.567; "about 0.65" includes ±5% of 0.65, or from 0.6175 to 0.6825; "about 0.84" includes ±5% of 0.84, or from 0.798 to 0.882; "about 0.86" includes 0.86... "About 0.89" includes ±5% of 0.89, or from 0.8455 to 0.9345; "About 1.24" includes ±5% of 1.24, or from 1.178 to 1.302; "About 1.30" includes ±5% of 1.30, or from 1.235 to 1.365; "About 1.42" includes ±5% of 1.42, or from 1.349 to 1.491; "About 1.70" includes ±5% of 1.70, or from 1.615 to 1.785; "About 1.83" includes ±5% of 1.83, or from 1.7385 to 1.9215; "About 1.90" includes ±5% of 1.90, or from 1.805 to 1.995.

[0169] In a preferred embodiment, when the detection wavelength is 284 nm between 0 and 10 minutes, 238 nm between 10 and 27 minutes, 370 nm between 27 and 35 minutes, and 254 nm between 35 and 70 minutes, peak 1 is 5-p-hydroxymaltol, peak 5 is geniposide, peak 6 is cimicifugoside, peak 7 is glycyrrhizin, peak 8 is cimicifugoside, peak 10 is 5-O-methylvisamidol glycoside, and peak 16 is glycyrrhizic acid.

[0170] In a preferred embodiment, the detection wavelength is 284 nm between 0 and 10 minutes, 238 nm between 10 and 27 minutes, 370 nm between 27 and 35 minutes, and 254 nm between 35 and 70 minutes. At nm, peak 1 comes from licorice root, gardenia seed, and saposhnikovia root; peak 2 comes from licorice root, gardenia seed, patchouli leaf, gypsum, and saposhnikovia root; peak 3 comes from gardenia seed; peak 4 comes from licorice root; peak 5 comes from gardenia seed; peak 6 comes from saposhnikovia root; peak 7 comes from licorice root; peak 8 comes from licorice root; peak 9 comes from saposhnikovia root; peak 10 comes from saposhnikovia root; peak 11 comes from licorice root; peak 12 comes from licorice root; peak 13 comes from licorice root; peak 14 comes from licorice root; peak 15 comes from licorice root; and peak 16 comes from licorice root.

[0171] According to another aspect of the present invention, a quality control method for a traditional Chinese medicine compound containing gardenia seeds is provided, the quality control method comprising the following steps:

[0172] (1) Establish standard fingerprint spectra of traditional Chinese medicine compound reference samples based on the above fingerprint spectra construction method;

[0173] (2) Take the test solution of the traditional Chinese medicine compound and perform detection according to the chromatographic conditions in the above fingerprint chromatogram construction method to obtain the fingerprint chromatogram of the traditional Chinese medicine compound sample; and

[0174] (3) Compare the fingerprint spectrum of the Chinese medicine compound sample to be tested obtained in step (2) with the standard fingerprint spectrum of the Chinese medicine compound reference sample obtained in step (1). If it meets the requirements, it is a qualified product; if it does not meet the requirements, it is an unqualified product.

[0175] In a preferred embodiment, the compliance requirement includes one or more of the following:

[0176] (1) The fingerprint spectrum of the Chinese herbal compound sample showed 16 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak was within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint spectrum of the Chinese herbal compound reference sample.

[0177] (2) Taking the 5-O-methylvisamidol glycoside peak as the S peak, the relative retention times of each characteristic chromatographic peak in the fingerprint chromatogram of the Chinese herbal compound sample to be tested and the S peak are within ±10% of the relative retention times of each characteristic chromatographic peak in the standard fingerprint chromatogram of the Chinese herbal compound reference sample; and

[0178] (3) According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the fingerprint of the sample to be tested of the traditional Chinese medicine compound and the standard fingerprint of the reference sample of the traditional Chinese medicine compound shall not be less than 0.90.

[0179] According to another aspect of the present invention, the above-described construction method or the above-described quality control method is provided for use in the quality testing and / or quality evaluation and / or quality control of traditional Chinese medicine compound containing gardenia seeds.

[0180] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or conditions recommended by the manufacturer.

[0181] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention. The preferred embodiments and materials described herein are for illustrative purposes only.

[0182] The features mentioned above in this invention, or the features mentioned in the embodiments, can be combined arbitrarily. All features disclosed in this patent specification can be used in any compositional form, and each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.

[0183] Example

[0184] 1. Instruments, reagents, and reference standards

[0185] 1.1 Experimental Apparatus

[0186] The experimental apparatus is shown in Table 1.

[0187] Table 1 Summary of Instruments and Equipment

[0188]

[0189] 1.2 Experimental Reagents and Chemicals

[0190] The reference standards and reagents are shown in Table 2.

[0191] Table 2 Summary of Reference Standards and Reagents

[0192]

[0193] The sample information table is shown in Table 3.

[0194] Table 3 Sample Information Table

[0195]

[0196] 2. Establishment of chromatographic conditions

[0197] The following experiments were conducted using the reference sample decoction 1.

[0198] 2.1 Screening of Gradient Elution Conditions

[0199] To comprehensively observe the characterization of each component in the reference sample, and considering the characterization of water-soluble and alcohol-soluble components, a preliminary method for preparing the reference sample containing Gardenia jasminoides powder and its characteristic chromatogram is proposed: Take approximately 1.0 g of the powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, seal tightly, weigh, sonicate (500 W, 40 kHz) for 30 minutes, cool, weigh again, replenish the lost weight with 50% methanol, shake well, filter, and collect the filtrate.

[0200] The characteristic chromatograms of the intermediates of the reference sample were explored, and appropriate chromatographic conditions were set. When acetonitrile (A)-0.1% phosphoric acid aqueous solution (B) was used as the mobile phase, gradient elution was performed according to the provisions in Tables 4 to 8, the flow rate was 1.0 ml per minute, the column temperature was 30℃, and the detection was performed with an ultraviolet detector.

[0201] Table 4 Elution gradient 1

[0202]

[0203] Under the elution gradient 1 conditions in Table 4, the chromatographic peaks are mainly concentrated between 10 and 35 minutes and the resolution of each chromatographic peak is poor. Therefore, the elution program is adjusted within this time period.

[0204] Table 5 Elution gradient 2

[0205]

[0206] Under the elution gradient 2 conditions in Table 5, the chromatographic peaks are still mainly concentrated in the 10-35 minute range and the resolution of each chromatographic peak is still poor. Therefore, the elution program is adjusted for this time period.

[0207] Table 6 Elution gradient 3

[0208]

[0209] Under elution gradient 3 in Table 6, there was significant co-elution of the chromatographic peak around 20 minutes and 30 minutes. Therefore, the elution program was adjusted for this time period.

[0210] Table 7 Elution gradient 4

[0211]

[0212] Under elution gradient 4 in Table 7, there was significant co-elution of the chromatographic peak (geniposide) around 17 minutes and the chromatographic peak (citric acid glycoside) around 20 minutes. Therefore, the elution program was adjusted for this time period.

[0213] Table 8 Elution gradient 5

[0214]

[0215] Under the elution gradient 5 conditions in Table 8, the chromatographic peaks responded well at 284 nm before 10 minutes, at 238 nm from 10 to 27 minutes, at 370 nm from 27 to 35 minutes, and at 254 nm from 35 to 75 minutes; the resolution of each chromatographic peak met the requirement of >1.5.

[0216] 2.2 Selection of detection wavelength

[0217] Elution was performed using the mobile phase gradient described above. The characteristic chromatogram of the traditional Chinese medicine composition containing gardenia kernel was detected using a PDA full-wavelength scan. The wavelength used for detecting the characteristic chromatogram of the traditional Chinese medicine composition containing gardenia kernel was determined based on the absorption wavelength (maximum) of each chromatographic peak.

[0218] 3D wavelength diagram of the characteristic spectral results of the reference sample containing gardenia seed of traditional Chinese medicine composition (e.g.) Figure 1 As shown in the figure, each chromatographic peak exhibits the maximum absorption wavelength at 284nm, 238nm, 370nm, and 254nm.

[0219] Under the preliminary experimental gradient conditions, the chromatographic peaks responded well at 284 nm for the first 10 minutes, at 238 nm for 10–27 minutes, at 370 nm for 27–35 minutes, and at 254 nm for 35–75 minutes. Since the content and characteristics share the same chromatographic conditions, the wavelength switching should be considered to characterize each index component to the greatest extent possible.

[0220] 2.3 Preliminary determination of chromatographic conditions

[0221] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.5 mm, particle size 5 µm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B, with gradient elution performed according to elution condition 5 in Table 8; the flow rate was 1.0 mL per minute, the column temperature was 30 °C, and the detection wavelengths were 284 nm for 0–10 minutes, 238 nm for 10–27 minutes, 370 nm for 27–35 minutes, and 254 nm for 35–75 minutes.

[0222] Preparation of the test solution: Take 1.0 g of the powder, accurately weigh it, place it in a stoppered conical flask, accurately add 25 ml of 50% methanol, weigh it, sonicate for 30 minutes (frequency 40 kHz, power 500 W), cool it, weigh it again, make up the weight loss with 50% methanol, shake well, filter it, and take the filtrate to obtain the test solution.

[0223] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0224] As can be seen from the above chromatograms, the chromatographic peaks are well separated and have good peak shapes in the acetonitrile-0.1% phosphoric acid solution mobile phase system. Therefore, this gradient method is tentatively selected as the chromatographic gradient method for the characteristic chromatogram of the reference sample.

[0225] 2.4 Selection of mobile phase composition

[0226] Elution was performed under the mobile phase conditions described above. The effects of different types of acids commonly found in the literature (phosphoric acid, formic acid) on the system suitability parameters (peak shape, resolution, symmetry factor, etc.) of the characteristic peaks of the reference sample containing gardenia kernel were investigated. Appropriate chromatographic parameters were selected based on the results.

[0227] Analysis of the above chromatograms and tables shows that the overall response of phosphoric acid and formic acid is not significantly different. Considering that the content determination and characteristic chromatograms share the same chromatographic conditions, and based on the differences in the content determination results, phosphoric acid was selected as the mobile phase.

[0228] 2.5 Investigation of different acid concentrations

[0229] Elution was performed under the mobile phase conditions described above. The effects of different phosphoric acid concentrations (0.2%, 0.1%, and 0.05%) on the characteristic chromatograms of the traditional Chinese medicine composition containing gardenia seeds were investigated, and appropriate chromatographic parameters were selected based on the results.

[0230] Analysis of the above spectral and table data shows that the overall responses of each characteristic peak are relatively close when the phosphoric acid concentration is 0.05%, 0.10%, and 0.20%, respectively. However, when the phosphoric acid concentration is 0.05%, the separation effect of each characteristic peak is better than that of other phosphoric acid concentrations. Therefore, 0.05% phosphoric acid is selected as the mobile phase analysis condition for this method.

[0231] 2.6 Investigation of different flow velocities

[0232] Elution was performed under the mobile phase conditions described above. The effects of different flow rates (0.8 ml / min, 1.0 ml / min, 1.2 ml / min) on the characteristic chromatograms of the traditional Chinese medicine composition containing gardenia seed (peak shape, resolution, symmetry factor, content determination results, etc.) were investigated, and appropriate chromatographic parameters were selected based on the results.

[0233] Analysis of the above chromatograms and table data shows that when the flow rate is 0.8 ml / min, the overall response of each characteristic peak is high, but the separation is poor. When the flow rate is 1.2 ml / min, the overall response of each characteristic peak is low. When the flow rate is 1.0 ml / min, the overall response is moderate, and the separation effect of each characteristic peak is better than that of other flow rates. Therefore, a flow rate of 1.0 ml / min is selected as the analytical condition for this method.

[0234] 2.7 Investigation of different column temperatures

[0235] Elution was performed using the mobile phase gradient described above. The effects of different column temperatures (25℃, 30℃, 35℃) on the characteristic chromatograms of the traditional Chinese medicine composition containing gardenia seeds (peak shape, resolution, symmetry factor, etc.) were investigated, and appropriate chromatographic parameters were selected based on the results.

[0236] Analysis of the above spectral and table data shows that when the column temperature is 25℃, the overall separation of each characteristic peak is poor, with peaks 8 and 9 exhibiting a peak-enclosing phenomenon. When the column temperature is 35℃, the overall separation of each characteristic peak is poor. When the column temperature is 30℃, the overall separation of each characteristic peak is good, and the overall response is moderate. Therefore, a column temperature of 30℃ is selected as the analytical condition for this method.

[0237] 2.8 Determination of chromatographic conditions

[0238] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.5 mm, particle size 5 µm); acetonitrile was used as mobile phase A, and 0.05% phosphoric acid was used as mobile phase B, with gradient elution performed according to elution condition 5 in Table 8; the flow rate was 1.0 mL per minute, the column temperature was 30 °C, and the detection wavelengths were 284 nm for 0–10 minutes, 238 nm for 10–27 minutes, 370 nm for 27–35 minutes, and 254 nm for 35–75 minutes.

[0239] 3. Investigation of sample processing methods

[0240] 3.1 Examination of Extraction Methods

[0241] Accurately weigh 25g of the decoction and place it in a round-bottom flask. Concentrate to near dryness, then accurately add 25ml of 50% methanol. Weigh the solution, reflux for 30 minutes, and then sonicate (500W, 40kHz) for 30 minutes. Cool, weigh again, and replenish the lost weight with 50% methanol. Shake well, filter, and collect the filtrate. Determine the chromatogram of the reference sample under the above chromatographic conditions, investigate the effect of different extraction methods on the characteristic chromatogram, and select appropriate chromatographic parameters based on the results.

[0242] The results of total peak area / sample amount show that the extraction effects of reflux and ultrasonic extraction on the decoction of traditional Chinese medicine composition containing gardenia kernel are similar, and the overall response of each characteristic peak is good. Considering the simplicity of the experiment, ultrasonic extraction method was selected.

[0243] 3.2 Ultrasonic Power Assessment

[0244] The effects of ultrasonic power on the characteristic chromatogram of the reference sample were investigated at 200W, 350W, and 500W. Approximately 25g of the decoction was accurately weighed and placed in a round-bottom flask. The solution was concentrated to near dryness, and 25ml of 50% methanol was accurately added. The mixture was weighed, and the solution was ultrasonically treated at 200W, 350W, and 500W for 30 minutes respectively. After cooling, the solution was weighed again, and the lost weight was replenished with 50% methanol. The mixture was mixed thoroughly, filtered, and the filtrate was collected. The effect of ultrasonic power on the characteristic chromatogram of the reference sample was investigated under the above chromatographic conditions. Appropriate parameters were selected based on the results.

[0245] The results showed that the RSD value of total peak area / sample size was less than 5.0% under different ultrasonic powers. Therefore, in order to reduce experimental error, the ultrasonic power was selected as 500W.

[0246] 3.3 Investigation of Extraction Solvents

[0247] The use of extraction solvents was investigated to evaluate the characteristic chromatogram of the decoction of the reference sample. Methanol, anhydrous ethanol, 50% methanol, and 50% ethanol were tested as extraction solvents, and a suitable extraction solvent was determined based on the results. Approximately 25.0 g of the decoction was accurately weighed and placed in a round-bottom flask. The solution was concentrated to near dryness, and 25 ml of each of the above-mentioned solvents was added. The mixture was treated for 30 minutes according to the determined extraction method. After cooling, the solution was weighed again, and the weight loss was replenished with the appropriate solvent. The mixture was mixed thoroughly and filtered to obtain the final product. The effect of different extraction solvents on the characteristic chromatogram of the reference sample decoction was investigated, and appropriate parameters were selected based on the results.

[0248] The results showed that, based on the total peak area / sample amount, anhydrous ethanol had a weaker extraction effect than the other three extraction solvents, while methanol, 50% methanol, and 50% ethanol had similar extraction effects. Considering that ethanol is less toxic than methanol, 50% ethanol was selected as the extraction solvent.

[0249] 3.4 Investigation into the amount of extraction solvent added

[0250] To investigate the effect of different amounts of extraction solvent on the characteristic chromatogram of the reference sample decoction, the amount of extraction solvent added was examined during the preparation of the test sample. Approximately 25.0 g of the decoction was accurately weighed, concentrated to near dryness, and then 25 ml, 50 ml, and 100 ml of 50% ethanol were accurately added respectively. The samples were weighed, sonicated for 30 minutes, cooled, and weighed again. The weight loss was replenished with 50% ethanol, mixed thoroughly, and filtered to obtain the final sample. The chromatogram was determined under the above chromatographic conditions to investigate the effect of different amounts of extraction solvent added on the characteristic chromatogram, and appropriate parameters were selected based on the results.

[0251] The results showed that when 25 ml, 50 ml, and 100 ml of 50% ethanol were added respectively, the total peak area × dilution factor was almost the same, indicating that the extraction effect was similar. In order to save experimental costs, the amount of solvent added was selected to be 25 ml.

[0252] 3.5. Examination of extraction time

[0253] To investigate the extraction time of the sample for preparing the characteristic chromatogram of the reference sample (decoction), approximately 25.0 g of the decoction was accurately weighed, concentrated to near dryness, and 25 mL of 50% ethanol was accurately added. The sample was then weighed and sonicated for 15, 30, and 60 minutes, respectively. After cooling, the sample was weighed again, and the weight loss was replenished with 50% ethanol. The mixture was then stirred and filtered to obtain the final sample. The chromatogram of the reference sample (decoction) was determined under the above chromatographic conditions to investigate the effect of different extraction times on the characteristic chromatogram of the reference sample (decoction), and appropriate parameters were selected based on the results.

[0254] The results showed that the total peak area / sample size ratios obtained by sonication for 15 min, 30 min, and 60 min were similar. Considering both factors, a sonication time of 30 min was selected to improve experimental efficiency and reduce experimental error.

[0255] 3.6 Sampling Quantity Examination

[0256] To investigate whether the reference sample (decoction) was completely extracted under different sampling amounts, approximately 20g, 25g, and 30g of the decoction were accurately weighed, concentrated to near dryness, and 25ml of 50% ethanol was accurately added. The mixture was then weighed, sonicated for 30 minutes, removed, cooled, and weighed again. The weight loss was replenished with 50% ethanol, mixed well, and filtered to obtain the final sample. The effect of different sampling amounts on the characteristic chromatogram of the reference sample (decoction) was investigated, and appropriate parameters were selected based on the results.

[0257] The results showed that when the sample amounts were 20g, 25g and 30g, the measured total peak area / sample amount results were similar. However, when the sample amount was 25g, the overall response of each characteristic peak was better than that of other sample amounts. Therefore, the sample amount of 25g was selected.

[0258] 3.7 Determination of System Adaptability Conditions and Test Sample Treatment Methods

[0259] Chromatographic conditions and system suitability tests were performed using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.5 mm, particle size 5 µm); acetonitrile was used as mobile phase A, and 0.1% phosphoric acid was used as mobile phase B, with gradient elution performed according to elution condition 5 in Table 8; the flow rate was 1.0 mL per minute, the column temperature was 30 °C, and the detection wavelengths were 284 nm for 0–10 minutes, 238 nm for 10–27 minutes, 370 nm for 27–35 minutes, and 254 nm for 35–70 minutes.

[0260] Preparation of reference solution: Accurately weigh appropriate amounts of geniposide, cimicifugain, glycyrrhizin, 5-O-methylvisamiloside, and ammonium glycyrrhizate reference standards. Add 50% methanol to prepare a mixed solution containing 20 μg of geniposide, 30 μg of cimicifugain, 30 μg of glycyrrhizin, 30 μg of 5-O-methylvisamiloside, and 0.15 mg of ammonium glycyrrhizate per 1 ml. Shake well. (Weight of glycyrrhizic acid = weight of ammonium glycyrrhizate / 1.0207).

[0261] Preparation of the test solution: Take 25.0g of the decoction of this product, accurately weigh it, concentrate it to near dryness, accurately add 25ml of 50% ethanol, weigh it, sonicate it for 30 minutes (frequency 40kHz, power 500W), cool it, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0262] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0263] 3.8. Assignment of major chromatographic peaks in characteristic chromatograms

[0264] Preparation of Mixed Reference Solution 1: Accurately weigh appropriate amounts of geniposide, cimicifugain, cimicifugain, 5-O-methylvisamilol, 5-p-hydroxymaltol, glycyrrhizin, and ammonium glycyrrhizate reference standards. Add 50% methanol to prepare a mixed solution containing 20 μg geniposide, 30 μg cimicifugain, 10 μg cimicifugain, 30 μg glycyrrhizin, 30 μg 5-O-methylvisamilol, 60 μg 5-p-hydroxymaltol, and 0.15 mg ammonium glycyrrhizate per 1 ml. Shake well to obtain the solution.

[0265] Preparation of Mixed Reference Solution 2: Take appropriate amounts of patchouli flavonol and radiuxin flavonol reference standards, accurately weigh them, add 50% methanol to prepare a mixed solution containing 10 μg of patchouli flavonol and 10 μg of radiuxin flavonol per 1 ml, shake well, and the solution is ready.

[0266] Prepare blank solution, reference sample solution, gypsum negative test solution, gypsum single-herb test solution, gardenia seed negative test solution, gardenia seed single-herb test solution, roasted saposhnikovia root negative test solution, roasted saposhnikovia root single-herb test solution, patchouli leaf negative test solution, patchouli leaf single-herb test solution, licorice negative test solution, licorice single-herb test solution, and reference solution according to the test sample preparation method. Inject and analyze using the [characteristic chromatogram] determination method and record the chromatograms.

[0267] The results showed that peaks 4, 7, 8 (glycyrrhizin), 11, 12, 13, 14, 15, 16, 17, 18 (glycyrrhizic acid), and 19 were specific components for the medicinal flavor of licorice; peaks 3 and 5 (geniposide) were specific components for the medicinal flavor of gardenia seed; peaks 6 (cipridin), 9 (cipridin), and 10 (5-O-methylvisamidol) were specific components for the medicinal flavor of saposhnikovia root; and peak 1 (5-p-hydroxymethyl... Peak 1 is a common peak of licorice, gardenia seed, and saposhnikovia root (a chromatographic peak produced after stir-frying with honey wine). Peak 2 is a common peak of licorice, gardenia seed, patchouli leaf, gypsum, and saposhnikovia root (a chromatographic peak produced after stir-frying with honey wine). Peak 20 is a common peak of licorice and saposhnikovia root. Considering that patchouli flavonol and radiucin flavonol have poor water solubility and did not transfer from the fine powder to the decoction, these components were not included in the characteristic chromatogram of the reference sample (decoction). There was no interference from the lack of negative reference samples and blank controls.

[0268] 4. Methodological Validation

[0269] 4.1 Specificity

[0270] Take the test solution, blank control solution, and reference solution, and perform HPLC analysis under the determined chromatographic conditions, and record the chromatograms.

[0271] The results showed that the blank solution had no interference, and the chromatographic peaks of the reference standard had good shape and excellent separation, thus the method has good specificity.

[0272] 4.1.1 Instrument precision test

[0273] Take the same sample solution and inject it 6 times consecutively under the conditions determined above, and determine the relative retention time and relative peak area of ​​20 characteristic peaks.

[0274] The results above show that, under six consecutive injections of the same sample, the relative retention time (RSD) of each characteristic peak in the characteristic spectrum of the traditional Chinese medicine composition (decoction) containing gardenia kernel was less than 5.0%, and the relative peak area (RSD) was between 0% and 9.8%. Considering that no peak area standard has been established, the standard for relative peak area (RSD) can be appropriately relaxed, thus indicating that the instrument precision of this method is good.

[0275] 4.1.2 Intermediate precision test (different personnel / different instruments)

[0276] Six samples (decoctions) of the same batch of traditional Chinese medicine composition containing gardenia kernels were taken by experimenters A and B respectively. They were processed according to the test sample preparation method, and the same chromatographic column was used on different instruments to determine the relative retention times of 20 peaks.

[0277] The results above show that the relative retention times of each characteristic peak measured by different personnel using different instruments are relatively small, and the RSD is less than 5.0%, indicating that the intermediate precision of this method is good.

[0278] 4.1.3 Repeatability Test

[0279] Take six reference samples (decoctions) of traditional Chinese medicine compositions containing gardenia seeds from the same batch, weigh them accurately, prepare them according to the preparation method of the test solution, and determine the relative retention time and relative peak area of ​​20 peaks.

[0280] The results showed that, in parallel determination of 6 test samples, the relative retention times (RSDs) of each characteristic peak in the characteristic spectrum of the traditional Chinese medicine composition (decoction) containing gardenia kernel were all less than 5.0%, indicating that the method has good repeatability.

[0281] 4.1.4 Durability Test

[0282] 4.1.4.1 Solution Stability

[0283] Take the same sample solution, prepare it, and inject it at 0, 4, 8, 12, 24, 36, 48, 60, and 72 hours to determine the relative retention time and relative peak area of ​​20 peaks.

[0284] The results showed that when the same test sample solution was injected and tested within 72 hours, the relative retention time (RSD) of each characteristic peak in the characteristic spectrum of the reference sample (decoction) containing gardenia seed was less than 2.0%, and the relative peak area (RSD) was between 0% and 13.6%. Considering that no peak area standard has been established, the standard for relative peak area (RSD) can be appropriately relaxed. Therefore, the test sample solution was stable within 72 hours.

[0285] 4.1.4.2 Durability under different flow rates

[0286] Take 25.0g of the same batch of Chinese herbal composition reference sample (decoction) containing gardenia seed, accurately weigh it, process it according to the test sample preparation method, and conduct the test at different flow rates of 0.9ml / min, 1.0ml / min and 1.1ml / min, and determine the relative retention time of 20 peaks.

[0287] The results showed that different flow rates had a significant impact on the relative retention time of each characteristic peak in the reference sample (decoction) of the traditional Chinese medicine composition containing gardenia kernel. It is recommended to fix the flow rate at 1.0 ml / min.

[0288] 4.1.4.3 Durability at different column temperatures

[0289] Take 25.0g of the same batch of Chinese herbal composition reference sample (decoction) containing gardenia kernel, weigh accurately, process according to the test sample preparation method, and conduct tests at different column temperatures of 27℃, 30℃ and 33℃ to determine the relative retention time of 20 peaks.

[0290] The results showed that the relative retention times of the characteristic peaks of the reference sample (decoction) of the traditional Chinese medicine composition containing gardenia kernel were relatively similar at different column temperatures, and the relative retention time RSD was less than 5.0%, indicating that the method has good column temperature robustness.

[0291] 4.1.4.4 Durability under different acid concentrations

[0292] Take 25.0g of the same batch of Chinese herbal composition reference sample (decoction) containing gardenia kernel, accurately weigh it, process it according to the test sample preparation method, and conduct the test under different acid concentrations of 0.04% phosphoric acid, 0.05% phosphoric acid and 0.06% phosphoric acid as mobile phase, and determine the relative retention time of 20 chromatographic peaks.

[0293] 4.1.4.5. Durability of different chromatographic columns

[0294] Accurately weigh 25.0g of the decoction containing gardenia seeds, process it according to the preparation method of the test sample, and conduct experiments under different chromatographic columns (Column 1: YMC-Pack ODS-A, 250*4.6mm lDS-5μm, 12nm, SN:129FB30377; Column 2: YMC-Pack ODS-A, 250*4.6mm lDS-5μm, 12nm, SN:109IB20208; Column 3: ZORBAXSB-C18 StsbleBond Analytical 4.6*250mm 5-Micron, SN:USCL122250), and determine the relative retention times of 20 peaks.

[0295] The results showed that different chromatographic columns had little effect on the relative retention time of each characteristic peak in the reference sample (decoction) of the traditional Chinese medicine composition containing gardenia kernel, and the relative retention time RSD was less than 5.0%. Therefore, the method has good robustness with different chromatographic columns.

[0296] 4.1.4.6 Determination of the number of theoretical plates

[0297] After specificity, robustness, solution stability, instrument precision, repeatability, and intermediate precision tests, this method has been proven suitable for the determination of the content of this product. The theoretical plate number of the test solution for 5-O-methylvisamidol glycoside was statistically analyzed for each validation item.

[0298] The results showed that the theoretical plate number of 5-O-methylvisamidol glycoside was above 94045 during the verification process. Therefore, the system applicability requirement of this method was adjusted to be that the theoretical plate number of 5-O-methylvisamidol glycoside is not less than 10000.

[0299] 4.1.4.7 Summary

[0300] Based on the above methodological investigation results, among the 20 common peaks of the established reference sample decoction [characteristic chromatogram], each chromatographic peak is affected to a certain extent by the change in flow rate.

[0301] 4.2 Selection of chromatographic peaks for reference samples

[0302] To ensure that no chromatographic peaks are lost during the transfer of the formulation, the characteristic chromatographic peaks of the reference sample (decoction) were selected, and unidentified peaks and peaks with excessively small peak areas (i.e., peaks 12, 14, 19, and 20) were removed. The characteristic chromatogram of the selected reference sample 1 (decoction) is shown below. Figure 2 As shown, 16 characteristic peaks were observed. Based on the actual situation, the chromatographic peak corresponding to the 5-O-methylvisamilol glycoside reference in the characteristic chromatogram of the traditional Chinese medicine composition containing gardenia seed showed a high response value, good symmetry, and excellent separation. Therefore, peak 10 (5-O-methylvisamilol glycoside) was determined as the S peak. The chemical properties of each common peak in the reference sample were analyzed, and peak 1 was identified as 5-p-hydroxymaltol, peak 5 as geniposide, peak 6 as cimicifugain glycoside, peak 8 as glycyrrhizin, peak 9 as cimicifugain, peak 10 as 5-O-methylvisamilol glycoside, and peak 16 as glycyrrhizic acid.

[0303] Based on the above conditions, in order to ensure the stability and feasibility of the method, the following method is determined:

[0304] Chromatographic conditions and system suitability tests were conducted using octadecylsilane-bonded silica gel as the packing material (column length 250 mm, inner diameter 4.5 mm, particle size 5 µm); acetonitrile was used as mobile phase A, and 0.05% phosphoric acid was used as mobile phase B, with gradient elution performed according to elution condition 5 in Table 8; the flow rate was 1.0 mL / min, the column temperature was 30 °C, and the detection wavelengths were 284 nm for 0–10 min, 238 nm for 10–27 min, 370 nm for 27–35 min, and 254 nm for 35–70 min. The theoretical plate number, calculated based on the 5-O-methylvisamidol glycoside peak, should be no less than 10,000.

[0305] Preparation of the reference solution: Accurately weigh appropriate amounts of geniposide, cimicifugain, 5-O-methylvisamiloside, glycyrrhizin, ammonium glycyrrhizate, cimicifugain, and 5-p-hydroxymaltol reference standards. Add 50% methanol to prepare a mixed solution containing 20 μg of geniposide, 30 μg of cimicifugain, 30 μg of glycyrrhizin, 30 μg of 5-O-methylvisamiloside, 0.15 mg of ammonium glycyrrhizate, 10 μg of cimicifugain, and 10 μg of 5-p-hydroxymaltol per 1 ml. Shake well. (Glycyrrhizic acid weight = ammonium glycyrrhizate weight / 1.0207).

[0306] Preparation of the test solution: Take 25.0g of the decoction of this product, accurately weigh it, concentrate it to near dryness, accurately add 25ml of 50% ethanol, weigh it, sonicate it for 30 minutes (frequency 40kHz, power 500W), cool it, weigh it again, make up the weight loss with 50% ethanol, shake it well, filter it, and take the filtrate to obtain the test solution.

[0307] The determination method involves precisely pipetting 10 μl of the reference solution and the test solution into the liquid chromatograph and measuring the results.

[0308] 5. Verification Experiment

[0309] Experimental results are as follows Figure 3 As shown.

[0310] Three samples were decocted in parallel according to the determined process. The characteristic chromatograms of the reference samples all showed the same characteristic peaks at the corresponding positions of the chromatographic peaks of geniposide, cimicifugain, 5-O-methylvisamidol, glycyrrhizin, ammonium glycyrrhizinate, cimicifugain, and 5-p-hydroxymaltol. The overall chromatograms were basically consistent and the batches were uniform and stable. The preparation process is stable and feasible.

[0311] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The descriptions of the embodiments above are only for the purpose of helping to understand the method and core ideas of the present invention. Furthermore, any changes or modifications made by those skilled in the art based on the ideas of the present invention, its specific implementation methods, and its application scope, are all within the scope of protection of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A method for constructing fingerprint profiles of a traditional Chinese medicine compound containing gardenia seeds, characterized in that, The construction method includes the following steps: Preparation of the test solution of the traditional Chinese medicine compound: Take an appropriate amount of the decoction of the traditional Chinese medicine compound containing gardenia seed, concentrate it, add the first solvent and weigh it, extract for a period of time, cool it and weigh it again, use the first solvent to make up for the lost weight, shake it well, filter it, and take the filtrate to obtain the test solution of the traditional Chinese medicine compound; wherein, the traditional Chinese medicine compound includes gypsum, gardenia seed, saposhnikovia root, patchouli leaf and licorice; Preparation of reference solutions: Weigh appropriate amounts of geniposide, cimicifugain, 5-O-methylvisamiloside, glycyrrhizin, ammonium glycyrrhizate, cimicifugain, and 5-p-hydroxymaltol reference standards, and add methanol solution to prepare reference solutions with concentrations of 5~200 μg / ml for each of geniposide, cimicifugain, 5-O-methylvisamiloside, glycyrrhizin, ammonium glycyrrhizate, cimicifugain, and 5-p-hydroxymaltol. Based on the results of high-performance liquid chromatography (HPLC) detection of the test solution and the reference solution of the traditional Chinese medicine compound, a fingerprint spectrum of the traditional Chinese medicine compound was obtained. The chromatographic conditions for high-performance liquid chromatography (HPLC) detection are as follows: a column packed with octadecylsilane-bonded silica gel is used; mobile phase A is selected from one or more of acetonitrile, methanol, and tetrahydrofuran; mobile phase B is an aqueous acid solution, an aqueous alkaline solution, and / or an aqueous buffer solution; the gradient elution program is as follows: 0–6 min, 97%B→85%B; 6–20 min, 85%B→79%B; 20–23 min, 79%B→76%B; 23–26 min, 76%B→75%B; 26–40 min, 75%B→62%B; 40–55 min, 62%B→32%B; 55–60 min, 32%B→20%B; 60–62 min, 20%B→97%B; 62–75 min, 97%B; flow rate is 0.92–1.08 ml / min; column temperature is 27–33 °C; and detection wavelength is 200–400 nm. nm, injection volume is 1~20 μl.

2. The construction method according to claim 1, characterized in that, The mass ratio of the gypsum, gardenia seed, saposhnikovia root, patchouli leaf and licorice is (15~25):(2~10):(130~200):(20~40):(80~160); Preferably, the mass ratio of the gypsum, gardenia seed, saposhnikovia root, patchouli leaf, and licorice is about 20.65: about 4.13: about 165.2: about 28.91: about 123.9; Preferably, the mass ratio of the gypsum, the gardenia seed, the saposhnikovia root, the patchouli leaf, and the licorice is (1~10):(0.5~5):(10~70):(2~20):(10~50); Preferably, the mass ratio of the gypsum, the gardenia seed, the saposhnikovia root, the patchouli leaf, and the licorice is about 5: about 1: about 40: about 7: about 30; Preferably, the windproof is a baked windproof; Preferably, the mass / volume (g / ml) ratio of the traditional Chinese medicine decoction containing gardenia seeds to the first solvent is 0.5~2, for example, about 1; Preferably, the mass of the traditional Chinese medicine decoction containing gardenia seeds is 20-30 g, for example, about 25 g; Preferably, the preparation method of the traditional Chinese medicine compound decoction includes the following steps: take appropriate amounts of each medicinal herb slices, crush them into coarse particles, weigh out the crushed gypsum, gardenia, patchouli leaves, saposhnikovia root (baked), and licorice root, mix them evenly, add honey wine to moisten, stir-fry, cool, pulverize, and pass through a No. 4 sieve; take one dose of the prescription amount, add solvent, cover, heat over high heat to boiling, then simmer over low heat to obtain a concentrated liquid, filter while hot to obtain the traditional Chinese medicine compound decoction; Preferably, the concentration of the mead is 10-40%; Preferably, the ratio of honey to wine in the mead is approximately 1:approximately 1; Preferably, the soaking time is 1-4 hours; Preferably, the frying temperature is 50°C to 80°C; Preferably, the frying time is 30-90 minutes; Preferably, the solvent is water; Preferably, the mass ratio of the prescription dose to the mass / volume ratio of the solvent is 0.02~0.03, in g / mL; Preferably, the simmering time is 30-60 minutes; Preferably, the volume ratio of the concentrate to the solvent is 0.4 to 0.6; Preferably, the volume of the first solvent is 20-30 ml, for example, about 25 ml; Preferably, the first solvent is an ethanol solution; More preferably, the volume percentage concentration of the ethanol solution is 30% to 70%, for example, about 50%; More preferably, the extraction method is ultrasonic extraction or reflux extraction, for example, ultrasonic extraction; Preferably, the frequency of the ultrasonic extraction is 20~60 kHz, for example, about 40 kHz; Preferably, the power of the ultrasonic extraction is 200~600 W, for example, about 500 W; Preferably, the ultrasonic extraction time is 15-60 minutes, for example, about 30 minutes.

3. The construction method according to claim 1, characterized in that, In the preparation of the reference solution, the volume percentage concentration of the methanol solution is 30% to 70%, for example, about 50%; More preferably, the concentration of geniposide in the reference solution is about 20 μg / ml; More preferably, the concentration of cimicifugoside in the reference solution is about 30 μg / ml; More preferably, the concentration of 5-O-methylvisamidol in the reference solution is about 30 μg / ml; More preferably, the concentration of glycyrrhizin in the reference solution is about 30 μg / ml; More preferably, the concentration of ammonium glycyrrhizate in the reference solution is about 150 μg / ml; More preferably, the concentration of cimicifugain in the reference solution is about 10 μg / ml; More preferably, the concentration of 5-p-hydroxymaltol in the reference solution is about 10 μg / ml.

4. The construction method according to claim 1, characterized in that, The flow rate for the high-performance liquid chromatography detection is 0.94~1.06 ml / min, for example, about 1.0 ml / min; More preferably, the column temperature is 28~32°C, for example, about 30°C; More preferably, the detection wavelength is approximately 284 nm between the 0th and 10th minute; More preferably, the detection wavelength is approximately 238 nm between the 10th and 27th minutes. More preferably, the detection wavelength is approximately 370 nm between the 27th and 35th minutes. More preferably, the detection wavelength is approximately 254 nm between the 35th and 70th minutes. More preferably, the injection volume is 5 to 15 μl, for example, about 10 μl; More preferably, the theoretical plate number of the chromatographic peak corresponding to the 5-O-methylvisamidol glycoside is not less than 10,000.

5. The construction method according to claim 1, characterized in that, The chromatographic column is a YMC-Pack ODS-A column or a ZORBAX SB-C18 column; Preferably, the chromatographic column has the following specifications: column length 250 mm, inner diameter 4.6 mm, and particle size 5 μm.

6. The construction method according to claim 1, characterized in that, Mobile phase A is acetonitrile; Preferably, the acid aqueous solution, alkaline aqueous solution and / or buffer salt aqueous solution are selected from one or more weak acids and their salts, weak bases and their salts of different concentrations; Preferably, the acidic aqueous solution, alkaline aqueous solution, and / or buffer salt aqueous solution are selected from formic acid, glacial acetic acid, phosphoric acid, trifluoroacetic acid, formic acid and ammonium formate, acetic acid and sodium acetate, acetic acid and ammonium acetate, disodium hydrogen phosphate and sodium dihydrogen phosphate, disodium hydrogen phosphate and potassium dihydrogen phosphate, disodium hydrogen phosphate and citric acid, citric acid and sodium citrate, glycine and hydrochloric acid, or phthalic acid and hydrochloric acid; More preferably, the acid aqueous solution is a 0.01% to 1% acid aqueous solution; More preferably, the acidic aqueous solution is a 0.01% to 0.2% phosphoric acid aqueous solution; More preferably, the acidic aqueous solution is an aqueous solution of about 0.05% phosphoric acid; Preferably, the buffer salt solution is a phosphate solution and / or a phosphate solution; Preferably, the pH value of the buffer salt solution is not greater than 7.

0.

7. The construction method according to claim 1, characterized in that, When the detection wavelength is 284 nm between 0 and 10 minutes, 238 nm between 10 and 27 minutes, 370 nm between 27 and 35 minutes, and 254 nm between 35 and 70 minutes, the fingerprint spectrum includes peaks 1-16, and the retention times of peaks 1-16 are approximately 7.288 min, 8.514 min, 11.058 min, 12.546 min, 13.254 min, 16.129 min, 20.699 min, 21.301 min, 22.030 min, 24.691 min, 30.582 min, 32.140 min, 35.108 min, 41.861 min, 45.170 min, and 46.890 min, respectively. Preferably, the detection wavelength is 284 nm between 0 and 10 minutes, 238 nm between 10 and 27 minutes, 370 nm between 27 and 35 minutes, and 254 nm between 35 and 70 minutes. At nm, the fingerprint spectrum includes peaks 1-16, with peak 10 serving as a reference peak. The average relative retention times of peaks 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, and 16 are approximately 0.30, 0.34, 0.45, 0.51, 0.54, 0.65, 0.84, 0.86, 0.89, 1.24, 1.30, 1.42, 1.70, 1.83, and 1.90, respectively. More preferably, when the detection wavelength is 284 nm between 0 minutes and 10 minutes, 238 nm between 10 minutes and 27 minutes, 370 nm between 27 minutes and 35 minutes, and 254 nm between 35 minutes and 70 minutes, peak 1 is 5-p-hydroxymaltol, peak 5 is geniposide, peak 6 is cimicifugoside, peak 7 is glycyrrhizin, peak 8 is cimicifugoside, peak 10 is 5-O-methylvisamidolol glycoside, and peak 16 is glycyrrhizic acid; More preferably, the detection wavelength is 284 nm between 0 minutes and 10 minutes, 238 nm between 10 minutes and 27 minutes, 370 nm between 27 minutes and 35 minutes, and 254 nm between 35 minutes and 70 minutes. At nm, peak 1 comes from licorice root, gardenia seed, and saposhnikovia root; peak 2 comes from licorice root, gardenia seed, patchouli leaf, gypsum, and saposhnikovia root; peak 3 comes from gardenia seed; peak 4 comes from licorice root; peak 5 comes from gardenia seed; peak 6 comes from saposhnikovia root; peak 7 comes from licorice root; peak 8 comes from licorice root; peak 9 comes from saposhnikovia root; peak 10 comes from saposhnikovia root; peak 11 comes from licorice root; peak 12 comes from licorice root; peak 13 comes from licorice root; peak 14 comes from licorice root; peak 15 comes from licorice root; and peak 16 comes from licorice root.

8. A quality control method for a traditional Chinese medicine compound containing gardenia seeds, characterized in that, The quality control method includes the following steps: (1) Establish a standard fingerprint spectrum of a traditional Chinese medicine compound reference sample using the fingerprint spectrum construction method according to any one of claims 1 to 7; (2) Take the test solution of the traditional Chinese medicine compound and detect it under the chromatographic conditions in the fingerprint spectrum construction method according to any one of claims 1 to 7 to obtain the fingerprint spectrum of the traditional Chinese medicine compound sample to be tested; as well as (3) Compare the fingerprint spectrum of the Chinese herbal compound sample to be tested obtained in step (2) with the standard fingerprint spectrum of the Chinese herbal compound reference sample obtained in step (1). If the fingerprint spectrum meets the requirements, the sample is a qualified product; if it does not meet the requirements, the sample is an unqualified product.

9. The quality control method according to claim 8, characterized in that, The compliance requirements include one or more of the following: (1) The fingerprint spectrum of the Chinese herbal compound sample to be tested shows 16 characteristic chromatographic peaks, and the retention time of each characteristic chromatographic peak is within ±10% of the retention time value of the corresponding reference chromatographic peak in the standard fingerprint spectrum of the Chinese herbal compound reference sample. (2) Taking the 5-O-methylvisamidol glycoside peak as the S peak, the relative retention time of each characteristic chromatographic peak in the fingerprint spectrum of the Chinese herbal compound sample to be tested and the S peak is within ±10% of the relative retention time value of each characteristic chromatographic peak in the standard fingerprint spectrum of the Chinese herbal compound reference sample. as well as (3) According to the similarity evaluation system of chromatographic fingerprint of traditional Chinese medicine, the similarity between the fingerprint of the test sample of the traditional Chinese medicine compound and the standard fingerprint of the reference sample of the traditional Chinese medicine compound shall not be less than 0.

90.

10. The use of the construction method according to any one of claims 1 to 7 or the quality control method according to claim 8 or 9 in the quality testing and / or quality evaluation and / or quality control of traditional Chinese medicine compound containing gardenia seeds.

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