A method for constructing and detecting HPLC fingerprint of a traditional Chinese medicine composition

Abstract

The present invention provides a method for establishing and detecting a fingerprint of a traditional Chinese medicine composition, and specifically relates to a method for establishing and detecting an HPLC fingerprint of a traditional Chinese medicine composition whose raw materials include spinach seeds and gardenia. The method comprises the following steps: preparing a sample of the traditional Chinese medicine composition, accurately weighing it, adding an extraction solvent, ultrasonically treating it, cooling it, centrifuging it, and then filtering it with suction; taking the filtrate and spinning it to obtain a residue; adding a solvent to dissolve the residue, fixing the volume, shaking it well, filtering it, and taking a subsequent filtrate to obtain a sample solution; preparing a reference solution: taking six reference substances, including genipin gentiobioside, gardeniaside, magnolamine, and spinosin, to prepare a mixed reference solution; and using HPLC to perform analysis and determination to complete the construction of a fingerprint. The method has strong specificity, high precision, good stability, and strong repeatability, and provides richer information for the intrinsic quality control of traditional Chinese medicine compositions whose raw materials include spinach seeds and gardenia.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to an HPLC fingerprint establishment method and a quality detection method of a traditional Chinese medicine composition whose raw materials include spinach seeds and gardenia jasmine. Background Art

[0002] The Chinese jujube seed and gardenia pairing comes from the book "Shi Jinmo's Pharmacopoeia." The Chinese jujube seed and gardenia are both medicinal and edible ingredients in this recipe. Chinese jujube seed nourishes the liver and calms the mind, nourishing the Yin blood of the heart and liver. Gardenia has a bitter and cold nature and enters the heart, liver, lung, stomach, and triple burner meridians, known for its ability to clear and purge triple burner heat. The two herbs work together, their properties complementing each other, to clear heart heat and nourish the mind. The combination, one tonic, one purgative, one clearing, one astringent, clears the heart and cools the liver, relieving restlessness and calming the mind. It is primarily used to treat symptoms such as excessive heart fire, restlessness, insomnia, and excessive dreaming.

[0003] Quality control of traditional Chinese medicine preparations often relies on quantitative analysis of a single or a few ingredients and qualitative identification of some of the ingredients in a formula, lacking comprehensive, comprehensive quality control research. To ensure and stabilize product quality, comprehensive research into quality testing and control methods is necessary. Currently, while the active ingredients of traditional Chinese medicines and their preparations have yet to be fully elucidated, fingerprint analysis, compared to the determination of index component content, can better objectively reflect the overall quality of traditional Chinese medicine preparations and is currently the mainstream method for effective quality control of traditional Chinese medicines and their preparations.

[0004] Currently, high-performance liquid chromatography (HPLC) methods are mostly used for the characterization and fingerprint analysis of single medicinal materials, such as jujube seeds or gardenia. Reports on the simultaneous determination of jujube seeds and gardenia are rare. Chinese patent CN113655163A discloses a method for fingerprinting an instant jujube seed solid beverage, Xinning Drink. The formula contains both jujube seeds and gardenia, but the three main components identified are chlorogenic acid, puerarin, and irisin, which are not the primary active ingredients of jujube seeds and gardenia. Xinshenning tablets are a typical traditional Chinese medicine composition of the jujube seed and gardenia pair, consisting of six Chinese herbs: jujube seeds, gardenia, polygala tenuifolia, poria, licorice, and Liushenqu. The current quality standard for Xinshenning tablets only includes geniposide. Song Jia et al. disclosed a method for determining the content of geniposide, liquiritin, or spinosin in Xinshenning tablets (Song Jia et al., 2016), but cannot qualitatively determine the multiple active ingredients of jujube seeds and gardenia.

[0005] Because the various components of traditional Chinese medicine compositions are interconnected, interact, and influence each other, a single component cannot fully control product quality. Therefore, it is necessary to establish a new HPLC fingerprint method to perform quality testing on traditional Chinese medicine compositions containing raw materials such as Ziziphus jujuba seeds and Gardenia jasminoides to ensure clinical safety. Summary of the Invention

[0006] In view of this, the present invention provides a method for establishing an HPLC fingerprint of a traditional Chinese medicine composition and a method for detecting its quality.

[0007] To achieve the above objectives, the present invention provides the following technical solutions:

[0008] The present invention provides a method for constructing an HPLC fingerprint of a traditional Chinese medicine composition, wherein the raw materials of the traditional Chinese medicine composition include spinach seeds and gardenia jasmine, and the HPLC fingerprint construction method comprises the following steps:

[0009] S1. Prepare the test solution: take a sample of the Chinese medicine composition, accurately weigh it, add the extraction solvent, ultrasonicate it, cool it, centrifuge it, and filter it with suction. Take the filtrate and spin-dry it to obtain a residue. Dissolve the residue in the solvent, make up to volume, shake it well, filter it, and take the filtrate to obtain the test solution.

[0010] The extraction solvent is methanol solution or ethanol solution, preferably 50% methanol.

[0011] S2 Preparation of reference solution: Genipin gentiobioside, geniposide, magnololamine, spinosin, crocin I, and crocin II as reference substances, add organic solvent to prepare a mixed reference solution;

[0012] The organic solvent is a methanol solution or an acetonitrile solution.

[0013] The test solution and the reference solution were respectively taken and measured by high performance liquid chromatography to obtain an HPLC fingerprint of the traditional Chinese medicine composition.

[0014] In some specific embodiments of the present invention, the chromatographic column used in the high performance liquid chromatography method is Agilent 5 TC-C18 (2), 250×4.6 mm, 5 μm; the mobile phase A is acetonitrile, the mobile phase B is 0.1% phosphoric acid aqueous solution, and the gradient elution is performed; the gradient elution method is as follows:

[0015]

[0016] In some specific embodiments of the present invention, the HPLC detection conditions are as follows: the chromatographic column is Agilent 5 TC-C18(2), 250×4.6 mm, 5 μm; the flow rate of the mobile phase is 0.8-1.2 mL / min; the injection volume is 5-20 μL, the column temperature is 25-45°C, and the detection wavelengths are: 0-24 min, 227 nm; 24-42 min, 335 nm; 42-63 min, 440 nm.

[0017] In some specific embodiments of the present invention, the HPLC detection conditions are as follows: the flow rate of the mobile phase is 1.0 mL / min; the injection volume is 10 μL, and the column temperature is 30°C.

[0018] In some specific embodiments of the present invention, the raw materials of the traditional Chinese medicine composition include 1-10 parts by weight of spinach seeds and 10-1 parts by weight of gardenia, preferably 6-10 parts by weight of spinach seeds and 4.5-6 parts by weight of gardenia.

[0019] In some specific embodiments of the present invention, the specific preparation method of the traditional Chinese medicine composition is: mixing the Chinese jujube seeds and the gardenia in a weight ratio of 6-10:4.5-6, extracting with water according to a conventional extraction method, and preparing into a conventional dosage form according to a conventional preparation process.

[0020] The conventional extraction methods include any one of decoction extraction, reflux extraction, maceration extraction, ultrasonic extraction or percolation extraction, or a combination of different extraction methods; the conventional dosage forms include granules, tablets, powders, capsules, oral solutions, pills, etc.

[0021] In some specific embodiments of the present invention, the specific preparation method of the traditional Chinese medicine composition is as follows: mixing spinach seeds and gardenia in a weight ratio of 6-10:4.5-6, extracting 2-4 times, combining the extracts, filtering, and rotary evaporating the filtrate at a temperature of 40-80°C and a vacuum degree of -0.06-0.08 MPa to obtain an extract with a relative density of 1.04-1.09; drying the extract under reduced pressure and vacuum at 60°C, and then grinding it into powder, and wet granulating it;

[0022] The extraction time for each of the above-mentioned extractions is 0.5-3 hours, and the extraction method is water extraction, including: adding 9 to 11 times the amount of water to the medicinal material mixture for the first time, soaking for 25 to 35 minutes, extracting and boiling for 1 to 2 hours, and filtering while hot; for the second and subsequent extractions, adding 8 to 10 times the amount of water to the medicinal material mixture.

[0023] In some specific embodiments of the present invention, the preparation steps of the test solution are as follows: take about 0.5 g of sample powder, accurately weigh, accurately add 20 ml of 50% methanol, ultrasonically treat for 30 minutes, take out, cool, centrifuge and filter, wash the residue once with 5 ml of 50% methanol, combine the washing liquid and the filtrate, spin dry, dissolve the residue in 50% methanol, transfer it to a 5 ml volumetric flask, add 50% methanol to the volume, shake well, filter, take the filtrate, filter through a 0.22 μm microporous filter membrane, and obtain the test solution.

[0024] In some specific embodiments of the present invention, the ultrasonic extraction power is 250W, the frequency is 40kHz, and the ultrasonic extraction time is 10 to 60 minutes. Preferably, the ultrasonic extraction time is 30 minutes.

[0025] In some specific embodiments of the present invention, the preparation steps of the reference solution are: taking appropriate amounts of genipin gentiobioside, gardenia glycoside, magnololamine, spinosum, crocin I, and crocin II reference substances, accurately weighing them, and adding methanol to prepare mixed reference solutions with concentrations of 225 μg / mL, 621 μg / mL, 38 μg / mL, 29 μg / mL, 190 μg / mL, and 47 μg / mL.

[0026] The HPLC fingerprint of the traditional Chinese medicine composition includes 15 common peaks (characteristic peaks). Taking gardenia glycoside as the reference peak (S peak), the relative retention times of common peaks 1 to 15 are calculated to be within ±5% of the specified values, namely: 0.51 to 0.56 (peak 1), 0.60 to 0.66 (peak 2), 0.62 to 0.69 (peak 3), 0.7 to 0.85 (peak 4), 0.95 to 1.05 (peak s), 1.19 to 1.32 (peak 5), 1.84 to 2.04 (peak 6), 1.92 to 2.12 (peak 8), 2.40 to 2.65 (peak 9), 2.65 to 2.93 (peak 10), 2.67 to 2.95 (peak 11), 2.69 to 2.98 (peak 12), 2.80 to 3.10 (peak 13), 3.06 to 3.39 (peak 14). (peak 14), 3.15~3.48 (peak 15).

[0027] The aforementioned method for constructing TCM composition profiles was used to establish HPLC fingerprints for 15 batches of TCM compositions. The Chinese Pharmacopoeia Committee's "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (Version 2012.130723)" was used to generate a standard HPLC fingerprint of TCM compositions consisting of 15 common peaks. Peak 4 is genipin gentiobioside, peak 5 is geniposide, peak 6 is magnolamine, peak 8 is spinosin, peak 10 is crocin I, and peak 13 is crocin II.

[0028] The present invention also provides a method for detecting the quality of a Chinese medicine composition, comprising the following steps:

[0029] (1) Establishing an HPLC fingerprint of a mixed reference substance according to the above-mentioned method for constructing an HPLC fingerprint of a traditional Chinese medicine composition;

[0030] (2) establishing an HPLC fingerprint of the Chinese medicine composition sample to be tested according to the above-mentioned method for constructing an HPLC fingerprint of a Chinese medicine composition;

[0031] (3) The HPLC fingerprint of the mixed reference substance and the HPLC fingerprint of the TCM composition sample were imported into the TCM chromatographic fingerprint similarity evaluation system to perform full spectrum peak matching, generate an overlay spectrum, and then generate a reference fingerprint spectrum. The similarity of each batch of samples was calculated to be above 0.95.

[0032] In some specific embodiments of the present invention, a sample of the Chinese medicine composition to be tested is taken and operated according to the same method as above to obtain the fingerprint of the sample of the Chinese medicine composition to be tested and the HPLC spectrum of the Chinese medicine composition reference substance. The standard fingerprint of the Chinese medicine composition is analyzed using the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 Edition)" software of the National Pharmacopoeia Committee, and the similarity is greater than 0.95.

[0033] Through experiments, 15 common peaks were assigned: peaks 6, 8, and 9 were assigned to Ziziphus jujuba seeds, and peaks 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, 14, and 15 were assigned to Gardenia jasminoides.

[0034] The present invention discloses the following technical effects:

[0035] The HPLC fingerprint of the present invention identifies 15 chromatographic peaks accounting for more than 90% of the total peak area as shared peaks. Among them, the identified six chromatographic peaks, namely, nepinobioside, geniposide, magnolamine, spinosin, crocin I, and crocin II, are the main active ingredients of Ziziphus jujuba seeds and Gardenia jasminoides. This method can more comprehensively and accurately reflect the characteristics of Ziziphus jujuba seeds and Gardenia jasminoides, and can be used for quality testing of traditional Chinese medicine compositions containing Ziziphus jujuba seeds and Gardenia jasminoides as raw materials. By setting a specific mobile phase composition, gradient elution procedure, flow rate, detection wavelength, chromatographic column, column temperature, etc., the HPLC fingerprint established has well-separated chromatographic peaks, a stable baseline, and a good peak shape. This method has excellent precision, stability, and repeatability. The similarities of the 15 batches of samples used to establish the HPLC fingerprint were all greater than 0.95, indicating a high similarity. This method can effectively characterize the quality of the traditional Chinese medicine composition and avoid the single-mindedness and one-sidedness of traditional Chinese medicine composition quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Overlay chromatograms of 15 batches of Chinese medicine composition samples.

[0037] Figure 2 These are the reference fingerprints of 15 batches of Chinese medicine composition samples.

[0038] Figure 3 This is the HPLC fingerprint of the mixed reference substance of the Chinese medicine composition. DETAILED DESCRIPTION

[0039] The following further details the above content of the present invention through specific embodiments. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention.

[0040] Example 1 Method for constructing fingerprint of Chinese medicine composition

[0041] 1. Instruments and raw materials

[0042] Instruments: High-performance liquid chromatograph: Waters 2695; chromatographic column: Agilent 5 TC-C18(2) chromatographic column (4.6×250 mm, 5 μm); AVW-220D 1 / 100,000 analytical balance: Shimadzu Corporation; KH5200 ultrasonic cleaner: Kunshan Hechuang Ultrasonic Instrument Co., Ltd.; RE-52A rotary evaporator: Shanghai Yarong Biochemical Instrument Factory; TDL-40B desktop centrifuge: Shanghai Anting Scientific Instrument Factory.

[0043] Reagents: acetonitrile and methanol were chromatographically pure reagents from RCI LabScan; phosphoric acid (batch number: C12198140, analytical grade, MACKLIN); and water was purified water from North China Pharmaceutical Jinshui Yikang.

[0044] Reference substances: genipin gentiobioside (purity: HPLC ≥ 98%, batch number: DST211114-092, Chengdu Desite Biotechnology Co., Ltd.); geniposide (purity: HPLC ≥ 95%, batch number: XY21-3169, Xinyao (Shenzhen) Biotechnology Co., Ltd.); magnolamine (purity: HPLC ≥ 98%, batch number: DSTDM000401, Chengdu Desite Biotechnology Co., Ltd.); spinosin (purity: HPLC ≥ 95%, batch number: XY21-3158, Xinyao (Shenzhen) Biotechnology Co., Ltd.); crocin I (purity: HPLC ≥ 98%, batch number: DST200412-011, Chengdu Desite Biotechnology Co., Ltd.); crocin II (purity: HPLC ≥ 98%, batch number: DST200527-012, Chengdu Desite Biotechnology Co., Ltd.).

[0045] Prescription medicinal materials: Ziziphus jujuba seeds were purchased from Hebei Hancaotang Pharmaceutical Co., Ltd., and Gardenia jasminoides were purchased from Jiangxi Zhangshu Tianqitang Chinese Medicine Pieces Co., Ltd.

[0046] Preparation of the Traditional Chinese Medicine Composition: 10 parts by weight of the prescribed medicinal materials, Semen Ziziphi Spinosae, and 6 parts by weight of Gardenia Fructus, were weighed and extracted three times with water. The first extraction was performed with 10.5 times the amount of water, soaked for 30 minutes, then heated to a boil and maintained at a slight boiling point for 1 hour. The second and third extractions were performed with 9 times the amount of water, heated to a slight boiling point for 1 hour. The three extracts were combined and filtered through a 300-mesh filter. The filtrate was rotary evaporated at 60°C and a vacuum of -0.06 to 0.08 MPa to obtain an extract with a relative density of 1.04 to 1.09 (at 60°C). The extract was then dried under reduced pressure at 60°C and a vacuum of -0.06 to 0.08 MPa to obtain a dry extract with a moisture content of ≤4.5%. The extract was pulverized and wet-granulated with 1.5 times the amount of dextrin to obtain the granular formulation of the pharmaceutical composition of this embodiment.

[0047] 2. Preparation of sample solution

[0048] Preparation of test solution:

[0049] Take a sample of the traditional Chinese medicine composition, grind it into powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, add 20 ml of 50% methanol, ultrasonically treat it for 30 minutes, take it out, let it cool, centrifuge it and filter it, wash the residue once with 5 ml of 50% methanol, combine the washing liquid and the filtrate, spin dry, dissolve the residue in 50% methanol, transfer it to a 5 ml volumetric flask, add 50% methanol to the scale, shake it well, filter it, take the filtrate, and filter it through a 0.22 μm microporous membrane to obtain it.

[0050] Preparation of reference solution:

[0051] Take appropriate amounts of genipin gentiobioside, geniposide, magnololamine, spinosum, crocin I, and crocin II reference substances, accurately weigh them, and add methanol to prepare mixed reference substance solutions with concentrations of 225 μg / mL, 621 μg / mL, 38 μg / mL, 29 μg / mL, 190 μg / mL, and 47 μg / mL.

[0052] 3. Investigation of chromatographic conditions

[0053] Investigation of chromatographic columns:

[0054] Even for C18 chromatographic columns of the same specifications, the peak elution time and peak shape of the same compound in products from different vendors are different. The present invention conducted a test comparison on four chromatographic columns, namely YMC C18 (250×4.6 mm, 5μm), Welch C18 (250×4.6 mm, 5μm), Agilent C18 (2) (250×4.6 mm, 5μm), and Sepax C18 (150×4.6 mm, 5μm). The results showed that the separation effect of using chromatographic columns YMC C18 (250×4.6 mm, 5μm), Welch C18 (250×4.6 mm, 5μm) and Sepax C18 (150×4.6 mm, 5μm) was not as good as that of using chromatographic column Agilent C18 (2) (250×4.6 mm, 5μm), and the retention time was not moderate. Considering the separation degree, the number of chromatographic peaks, the peak shape and the stability of the baseline, Agilent C18 (2) (250×4.6 mm, 5μm) was finally selected. mm, 5 μm).

[0055] Detection wavelength:

[0056] The present invention initially selected a single wavelength detection method to conduct an experiment, such as a detection wavelength of 270nm. However, due to the large difference in the ultraviolet absorption spectrum of the diterpenoid component (crocin) in Gardenia Fructus Gardeniae under this wavelength, and the fact that the diterpenoid component elution time did not overlap with the iridoid ether glycosides component in Gardenia Fructus Gardeniae and the flavonoids and alkaloid components in Semen Ziziphi Spinosae, a fingerprint spectrum was established by using a time-sharing method to collect chromatograms of different absorption wavelengths. A diode array detector was used to detect the sample, and the chromatograms under each wavelength were compared and analyzed. The results showed that the number of peaks detected under 227, 335, and 440nm wavelengths was large, and each peak was well separated, with characteristic peaks being obvious and having a better peak shape. From the spectrum, chromatographic component information could be obtained as much as possible to reflect the overall picture of the system composition. Furthermore, after extensive experimental research, the present invention has found that when a specific detection wavelength is selected for the following specific time periods, i.e., 227 nm is selected for 0-24 min, 335 nm is selected for 24-42 min, and 440 nm is selected for 42-63 min, the peaks obtained are best separated, the characteristic peaks are most obvious, and the peak shapes are the best.

[0057] Flow rate:

[0058] The present invention screened flow rates of 0.8 ml / min, 0.9 ml / min, 1.0 ml / min, 1.1 ml / min, and 1.2 ml / min, and found that the peak shape and separation were better at a flow rate of 1.0 ml / min. Finally, 1.0 ml / min was selected as the flow rate for the gradient elution program.

[0059] Column temperature:

[0060] The present invention screened column temperatures of 25°C, 30°C, 35°C, and 40°C, and found that the peak shape and separation were better at 30°C, and finally 30°C was selected as the temperature for the gradient elution program.

[0061] Injection volume:

[0062] The present invention screened injection volumes of 5 μL, 10 μL, 15 μL, and 20 μL, and found that the peak area was more appropriate when the injection volume was 10 uL. Finally, 10 μL was selected as the injection volume for the gradient elution program.

[0063] Determination of mobile phase elution program:

[0064] The following five elution procedures were screened using an Agilent C18 (2) chromatographic column (250×4.6 mm, 5 μm), column temperature: 30°C, injection volume: 10 μL, and flow rate: 1.0 mL / minn.

[0065] Elution procedure 1

[0066] Mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution; detection wavelength: 0-4 min, 227 nm; 24-42 min, 335 nm; 42-63 min, 440 nm; elution mode:

[0067] 0~5min, 5%~10% acetonitrile; 5~6min, 10%~15% acetonitrile; 6~20min, 15% acetonitrile; 20~26min, 15%~16% acetonitrile; 26~27min, 16%~20% acetonitrile; 27-37min, 20%~23% acetonitrile; 37~47min, 23%~37% acetonitrile; 47~49min, 37%~39% acetonitrile; 49-60min, 39%~100% acetonitrile. At this time, the retention time, separation, number of peaks and baseline of the spectrum are good, which can meet the requirements of the analysis.

[0068] Elution procedure 2

[0069] Mobile phase A: acetonitrile, mobile phase B: 0.1% acetic acid in water; detection wavelength 270 nm, elution mode:

[0070] 0~10min, 19%~23% acetonitrile; 10~15min, 23%~45% acetonitrile; 15~20min, 45%~50% acetonitrile; 20~25min, 50%~70% acetonitrile; 25~30min, 70%~100% acetonitrile; 0-35min, 100%~19% acetonitrile. At this time, the separation effect of the component peaks in the chromatogram is poor.

[0071] Elution procedure 3

[0072] Mobile phase A: acetonitrile, mobile phase B: water; detection wavelength 220 nm, elution method:

[0073] 0~3min, 6% acetonitrile; 3~13min, 6%~18% acetonitrile; 13~22min, 18%~40% acetonitrile; 22~23 min, 40%~6% acetonitrile; 23~30min, 6% acetonitrile. At this time, the chromatographic peaks cannot be well separated.

[0074] Elution procedure 4

[0075] Mobile phase A: acetonitrile, mobile phase B: 0.5% phosphoric acid water; detection wavelength: 238 nm for 0-22 min; 440 nm for 22-31 min; elution method:

[0076] Elution conditions: 0-11 min, 15% acetonitrile; 11-22 min, 15→28 acetonitrile; 22-31 min, 28→45 acetonitrile; 31-42 min, 45→70 acetonitrile; 42-50 min, 70→15 acetonitrile. The number of peaks at this time is relatively small.

[0077] Elution procedure 5

[0078] Mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid water; detection wavelength: 227 nm for 0-24 min; 335 nm for 24-36 min; 440 nm for 36-60 min; elution mode:

[0079] The elution conditions are: 0-5 min, 5→10 acetonitrile; 5-6 min, 10→13 acetonitrile; 6-20 min, 13→15 acetonitrile; 20-26 min, 15→16 acetonitrile; 26-27 min, 16→20 acetonitrile; 27-37 min, 20→23 acetonitrile; 37-47 min, 23→35 acetonitrile; 47-49 min, 35→39 acetonitrile; 49-60 min, 39→100 acetonitrile. At this time, the number of peaks is slightly less, and the peak shape of spinosin is slightly poor.

[0080] After screening multiple gradient elution programs, elution program 1 was finally selected.

[0081] 4. Ingredient testing

[0082] The chromatographic column was Agilent 5 TC-C18(2) (250×4.6 mm, 5 μm); mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid in water; gradient elution was performed; flow rate was 1.0 mL / min; detection wavelength was 227 nm for 0 to 4 min, 335 nm for 24 to 42 min, and 440 nm for 42 to 63 min; injection volume was 10 μL; column temperature was 30°C; the gradient elution method was shown in Table 1.

[0083] Table 1 Gradient elution method

[0084]

[0085] Prepare the test solution according to the method in 2.

[0086] Determination: Accurately draw 10 μL of the test solution and inject it into the liquid chromatograph, and determine it according to the above chromatographic conditions to obtain the fingerprint of the traditional Chinese medicine composition.

[0087] 5. Precision experiment

[0088] Take an appropriate amount of the test solution and inject 6 times in succession. Using the geniposide peak as the reference peak, the relative retention time RSD of the common chromatographic peak is calculated to be less than 0.48%, and the relative peak area RSD is less than 3.73%, both less than 5%, indicating good instrument precision.

[0089] Table 2 Fingerprint precision investigation (relative retention time)

[0090]

[0091] Table 3 Fingerprint precision investigation (relative peak area)

[0092]

[0093] 6. Stability test

[0094] The test solution of this product was injected at 0, 2, 4, 6, 8, 10, 12, and 24 hours. The calculated relative retention time RSD of the common peak was <0.52%, and the relative peak area RSD was <3.73%, both less than 5%, indicating that the test solution has good stability within 24 hours.

[0095] Table 4 Fingerprint stability study (relative retention time)

[0096]

[0097] Table 5 Fingerprint stability study (relative peak area)

[0098]

[0099] 7. Repeatability Experiment

[0100] Six portions of the same batch of powdered traditional Chinese medicine composition containing spinach seeds were precisely weighed to prepare the test solution, which was then injected. The calculated relative retention time RSD for the common peak was less than 0.52%, and the relative peak area RSD was less than 3.83%, both less than 5%, demonstrating good reproducibility of the method.

[0101] Table 6 Repeatability of fingerprints (relative retention time)

[0102]

[0103] Table 7 Repeatability of fingerprint (relative peak area)

[0104]

[0105] 8. Fingerprint establishment

[0106] According to the above method, 15 batches of Chinese medicine composition granule samples containing Ziziphus jujuba seeds and Gardenia jasminoides were prepared and prepared into test solution. The chromatographic conditions of 4 were used for testing. The chromatograms of 15 batches of Chinese medicine composition granule samples were obtained. The relative peak area and relative retention time of each chromatographic peak are shown in Table 8-9. The data was processed by importing the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012.130723 Edition)" software. The superimposed spectrum was generated by full spectrum peak matching ( Figure 1 ), and the median method was used to generate a control chromatogram ( Figure 2 Based on the consistency of the relative retention time of each component in 15 batches of samples, 15 common peaks were selected as the characteristic peaks of the fingerprint spectrum and compared with the fingerprint spectrum of the mixed reference substance ( Figure 3 ) by comparison, six shared peaks were identified: peaks 4, 5, 6, 8, 10, and 13, representing genipin gentiobioside, geniposide, magnolamine, spinosin, crocin I, and crocin II, respectively. Peak 5 (geniposide) was selected as the reference peak (S) (t = 16.221 min) because it had the largest peak area, good separation, and a moderate and stable retention time.

[0107] Table 8 Relative retention time of fingerprint samples

[0108]

[0109] Table 9 Relative peak areas of fingerprint samples

[0110]

[0111] Similarity evaluation of HPLC fingerprints of batches 9 and 13 of finished products

[0112] The similarity evaluation software for Chinese medicine fingerprints was used to evaluate the similarity of 15 batches of samples. The results are shown in Table 10.

[0113] Table 10 Calculation results of fingerprint similarity of 15 batches of Chinese medicine compositions containing spinach seeds

[0114]

[0115] The test results showed that the similarity of the fingerprints of 15 batches of traditional Chinese medicine compositions containing Chinese jujube seeds was greater than 0.95, which met the requirements of the State Food and Drug Administration for the similarity of traditional Chinese medicine fingerprints.

[0116] 10. Correlation between preparations and various raw medicinal materials and attribution of characteristic peaks

[0117] Referring to the above-mentioned method for preparing the sample solution, solutions of the prescription medicinal materials, Chinese jujube seeds and Gardenia jasminoides, as well as reference and test solutions were prepared respectively. Under the optimal chromatographic conditions, the fingerprints of the corresponding amounts of each prescription medicinal material and the reference solution were measured. The chromatograms of the obtained samples and prescription medicinal materials were compared to confirm the attribution of each characteristic peak in the sample fingerprint. Of the 15 chromatographic peaks in the chromatogram of the test solution of the obtained sample, 15 chromatographic peaks were detected that were identical to the reference fingerprints of the prescription medicinal materials, Chinese jujube seeds and Gardenia jasminoides. Among them, peaks 6, 8, and 9 were attributed to Chinese jujube seeds, and peaks 1, 2, 3, 4, 5, 7, 9, 10, 11, 12, 13, 14, and 15 were attributed to Gardenia jasminoides.

[0118] Through these studies, an HPLC fingerprint was established for a traditional Chinese medicine composition containing Semen Ziziphi Spinosae and Fructus Gardeniae. The method demonstrated excellent reproducibility and specificity across multiple batches of samples, providing a deeper level of quality assessment than conventional quality standards.

[0119] Example 2 Fingerprint Detection of Traditional Chinese Medicine Composition

[0120] Chromatographic conditions: Chromatographic column: Agilent 5 TC-C18(2) (250×4.6mm, 5μm); mobile phase A: acetonitrile, mobile phase B: 0.1% phosphoric acid aqueous solution; gradient elution; flow rate: 1.0mL / min; detection wavelength: 0-24min, 227nm; 24-42min, 335nm; 42-63min, 440nm; injection volume: 10μL; column temperature: 30℃; gradient elution method is as follows:

[0121]

[0122] Preparation of test solution:

[0123] Take a sample of the Chinese medicine composition granules (batch number: 20221026), grind it into powder, take about 0.5 g, accurately weigh it, place it in a stoppered conical flask, add 20 ml of 50% methanol, ultrasonically treat it for 30 minutes, take it out, cool it, centrifuge it and filter it, wash the residue once with 5 ml of 50% methanol, combine the washing liquid and the filtrate, spin dry it, dissolve the residue in 50% methanol, transfer it to a 5 ml volumetric flask, add 50% methanol to the scale, shake it well, filter it, take the filtrate, and filter it through a 0.22 μm microporous filter membrane to obtain it.

[0124] Preparation of reference solution:

[0125] Take appropriate amounts of genipin gentiobioside, geniposide, magnololamine, spinosum, crocin I, and crocin II reference substances, accurately weigh them, and add methanol to prepare mixed reference substance solutions with concentrations of 225 μg / mL, 621 μg / mL, 38 μg / mL, 29 μg / mL, 190 μg / mL, and 47 μg / mL.

[0126] Sampling test: Aspirate the reference solution and test solution separately, inject them into the liquid chromatograph and measure.

[0127] By comparing with the reference fingerprint, it can be seen that the similarity between the fingerprint of the test sample and the reference fingerprint is greater than 0.95, and it can be considered that the test sample meets the established fingerprint standard.

[0128] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for constructing an HPLC fingerprint of a traditional Chinese medicine composition, characterized in that: The raw materials of the traditional Chinese medicine composition include Ziziphus jujuba seeds and Gardenia jasminoides, and the HPLC fingerprint construction method includes the following steps: S1. Prepare the test solution: take a sample of the Chinese medicine composition, accurately weigh it, add an extraction solvent, ultrasonically treat it, cool it, centrifuge it, and then filter it with suction. Take the filtrate and spin-dry it to obtain a residue. Dissolve the residue in a solvent, make up to volume, shake it well, filter it, and take the filtrate to obtain the test solution; wherein the extraction solvent is a methanol solution or an ethanol solution; S2 Preparation of reference solution: Genipin gentiobioside, geniposide, magnololamine, spinosin, crocin I, and crocin II are taken as reference substances, and organic solvent is added to prepare a mixed reference solution; The test solution and the mixed reference solution are respectively taken and measured by high performance liquid chromatography to obtain an HPLC fingerprint of the traditional Chinese medicine composition; the common peaks of the fingerprint include chromatographic peaks of genipin gentiobioside, geniposide, magnolamine, spinosin, crocin I and crocin II; The HPLC method uses an Agilent TC-C18 (2) column, 250 × 4.6 mm, 5 μm; the column temperature is 25 to 45 ° C; Mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, the flow rate of the mobile phase is 0.8-1.2 mL / min, and the injection volume is 5-20 μL; The detection wavelengths were 227 nm for 0-24 min, 335 nm for 24-42 min, and 440 nm for 42-63 min. Gradient elution; the gradient elution method is as follows: From 0 to 5 min, the volume percentage of the mobile phase A was changed from 5 to 10, and the volume percentage of the mobile phase B was changed from 95 to 90; 5-6 min, the volume percentage of the mobile phase A was changed from 10 to 15, and the volume percentage of the mobile phase B was changed from 90 to 85; 6-20 min, the volume percentage of the mobile phase A is maintained at 15, and the volume percentage of the mobile phase B is maintained at 85, From 20 to 26 min, the volume percentage of the mobile phase A was changed from 15 to 16, and the volume percentage of the mobile phase B was changed from 85 to 84; From 26 to 27 min, the volume percentage of the mobile phase A was changed from 16 to 20, and the volume percentage of the mobile phase B was changed from 84 to 80; From 27 to 37 min, the volume percentage of the mobile phase A was changed from 20 to 23, and the volume percentage of the mobile phase B was changed from 80 to 77; From 37 to 47 min, the volume percentage of the mobile phase A was changed from 23 to 37, and the volume percentage of the mobile phase B was changed from 77 to 63; From 47 to 49 min, the volume percentage of the mobile phase A was changed from 37 to 39, and the volume percentage of the mobile phase B was changed from 63 to 61; From 49 to 63 min, the volume percentage of the mobile phase A changed from 39 to 100, and the volume percentage of the mobile phase B changed from 61 to 0.

2. The method for constructing an HPLC fingerprint of a Chinese medicine composition according to claim 1, characterized in that: The flow rate of the mobile phase was 1.0 mL / min; the injection volume was 10 μL, and the column temperature was 30° C.

3. The method for constructing an HPLC fingerprint of a Chinese medicine composition according to claim 1, characterized in that: The raw materials of the traditional Chinese medicine composition include 1 to 10 parts by weight of spinach seeds and 10 to 1 parts by weight of gardenia.

4. The method for constructing an HPLC fingerprint of a Chinese medicine composition according to claim 1, wherein: In step S2, the organic solvent for preparing the reference solution is a methanol solution or an acetonitrile solution.

5. The method for constructing an HPLC fingerprint of a Chinese medicine composition according to claim 1, wherein: The preparation steps of the reference solution are as follows: taking accurately weighed reference substances of genipin gentiobioside, gardenoside, magnolamine, spinoside, crocin I, and crocin II, adding methanol solution, and preparing a mixed reference solution containing 225 μg / mL genipin gentiobioside, 621 μg / mL gardenoside, 38 μg / mL magnolamine, 29 μg / mL spinoside, 190 μg / mL crocin I, and 47 μg / mL crocin II.

6. A method for detecting a Chinese medicine composition, characterized in that: The raw materials of the Chinese medicine composition include spinach seeds and gardenia, and the detection method includes the following steps: (1) establishing an HPLC fingerprint of a mixed reference substance according to the method for constructing an HPLC fingerprint of a traditional Chinese medicine composition as described in any one of claims 1 to 5; (2) establishing an HPLC fingerprint of a sample of the Chinese medicine composition to be tested according to the method for constructing an HPLC fingerprint of a Chinese medicine composition as described in any one of claims 1 to 5; (3) The HPLC fingerprint of the mixed reference and the HPLC fingerprint of the Chinese medicine composition sample to be tested are imported into the Chinese medicine chromatographic fingerprint similarity evaluation system to perform full spectrum peak matching, generate an overlay spectrum, and then generate a reference fingerprint spectrum. The similarity of each batch of samples is calculated to be above 0.95.

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