Construction method of gardenoside-cinnamomum cassia pair fingerprint
Patent Information
- Application Number
- CN202610496094.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-15
- Publication Date
- 2026-06-12
Smart Images
Figure CN122193457A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of traditional Chinese medicine quality control technology, and in particular relates to a method for constructing a fingerprint spectrum of the gardenia-cinnamon herb pair. Background Technology
[0002] Insomnia symptoms include difficulty falling asleep, frequent awakenings, and early awakenings. These symptoms are often related to factors such as stress and anxiety. Currently, one of the effective treatments for insomnia is Western medicine, which has the advantages of rapid onset, clear effects, and high controllability. Western medicine treatment for insomnia mainly involves drug intervention, represented by benzodiazepines, newer non-benzodiazepines, and barbiturates. Their mechanisms of action are clear and their effects are good. However, long-term use of Western medicine has adverse reactions such as addiction and drug tolerance, and rebound symptoms are common after withdrawal, causing considerable distress to patients. Traditional Chinese medicine (TCM) has been treating insomnia for thousands of years and has the advantages of stable efficacy and fewer side effects, making it more acceptable to patients. In recent years, TCM has achieved certain therapeutic effects in its syndrome differentiation and treatment. Most TCM views believe that insomnia stems from an imbalance of Yin and Yang, with Yang Qi unable to enter Yin (i.e., inability to fall asleep). Excessive heart fire and insufficient kidney water lead to "restlessness of the mind," which in turn causes disharmony between the heart and kidneys, ultimately resulting in insomnia. In Traditional Chinese Medicine (TCM) theory, common patterns of insomnia include: liver stagnation turning into fire, phlegm-heat disturbance, yin deficiency with internal heat, deficiency of both heart and spleen, and disharmony between heart and kidney. Jiaotai Pill is a classic formula for treating insomnia caused by disharmony between heart and kidney. However, the extremely bitter and cold nature of Coptis chinensis (Huanglian) leads to adverse gastrointestinal reactions with long-term use, limiting its application in the health food industry. Gardenia jasminoides (Zhizi) clears the heart and relieves irritability, with similar effects to Coptis chinensis. Based on this, Professor Deng Jiagang innovatively proposed a Gardenia-Cinnamon combination based on long-term clinical experience. This improvement is based on the following scientific principles: using Gardenia jasminoides instead of Coptis chinensis based on the principle of optimizing medicinal properties, it retains the heat-clearing and fire-purging effects while avoiding the stomach-damaging effects of Coptis chinensis's bitterness and coldness, and retains Cinnamon arsenic to maintain its core function of "guiding fire back to its source." This Gardenia-Cinnamon combination is used as an adjunct treatment for insomnia caused by disharmony between heart and kidney and excessive heart fire.
[0003] The application of Traditional Chinese Medicine (TCM) is becoming increasingly widespread both domestically and internationally, making quality control a key factor influencing the development of the TCM industry. TCM components are complex, and traditional quality control methods have limitations, failing to comprehensively reflect their quality. TCM fingerprinting, as a comprehensive and quantifiable chromatographic identification method, can comprehensively present the chemical components and characteristics of TCM materials and their preparations, thereby evaluating their quality stability and consistency. The TCM pharmacodynamic relationship is constructed based on fingerprinting. It uses a chemometric model to correlate fingerprinting with pharmacodynamics, constructing a comprehensive quality evaluation model integrating chemical component and pharmacodynamic activity evaluation. This model can reveal the correlation between chemical components and pharmacodynamics, guiding and accelerating research on the active material basis of TCM, and providing a guarantee for improving the intrinsic quality of TCM. In recent years, it has been widely used in the research on the pharmacodynamic material basis of TCM and compound prescriptions. Cinnamon and gardenia have complex components; qualitative and quantitative analysis of one or two components is insufficient to comprehensively reflect their information, and to date, there is no relevant research on the herbal fingerprinting of cinnamon and gardenia. Summary of the Invention
[0004] To address the above problems, this invention provides a method for constructing a fingerprint spectrum of the gardenia-cinnamon drug pair.
[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: This invention provides a method for constructing a fingerprint spectrum of a gardenia-cinnamon herb pair, comprising the following steps: First, 12 batches of gardenia-cinnamon herb pair samples are prepared, and then test solutions and reference solutions are prepared; the reference solution and the test solution of the 12 batches of gardenia-cinnamon herb pair samples are precisely pipetted into a high-performance liquid chromatograph, chromatograms are collected, and the fingerprint spectrum of the gardenia-cinnamon herb pair is generated from the obtained chromatographic data using the "Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine" and the mean method, and the mean relative retention time of each common peak is calculated; The chromatographic conditions of the high-performance liquid chromatograph are as follows: Column: SymmetryShield™ RP18 5μm; Detector: Ultraviolet detector; Mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile; Elution gradient: 0-15 min: 6%A-8%A; 15-29 min: 8%A-9%A; 29-90min: 9%A-30%A; 90-110min: 30%A-55%A; 110-110.01 min: 55%A-6%A; 110.01-120min: 6%A; Detection wavelength: 254 nm; Column temperature: 30℃; Flow rate: 1.000 mL / min; Injection volume: 20 µL.
[0006] Furthermore, the method for preparing the test solution includes the following steps: S1. Place the cinnamon in a round-bottom flask, add 10 times the amount of water, soak for 1 hour, attach the volatile oil extractor, add water until it fills the marked part and overflows into the flask, then connect the reflux condenser; heat to boiling and maintain a gentle boil for 4 hours until the amount of oil in the volatile oil extractor no longer increases, stop heating, let it stand for a while, open the stopcock at the bottom of the measuring device to release the water, collect the volatile oil, filter the liquid in the round-bottom flask, collect the filtrate, i.e. filtrate A, and keep the cinnamon residue for later use; S2. Mix the gardenia powder and the cinnamon residue, add 10 times the amount of water, soak for 1 hour, decoct and extract 3 times, each time for 0.5 hours, collect the filtrate from each decoction, i.e. filtrate B, combine filtrate A and B, concentrate under reduced pressure to obtain a medicinal solution with a drug content of 1g / mL, add volatile oil, mix well, and obtain the gardenia-cinnamon drug pair sample solution; S3. Accurately measure 5 mL of the Gardenia-Cinnamon herb pair sample solution, add methanol to make up to 10 mL, filter, and prepare the test solution.
[0007] Furthermore, the mass ratio of the coarse cinnamon powder to gardenia powder is 1:6.
[0008] Furthermore, the method for preparing the reference solution includes the following steps: weighing geniposide reference standard, cinnamaldehyde reference standard and genipin gentiopicroside reference standard respectively, using methanol as solvent, and preparing the reference solution, wherein the concentrations of geniposide, cinnamaldehyde and genipin gentiopicroside are 0.515 mg / mL, 0.406 mg / mL and 0.43 mg / mL respectively.
[0009] Furthermore, the fingerprint spectrum of the gardenia-cinnamon drug pair includes 8 common peaks, of which peak 5 corresponds to geniposide, peak 7 corresponds to cinnamaldehyde, and peak 4 corresponds to genipin gentiopicroside.
[0010] Furthermore, using the geniposide chromatographic peak as a reference peak, the average relative retention times of each common peak are: Peak 1 has a mean relative retention time of 0.263. Peak 2 has a mean relative retention time of 0.308. Peak 3 has a mean relative retention time of 0.462. Peak 4 has a mean relative retention time of 0.756. Peak 5 has a mean relative retention time of 1. Peak 6 has a mean relative retention time of 2.822. Peak 7 has a mean relative retention time of 3.10. Peak 8 has a mean relative retention time of 3.297.
[0011] Compared with the prior art, the present invention has the following advantages and technical effects: The HPLC fingerprinting method for the gardenia-cinnamon herb pair established in this invention is accurate, stable, and reproducible. To accurately characterize the chemical components of the gardenia-cinnamon herb pair, this invention employs high-performance liquid chromatography (HPLC) to construct fingerprint spectra for 12 herb pair samples. By optimizing chromatographic conditions to ensure detection accuracy, and combined with precise comparison with chemical reference standards, the characteristic peaks of the herb pair were successfully identified. Attached Figure Description
[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the 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 creative effort.
[0013] Figure 1 HPLC reference fingerprints of 12 batches of Gardenia-Cinnamon herbal pair samples; Figure 2 This is a comparison chart of the fingerprint spectrum and the reference standard; Figure 3 The superimposed fingerprint spectra and control fingerprint spectra of 12 batches of Gardenia-Cinnamon herbal pairs are shown. Detailed Implementation
[0014] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.
[0015] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.
[0016] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.
[0017] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.
[0018] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.
[0019] The instruments used in the embodiments of the present invention are shown in Table 1, the reagents used are shown in Table 2, the sources of medicinal materials used are shown in Table 3, and the sample numbers are shown in Table 4.
[0020] Table 1 Experimental Apparatus Table 2 Experimental Reagents Table 3 Sources of medicinal materials Table 4 Sample Number This invention provides a method for establishing an HPLC fingerprint of the gardenia-cinnamon drug pair: Preparation method of test solution: Prepare different batches of gardenia-cinnamon samples, wherein the mass ratio of cinnamon to gardenia is 1:6. The specific preparation method is as follows: S1. Place the cinnamon in a round-bottom flask, add 10 times the amount of water, soak for 1 hour, attach the volatile oil extractor and add water until it fills the marked part and overflows into the flask, then connect the reflux condenser, slowly heat to boiling, and maintain a gentle boil for 4 hours until the amount of oil in the volatile oil extractor no longer increases, stop heating, let it stand for a while, open the stopcock at the bottom of the measuring device, slowly release the water, collect the amount of volatile oil, filter the medicine in the round-bottom flask, collect the filtrate, i.e., filtrate A, and obtain the cinnamon residue for later use; S2. Weigh the coarse gardenia powder (passed through a 20-mesh sieve) according to the proportion, add the above-mentioned cinnamon residue, add 10 times the amount of water, soak for 1 hour, decoct and extract 3 times, each time for 0.5 hours, collect the filtrate from each decoction, i.e. filtrate B, combine filtrates A and B, concentrate under reduced pressure to obtain a medicinal solution with a drug content of 1g / mL, add volatile oil, mix well, and you will get the gardenia-cinnamon herb pair sample solution, which should be stored at 2-8℃ for later use. S3. Accurately measure 5 mL of the Gardenia-Cinnamon herb pair sample solution prepared in S2, add methanol to make up to 10 mL, filter, and filter the filtrate through a 0.22 μm microporous membrane (organic system) to prepare the test solution.
[0021] Preparation of reference solutions: Weigh out geniposide reference standard, cinnamaldehyde reference standard and genipin gentiopicroside reference standard respectively, and use methanol as solvent to prepare reference solutions containing geniposide, cinnamaldehyde and genipin gentiopicroside, with concentrations of 0.515 mg / mL, 0.406 mg / mL and 0.43 mg / mL respectively.
[0022] Establishment of chromatographic conditions: This invention screened wavelengths (235 nm to 380 nm) for different time periods targeting the characteristic peaks and tested them in various mobile phases (including 0.1% formic acid aqueous solution-acetonitrile, methanol-water, etc.). It was found that 0.1% formic acid aqueous solution-acetonitrile, with a wavelength of 254 nm, provided the best separation effect for the characteristic peaks. Further investigation was conducted using 0.1% formic acid aqueous solution (A)-acetonitrile (B) as the mobile phase. The elution program was as follows: 0-15 min: 6%A-8%A; 15-29 min: 8%A-9%A; 29-90 min: 9%A-30%A; 90-110 min: 30%A-55%A; 110-110.01 min: 55%A-6%A; 110.01-120 min: 6%A. The elution time of the characteristic peaks was suitable, and the peak shape was good. The chromatographic conditions during the experiment are shown in Table 5.
[0023] Table 5 Chromatographic conditions for the Gardenia-Cinnamon herb pair Example 1: Investigation of Fingerprint Mapping Methodology Precision assessment: Accurately pipette the S1 test solution and inject it 6 times consecutively under the above chromatographic conditions. Use peak 5 (geniposide) as the reference peak, calculate the relative retention time and relative peak area of each common peak, and calculate the RSD (%). The results are shown in Table 6.
[0024] Stability assessment: Accurately pipette the S1 test solution and inject it sequentially at 0, 2, 4, 8, 12, and 24 hours. Using peak 5 (geniposide) as the reference peak, calculate the relative retention time and relative peak area of each common peak, and calculate the RSD (%). The results are shown in Table 6.
[0025] Repeated examination: Six parallel S1 test solutions were prepared and analyzed under the above chromatographic conditions. Peak 5 (geniposide) was used as the reference peak. The relative retention time and relative peak area of each common peak were calculated, and the RSD (%) was calculated. The results are shown in Table 6.
[0026] Table 6. Results of Relative Retention Time and Relative Peak Area (RSD) for Precision, Stability and Repeatability Table 6 shows that: In the precision test, the RSD values of the relative retention times of the eight common peaks ranged from 0.054% to 0.558%, and the RSD values of the relative retention peak areas ranged from 0.217% to 0.807%, all less than 3%, meeting the relevant requirements and indicating good instrument precision. In the stability test, the RSD values of the relative retention times of the eight common peaks ranged from 0.088% to 0.520%, and the RSD values of the relative retention peak areas ranged from 0.236% to 2.604%, all less than 3%, demonstrating the good stability of the test solution within 24 hours. In the repeatability test, the RSD values of the relative retention times of the eight common peaks ranged from 0.080% to 0.429%, and the RSD values of the relative retention peak areas ranged from 0.220% to 2.780%, all less than 3%, meeting the relevant requirements and indicating good method repeatability.
[0027] Example 2: Generation and Similarity Analysis of Comparative Fingerprint Maps Twelve batches of Gardenia-Cinnamon herbal pair samples were prepared into test solutions, which were then injected sequentially for analysis. HPLC chromatograms of different batches were obtained through chromatographic conditions. Using the HPLC chromatogram of sample S1 as a reference, three characteristic peaks were identified by comparing it with reference solutions of genipin, gentiopicrin, geniposide, and cinnamaldehyde. The HPLC chromatograms of the 12 batches of Gardenia-Cinnamon herbal pair test solutions were imported into the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)" in AIA format. Using the mean method, with the chromatogram of sample S1 as a reference, and a time pane width of 0.1 min, multi-point correction and Mark peak matching were performed to generate superimposed fingerprint chromatograms of the 12 batches of Gardenia-Cinnamon herbal pair samples and reference fingerprint chromatograms. Eight common peaks were identified, similarity was calculated, and standard chromatograms were created. Figure 1 HPLC reference fingerprints of 12 batches of Gardenia-Cinnamon herbal pair samples; Figure 2This is a comparison chart of the fingerprint spectrum and the reference standard; Figure 3 The superimposed fingerprint spectra and control fingerprint spectra of 12 batches of Gardenia-Cinnamon herbal pairs are shown.
[0028] Table 7 shows the similarity of 12 batches of Gardenia-Cinnamon herbal sample pairs, and Table 8 shows the similarity results of the fingerprint chromatograms of 12 batches of Gardenia-Cinnamon herbal sample pairs and the control. In this invention, the chromatographic peak of geniposide is used as the reference peak to calculate the average relative retention time of the common peak of the 12 batches of Gardenia-Cinnamon herbal sample pairs. Table 9 shows the average relative retention time of the common peak of the test solution of the 12 batches of Gardenia-Cinnamon herbal sample pairs.
[0029] Table 7. Similarity of Gardenia-Cinnamon Herb Pair Samples Table 8. Similarity of fingerprint spectra between 12 batches of Gardenia-Cinnamon herb pairs and the control. Table 9. Mean relative retention times of common peaks in sample solutions of Gardenia-Cinnamon herb pair The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for constructing a fingerprint spectrum of a gardenia-cinnamon herbal pair, characterized in that, Includes the following steps: First, 12 batches of gardenia-cinnamon herb pair samples were prepared, and then test solution and reference solution were prepared. Accurately pipette the reference solution and the test solution of the 12 batches of Gardenia-Cinnamon herb pair samples, respectively, and inject them into the high performance liquid chromatograph. Collect the chromatograms, use the "Traditional Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" and the mean method to generate the Gardenia-Cinnamon herb pair fingerprint chromatogram from the obtained chromatographic data, and calculate the mean relative retention time of each common peak. The chromatographic conditions of the high-performance liquid chromatograph are as follows: Column: SymmetryShield™ RP18 5μm; Detector: Ultraviolet detector; Mobile phase A is a 0.1% formic acid aqueous solution, and mobile phase B is acetonitrile; Elution gradient: 0-15 min: 6%A-8%A; 15-29 min: 8%A-9%A; 29-90 min: 9%A-30%A; 90-110 min: 30%A-55%A; 110-110.01 min: 55%A-6%A; 110.01-120 min: 6%A; Detection wavelength: 254 nm; Column temperature: 30℃; Flow rate: 1.000 mL / min; Injection volume: 20 µL.
2. The method for constructing the fingerprint spectrum of the gardenia-cinnamon herbal pair according to claim 1, characterized in that, The preparation method of the test solution includes the following steps: S1. Place the cinnamon in a round-bottom flask, add 10 times the amount of water, soak for 1 hour, attach the volatile oil extractor, add water until it fills the marked part and overflows into the flask, then connect the reflux condenser; heat to boiling and maintain a gentle boil for 4 hours until the amount of oil in the volatile oil extractor no longer increases, stop heating, let it stand for a while, open the stopcock at the bottom of the measuring device to release the water, collect the amount of volatile oil, filter the medicine in the round-bottom flask, collect the filtrate, i.e. filtrate A, and use the obtained cinnamon residue for later use; S2. Mix the coarse gardenia powder and the cinnamon residue, add 10 times the amount of water, soak for 1 hour, decoct and extract 3 times, each time for 0.5 hours, collect the filtrate from each decoction, i.e. filtrate B, combine filtrate A and filtrate B, concentrate under reduced pressure to obtain a medicinal solution with a drug content of 1g / mL, add volatile oil, mix well, and obtain the gardenia-cinnamon drug pair sample solution; S3. Accurately measure 5 mL of the Gardenia-Cinnamon herb pair sample solution, add methanol to make up to 10 mL, filter, and obtain the test solution.
3. The method for constructing the fingerprint spectrum of the gardenia-cinnamon herbal pair according to claim 2, characterized in that, The mass ratio of the coarse cinnamon powder to gardenia powder is 1:
6.
4. The method for constructing the fingerprint spectrum of the gardenia-cinnamon herbal pair according to claim 1, characterized in that, The preparation method of the reference solution includes the following steps: weighing geniposide reference standard, cinnamaldehyde reference standard and genipin gentiopicroside reference standard respectively, using methanol as solvent, and preparing the reference solution, wherein the concentrations of geniposide, cinnamaldehyde and genipin gentiopicroside are 0.515 mg / mL, 0.406 mg / mL and 0.43 mg / mL respectively.
5. The method for constructing the fingerprint spectrum of the gardenia-cinnamon herbal pair according to claim 1, characterized in that, The fingerprint spectrum of the gardenia-cinnamon drug pair includes 8 common peaks, of which peak 5 corresponds to geniposide, peak 7 corresponds to cinnamaldehyde, and peak 4 corresponds to genipin gentiopicroside.
6. The method for constructing the fingerprint spectrum of the gardenia-cinnamon herbal pair according to claim 5, characterized in that, Using the geniposide chromatographic peak as a reference peak, the average relative retention times of each common peak are: Peak 1 has a mean relative retention time of 0.
263. Peak 2 has a mean relative retention time of 0.
308. Peak 3 has a mean relative retention time of 0.
462. Peak 4 has a mean relative retention time of 0.
756. Peak 5 has a mean relative retention time of 1. Peak 6 has a mean relative retention time of 2.
822. Peak 7 has a mean relative retention time of 3.
10. Peak 8 has a mean relative retention time of 3.297.