Establishment method of multi-wavelength fusion fingerprint spectrum of herba inulae and caulis lonicerae particles for clearing arthralgia

Abstract

The invention belongs to the field of analysis of traditional Chinese medicine preparations, and particularly discloses a method for establishing a multi-wavelength fusion fingerprint spectrum of Jinxiaoqingbi granules, which comprises the following steps: preparing reference substance and test solution, determining HPLC (High Performance Liquid Chromatography) chromatographic conditions and establishing a standard fingerprint spectrum. The invention also discloses a multi-wavelength fused HPLC standard fingerprint spectrum of the Jinghuangqingbi granules obtained by the method, and the fingerprint spectrum has 22 characteristic peaks. The method disclosed by the invention is simple and convenient to operate, high in stability and good in reproducibility, can comprehensively evaluate and control the quality of the Jinghuangqing granules by comparing characteristic peaks in a standard fingerprint spectrum, is beneficial to stabilizing the product quality, ensures the uniformity and stability of the internal quality of the Jinghuangqing granules, and ensures the safety and effectiveness of clinical medication.

Description

Technical Field

[0001] The invention belongs to the field of analysis of traditional Chinese medicine preparations, and specifically relates to a method for establishing a multi-wavelength fusion fingerprint spectrum of Jinteng Qingbi granules and a standard fingerprint spectrum thereof. Background Art

[0002] Jinteng Qingbi Granule is a Chinese patent medicine of Lunan Houpu Pharmaceutical Co., Ltd. (Standard of the State Food and Drug Administration YBZ00332008). It is made of 11 Chinese medicinal herbs including honeysuckle, sinomenine, oldenlandia diffusa, Scrophularia, Rehmannia root, Trillium, Herba Luzi, Licorice, Angelica, White Peony Root, and Scolopendra. Its main effects are clearing away heat and detoxifying, promoting blood circulation and reducing swelling, and relieving pain. It is used for the active stage of rheumatoid arthritis, which belongs to the syndrome of internal toxic heat and damp-heat blocking the collaterals. Symptoms include swollen joints, pain that refuses to be pressed, morning stiffness, fever when touched, or red skin, fever, sweating, thirst, dry stool and yellow urine, red tongue, yellow and greasy fur, and slippery and rapid pulse.

[0003] The whole formula of Jinteng Qingbi Granules uses honeysuckle and sinomenine as the main drugs; Hedyotis diffusa, Trillium, Herba Lycopodii, Angelica sinensis, and Paeonia lactiflora as the assistant drugs; Scrophularia ningpoensis, Rehmannia glutinosa, and Licorice as the adjuvant drugs; and Scolopendra subspinipes as the messenger drugs. The drugs are matched and have the effects of clearing away heat and detoxifying, promoting blood circulation and reducing swelling, and relieving numbness and pain. Honeysuckle is rich in volatile oils, flavonoids, and organic acids. Experimental research results show that its decoction, oral solution, and injection have the effects of antipyretic, anti-inflammatory, and regulating the body's immune function. Sinomenine mainly contains active ingredients such as sinomenine and magnolia alkaloids, which have anti-inflammatory, analgesic, anti-rheumatic, and immunosuppressive effects. It is a commonly used Chinese medicine for the treatment of rheumatoid arthritis. Angelica, white peony root, Hedyotis diffusa, and Herba Deerbearingae can nourish blood and invigorate blood circulation, relieve acute pain, and strengthen tendons and bones; Scrophularia, Dry Rehmannia root, Licorice, and Tripterygium wilfordii can nourish yin and cool blood, and purge fire and detoxify; Centipede can invigorate blood circulation and eliminate blood stasis, attack toxins and dissipate stagnation, and dredge meridians and relieve pain. Pharmacological studies have shown that centipede can improve microcirculation and has strong analgesic and anti-inflammatory effects. All the medicines are used together to achieve the effects of clearing away heat and detoxification, invigorating blood circulation, eliminating blood stasis and swelling, and dredge meridians and relieve pain.

[0004] Since Jinteng Qingbi Granules is a Chinese patent medicine composed of 11 raw medicinal materials, the determination of one or several index components alone often has limitations and cannot fully and effectively reflect its overall quality. As a quality control technology that can reflect the overall picture of Chinese medicine as much as possible, Chinese medicine fingerprint is currently widely accepted in China and has become a hot spot in the modernization of Chinese medicine research.

[0005] At present, there is no research on the fingerprint of Jinteng Qingbi Granules. Based on this, this study established the HPLC fingerprint of Jinteng Qingbi Granules and used multi-wavelength fusion technology to establish the fingerprint of Jinteng Qingbi Granules, in order to grasp the quality of Jinteng Qingbi Granules as a whole and fully reflect its chemical information, thereby providing a reference for the quality control of Jinteng Qingbi Granules. Summary of the invention

[0006] The invention provides a method for establishing a multi-wavelength fusion fingerprint of Jinteng Qingbi granules. Gallic acid, neochlorogenic acid, sinomenine, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B and isochlorogenic acid C are used as reference substances to prepare mixed reference substance solutions and multiple batches of Jinteng Qingbi granule test sample solutions, which are respectively injected into an HPLC chromatography system, and an octadecylsilane bonded silica gel chromatography column and a methanol-phosphoric acid aqueous solution are used as mobile phases for determination. The corresponding data of the Jinteng Qingbi granule test sample are imported into a software of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) for analysis and comparison, so as to establish a fingerprint.

[0007] Jinteng Qingbi Granules contain gallic acid, neochlorogenic acid, sinomenine, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid, isochlorogenic acid C and other chemical components. Due to the differences in the absorption wavelengths of different components, it is difficult to detect them simultaneously at a single wavelength. Therefore, the present invention adopts multi-wavelength fusion technology so that more components contained in Jinteng Qingbi Granules can display better chromatographic peak signals, avoiding the defect of missing chromatographic peak information at a single wavelength, thereby enriching the chromatographic peak signals and making the fingerprint spectrum better reflect the intrinsic quality of Jinteng Qingbi Granules.

[0008] A method for establishing a multi-wavelength fusion fingerprint spectrum of Jintengqingbi, comprising the following steps:

[0009] (1) Accurately weigh gallic acid, neochlorogenic acid, sinomenine, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C reference substances, add methanol to completely dissolve them, prepare a mixed reference substance solution, filter through a 0.22 μm microporous membrane, and take the filtrate for later use;

[0010] (2) Preparation of test solution

[0011] Accurately weigh Jinteng Qingbi granules, accurately add ethanol solution, dissolve by ultrasonication, filter through a 0.22 μm microporous membrane, and take the filtrate to obtain the test solution;

[0012] (3) Chromatographic conditions

[0013] Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: methanol-phosphoric acid aqueous solution;

[0014] Elution was performed according to the following gradient elution program:

[0015]

[0016] (4) Preparation of fingerprint: The corresponding data of the Jinteng Qingbi Granules test sample were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) software for analysis and comparison to obtain the standard fingerprint of Jinteng Qingbi Granules composed of the common characteristic peaks of the samples.

[0017] The solvent used in the step (2) for preparing the Jinteng Qingbi granules test solution is 70% ethanol solution.

[0018] The chromatographic column is selected from one of Agilent ZORBAX SB-C18, Agilent Eclipse XDB-C18, and ThermoSecientific BDS HYPERSIL C18; the specification of the chromatographic column is 4.6 mm×250 mm, 5 μm.

[0019] The concentration of the phosphoric acid aqueous solution in the mobile phase is 0.1%-1.0%; preferably, the concentration of the phosphoric acid aqueous solution is 0.3%-0.5%; more preferably, it is 0.5%.

[0020] The temperature of the chromatographic column is 25-35°C, and more preferably the temperature of the chromatographic column is 30°C.

[0021] The flow rate of the mobile phase is 0.7-1.1 ml / min, preferably, the flow rate of the mobile phase is 1.0 ml / min.

[0022] Preferably, the detection wavelength is 262 nm or 330 nm.

[0023] The multi-wavelength fusion adopts the following method: according to the full wavelength scan, the chromatographic peak at a wavelength of 262nm is selected from 0 to 40min, the chromatographic peak at a wavelength of 330nm is selected from 40 to 72min, and the chromatographic peak at a wavelength of 262nm is selected from 72 to 105min. The data is programmed through Matlab software to perform multi-wavelength fusion for the entire time period.

[0024] The second object of the present invention is to provide a standard fingerprint of Jinteng Qingbi Granules obtained by the method.

[0025] The standard fingerprint of Jinteng Qingbi Granules is obtained by matching 19 batches of fingerprints to obtain 22 common peaks, with peak 4 of sinomenine as the reference peak, and its retention times are 0.430, 0.700, 0.840, 1.000, 1.291, 1.332, 1.361, 1.397, 1.663, 1.708, 1.773, 2.136, 2.547, 2.640, 3.012, 3.088, 3.194, 3.262, 4.288, 4.317, 4.460, 4.507. Among them, peak 1 is gallic acid, peak 3 is neochlorogenic acid, peak 5 is chlorogenic acid, peak 7 is cryptochlorogenic acid, peak 13 is isochlorogenic acid B, and peak 16 is isochlorogenic acid C.

[0026] The third object of the present invention is to provide application of the method in quality evaluation of Jinteng Qingbi Granules.

[0027] Compared with the prior art, the technical solution of the present invention has achieved the following beneficial technical effects:

[0028] (1) The present invention adopts multi-wavelength fusion technology to establish the fingerprint of Jinteng Qingbi Granules. Jinteng Qingbi Granules contain multiple chemical components such as gallic acid, neochlorogenic acid, sinomenine, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C. Due to the differences in the absorption wavelengths of different components, it is difficult to detect them simultaneously under a single wavelength. Therefore, this experiment adopts multi-wavelength fusion technology, which can make more components contained in Jinteng Qingbi Granules show better chromatographic peak signals, avoiding the defect of missing chromatographic peak information under a single wavelength, thereby enriching the chromatographic peak signals, so that the fingerprint can better reflect the intrinsic quality of Jinteng Qingbi Granules.

[0029] (2) The fingerprints of multiple batches of Jinteng Qingbi Granules were established by HPLC and the similarity evaluation system of Chinese medicine chromatographic fingerprints, 22 common peaks were extracted, and the chemical components of 7 of the common peaks were identified, which preliminarily reflected the overall chemical characteristics of Jinteng Qingbi Granules. The similarity between each batch of samples and the common pattern chromatogram of the present invention is high, which meets the requirements for the construction of the fingerprint, and the overall similarity of the fingerprint reaches more than 0.990, the similarity between samples is high, and there is no obvious difference in the composition of each chemical component.

[0030] (3) The established fingerprint spectrum is convenient, simple, and feasible, with good repeatability and stability, providing a scientific evaluation model for the overall quality control of Jinteng Qingbi Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the overlay of multi-wavelength fusion HPLC fingerprints of 19 batches of Jinteng Qingbi Granules;

[0032] Figure 2 This is the HPLC standard fingerprint of Jinteng Qingbi Granules (peaks 1-22 are 22 common characteristic peaks);

[0033] Among them, peak 1 is gallic acid; peak 3 is neochlorogenic acid; peak 4 is sinomenine; peak 5 is chlorogenic acid; peak 7 is cryptochlorogenic acid; peak 13 is isochlorogenic acid B; peak 16 is isochlorogenic acid C;

[0034] Figure 3 It is the HPLC fingerprint of the mixed reference substance;

[0035] Among them, peak 1 is gallic acid; peak 2 is neochlorogenic acid; peak 3 is sinomenine; peak 4 is chlorogenic acid; peak 5 is cryptochlorogenic acid; peak 6 is isochlorogenic acid B; peak 7 is isochlorogenic acid C. DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific embodiments, and it should be understood that the following embodiments are only used to describe the technical solution of the present invention, and are not used to limit the protection scope of the present invention. For those skilled in the art, obvious modifications and improvements made without departing from the concept of the present invention also belong to the protection scope of the present invention.

[0037] Example 1 Establishment of HPLC standard fingerprint of Jinteng Qingbi granules

[0038] 1. Instruments and reagents

[0039] 1.1 Instrument

[0040] SHIMADZU Essentia SIL-16 liquid chromatograph (Shimadzu Corporation, Japan), SK5210LHC ultrasonic cleaning machine (Shanghai Kedao Ultrasonic Instrument Co., Ltd.), SECURA125-1CN electronic balance (Sartorius Scientific Instrument Co., Ltd.).

[0041] 1.2 Materials

[0042] The reference substances gallic acid (110831-201204) and chlorogenic acid (110753-201314) were purchased from China Food and Drug Inspection Institutes; the reference substances neochlorogenic acid (DSTDX001501), cryptochlorogenic acid (DSTDY003501), isochlorogenic acid B (DSTDY003701), and isochlorogenic acid C (DSTDY003801) were purchased from Chengdu Desit Biotechnology Co., Ltd.; the reference substance sinomenine (22080212) was purchased from Beijing Betterkang Biotechnology Co., Ltd.

[0043] Jinteng Qingbi granules were provided by Lunan Houpu Pharmaceutical Co., Ltd., and the sample information is shown in Table 1. Methanol was chromatographically pure and purchased from Merck, Germany; anhydrous ethanol was purchased from Sinopharm Chemical Reagent Co., Ltd.; phosphoric acid was purchased from Laiyang Kangde Chemical Co., Ltd.; and purified water was purchased from Hangzhou Wahaha Group Co., Ltd.

[0044] Table 1 Jinteng Qingbi Granules Sample Information

[0045]

[0046] 2 Methods and Results

[0047] 2.1 Preparation of mixed reference solution

[0048] Accurately weigh gallic acid, neochlorogenic acid, sinomenine, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C reference substances in turn and place them in a volumetric flask. Add methanol to completely dissolve them to prepare a mixed reference solution containing 0.450 mg, 0.400 mg, 0.808 mg, 0.404 mg, 0.400 mg, 0.406 mg, and 0.400 mg of gallic acid, neochlorogenic acid, sinomenine, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C per 1 mL. Pass the solution through a 0.22 μm microporous filter membrane and take the filtrate for later use.

[0049] 2.2 Preparation of test solution

[0050] Accurately weigh about 0.5 g of Jinteng Qingbi Granules, place in a stoppered conical flask, accurately add 10 mL of 70% ethanol, seal, dissolve by ultrasound, pass through a 0.22 μm microporous filter membrane, and take the filtrate to obtain the test solution.

[0051] 2.3 Chromatographic conditions

[0052] Chromatographic column: Agilent ZORBAX SB-C18 (4.6×250 mm, 5 μm); mobile phase: water (0.5% phosphoric acid, A)-methanol (B); gradient elution, detection wavelength: 262 nm, 330 nm; column temperature 30° C.; injection volume 10 μL.

[0053] Perform gradient elution according to the following table:

[0054]

[0055] 2.4 Determination of HPLC fingerprint of Jinteng Qingbi granules

[0056] 2.4.1 Establishment of multi-wavelength fusion fingerprint

[0057] Take 19 batches of Jinteng Qingbi Granules samples, prepare them into test solutions according to the method under "2.2", and perform detection and analysis according to the chromatographic conditions under "2.3". According to the full wavelength scanning results, the chromatographic peak at a wavelength of 262nm is selected from 0 to 40min, the chromatographic peak at a wavelength of 330nm is selected from 40 to 72min, and the chromatographic peak at a wavelength of 262nm is selected from 72 to 105min. The data is programmed through Matlab software to perform full-time multi-wavelength fusion, and the chromatographic overlay diagram of 19 batches of samples is obtained, see Figure 1 .

[0058] 2.4.2 Calibration of main characteristic peaks

[0059] The corresponding data of 19 batches of Jinteng Qingbi granules samples were imported into the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" (2012 Edition) software for analysis and comparison, and the standard fingerprint was generated by fitting. Figure 2. A total of 22 common peaks were summarized and established in the obtained standard fingerprint. The chromatogram of the reference substance was measured under the same chromatographic conditions as the test substance, and the attribution of 7 characteristic peaks including gallic acid, neochlorogenic acid, sinomenine, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C was calibrated according to the corresponding peak positions of the chromatogram of the reference substance. Peak No. 4 (sinomenine) was set as the reference peak, and the relative peak area of ​​the common peaks was calculated as shown in Table 2

[0060]

[0061] 2.4.3 Similarity Evaluation

[0062] The chromatograms of 19 batches of Jinteng Qingbi Granules were compared with the standard fingerprints, and the similarity of the fingerprints of 19 batches of Jinteng Qingbi Granules was calculated by correlation coefficient method. The similarities of the 19 batches of Jinteng Qingbi Granules were: 0.999, 0.996, 0.998, 0.997, 0.999, 1.000, 0.995, 0.994, 0.998, 0.999, 0.999, 0.999, 0.999, 0.999, 0.998, 0.998, 0.999, 1.000, 0.999, 0.999, and the similarities were all greater than 0.990, which met the requirements for establishing fingerprints, indicating that the samples between batches had high similarity, good correlation, and small differences. It also shows that the quality stability and uniformity of each sample batch are good.

[0063] 2.5 Methodological Review

[0064] 2.5.1 Precision test

[0065] Accurately weigh 0.5 g of Jinteng Qingbi Granules (sample number S1), prepare the test solution according to the method in item "2.2", and inject the sample 6 times continuously according to the chromatographic conditions in item "2.3". The results show that the RSD of retention time of each common peak is less than 1%, and the RSD of peak area is less than 3%, which meets the requirements of fingerprint spectrum and indicates that the precision of the instrument is good.

[0066] 2.5.2 Repeatability test

[0067] Six portions of Jinteng Qingbi Granules from the same batch (sample number S1) were randomly taken out, each weighing about 0.5 g. The test solutions were prepared according to the method in item "2.2" and injected according to the chromatographic conditions in item "2.3". The results showed that the RSD of the retention time of each common peak was less than 1%, and the RSD of the peak area was less than 3%, indicating that the method had good repeatability.

[0068] 2.5.3 Stability study

[0069] Accurately weigh 0.5 g of Jinteng Qingbi Granules (sample number S1), prepare the test solution according to the method in "2.2", and according to the chromatographic conditions in "2.3", inject and detect the test solution in sequence at 0h, 2h, 4h, 8h, 12h, and 24h after the preparation of the test solution. The results show that the RSD of the retention time of each common peak is less than 1%, and the RSD of the peak area is less than 3%, indicating that the test sample has good stability within 24h.

Claims

1. A method for establishing a multi-wavelength fusion fingerprint spectrum of Jinteng Qingbi granules, characterized in that: Mixed reference solution and Jinteng Qingbi Granules test solution were prepared with gallic acid, neochlorogenic acid, sinomenine, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C reference standards, and injected into HPLC chromatography system respectively. The determination was carried out with octadecylsilane bonded silica gel chromatography column and methanol-phosphoric acid aqueous solution as mobile phase. The corresponding data of Jinteng Qingbi Granules test sample were imported into the 2012 edition of "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software for analysis and comparison, and the fingerprint was established.

2. The method according to claim 1, characterized in that: The steps include: (1) Accurately weigh gallic acid, neochlorogenic acid, sinomenine, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid B, and isochlorogenic acid C reference substances, add methanol to completely dissolve them, prepare a mixed reference substance solution, filter through a microporous membrane, and take the filtrate for later use; (2) Preparation of test solution Accurately weigh Jinteng Qingbi granules, accurately add ethanol solution, dissolve by ultrasound, pass through a microporous filter membrane, and take the filtrate to obtain the test solution; (3) Chromatographic conditions Chromatographic column: octadecylsilane bonded silica gel as filler; mobile phase: methanol and 0.1%-1.0% phosphoric acid aqueous solution; Elution was performed according to the following gradient elution program: (4) Preparation of fingerprint The corresponding data of the Jinteng Qingbi Granules test sample were imported into the 2012 edition of the "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System" software for analysis and comparison, and the standard fingerprint of Jinteng Qingbi Granules composed of the common characteristic peaks of the samples was obtained.

3. The method according to claim 2, characterized in that The chromatographic column described in step (3) is selected from one of AgilentZORBAX SB-C18, Agilent Eclipse XDB-C18, and Thermo Scientific BDS HYPERSIL C18; the specifications of the chromatographic column are 4.6 mm×250 mm, 5 μm.

4. The method according to claim 2, characterized in that The concentration of the phosphoric acid aqueous solution in the mobile phase of step (3) is 0.3%-0.5%.

5. The method according to claim 4, characterized in that The concentration of the phosphoric acid aqueous solution in the mobile phase of step (3) is 0.5%.

6. The method according to claim 2, characterized in that The temperature of the chromatographic column in step (3) is 25-35°C.

7. The method according to claim 2, characterized in that: The flow rate of the mobile phase in step (3) is 0.7-1.1 ml / min.

8. The method according to claim 2, characterized in that: The detection wavelengths in step (3) are 262nm and 330nm.

9. The method according to claim 2, characterized in that: The standard fingerprint of Jinteng Qingbi is obtained by matching the fingerprints of 19 batches of Jinteng Qingbi granules to obtain 22 common characteristic peaks, with the sinomenine No. 4 peak as the reference peak, and the relative retention times are 0.430, 0.700, 0.840, 1.000, 1.291, 1.332, 1.361, 1.397, 1.663, 1.708, 1.773, 2.136, 2.547, 2.640, 3.012, 3.088, 3.194, 3.262, 4.288, 4.317, 4.460, and 4.507, respectively.

10. Use of the method according to any one of claims 1 to 9 in the quality evaluation of Jinteng Qingbi Granules.

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