Fingerprint spectrum of granules for treating asthenia syndrome and its establishment method

Abstract

The present invention relates to the field of pharmacological research, specifically a granule fingerprint for treating asthenia syndrome and a method for establishing the same, the method comprising determining a reference substance and HPLC chromatographic conditions and establishing an HPLC standard fingerprint. The present invention discloses an HPLC standard fingerprint of granules for treating asthenia syndrome obtained by the method, the fingerprint calibrates characteristic peaks, and preliminarily reveals the effective components and mechanism of action of the compound Qirong granules in combination with network pharmacology and molecular docking technology. The method of the present invention has strong practicability, high stability and good reproducibility. By comparing the characteristic peaks in the standard fingerprint, the quality of the granules for treating asthenia syndrome can be comprehensively evaluated and controlled, which is conducive to grasping the stability of product quality, ensuring the uniformity and stability of its intrinsic quality, ensuring the safety and effectiveness of clinical medication, and providing a reference for the quality control, in-depth development and clinical application of granules for treating asthenia syndrome.

Description

Technical Field

[0001] The invention relates to the technical field of pharmacological research, and in particular to a granule fingerprint spectrum for treating asthenia syndrome and a method for establishing the fingerprint spectrum. Background Art

[0002] Frailty syndrome is a clinical disease closely related to aging, with fatigue, unexplained weight loss, repeated infections, impaired limb balance, impaired gait integrity, and decreased brain function as the main clinical manifestations. Against the backdrop of the deepening global aging, the number of frail people is growing rapidly, and the progressive decline in multi-system physiological functions and reserves increases the vulnerability of the elderly during stress, and increases the risk of disability, functional decline, hospitalization, and death, leading to increasingly prominent events such as disability, hospitalization, and death. At present, clinical guidelines mainly use exercise methods (yoga, balance training, Tai Chi, Ba Duan Jin, etc.), nutritional support, and other methods for intervention. Although there are certain positive effects, there are great limitations when facing frail patients who are not malnourished or have exercise restrictions, and they cannot fundamentally improve the patient's physical fitness. How to reverse the pre-frail state to a healthy state or the frail state to the pre-frail state through early clinical intervention is currently a very important public health issue.

[0003] Traditional Chinese medicine intervention for frailty based on syndrome differentiation and treatment is also effective, with the advantages of overall adjustment and individualized treatment. However, most of them are small sample case studies, and there is a basic lack of standardized clinical treatment drugs. It can be said that there is no specific treatment for the intervention measures for patients in the early stage of frailty in the elderly. Fufang Qirong Granules are made of raw astragalus, Cistanche deserticola, Polygonatum sibiricum, Leech, processed Polygonum multiflorum, red peony root, Acorus tatarinowii and other Chinese medicines, which have the effects of invigorating qi and kidney and promoting blood circulation and removing blood stasis. Fufang Qirong Granules are used in the clinic as an in-hospital preparation of the General Hospital of the People's Liberation Army to treat frailty syndrome, prethrombotic state of diabetes, and immune dysfunction in the elderly. Fufang Qirong Granules are rich in types of ingredients, containing compounds such as flavonoids, phthalides, alkaloids and phenolic acids, and have the characteristics of multi-component, multi-target and multi-pathway synergistic effects, which brings difficulties to the quality control and in-depth development of Fufang Qirong Granules. The fingerprint of traditional Chinese medicine can comprehensively characterize the chemical components of traditional Chinese medicine, comprehensively and comprehensively reflect the quality of traditional Chinese medicine, and is an important means to evaluate the quality of traditional Chinese medicine. However, there is no fingerprint of Compound Qirong Granules yet. Summary of the invention

[0004] Network pharmacology is based on the theory of systems biology and recognizes the interaction between drugs and the body from a holistic perspective. It coincides with the holistic theory of traditional Chinese medicine, especially the research on traditional Chinese medicine compound prescriptions. By constructing a "disease-gene-target-drug" collective network, the overall mechanism of action of traditional Chinese medicine can be quickly discovered. The joint analysis of traditional Chinese medicine fingerprints and network pharmacology has been widely used in the research of traditional Chinese medicine and traditional Chinese medicine compound prescriptions. By combining the multiple components in the fingerprints with the multi-target and multi-signal pathways of network pharmacology to explain the occurrence and development of diseases, the effective substances of traditional Chinese medicine and potential targets and mechanisms of action are predicted and analyzed. Therefore, the present invention combines fingerprints and network pharmacology to establish a particle fingerprint for the treatment of frailty syndrome, conducts network pharmacology research on the main components in the compound Qirong granules, and preliminarily explores the pharmacodynamic mechanism of the compound Qirong granules in the treatment of frailty.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for establishing a fingerprint spectrum of granules for treating asthenia syndrome comprises the following steps:

[0007] Grind 1g of compound Qirong granules and place in a conical flask, add 18-23mL of methanol solution, weigh and ultrasonicate, cool and weigh again, make up the reduced weight with methanol solution, shake well and filter, take the filtrate, which is the test solution;

[0008] Weigh the reference substance and add methanol solution. The reference substance contains echinacoside, peoniflorin, verbascoside, liquiritin, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, verbascoside and β-asarone. Prepare solutions containing 20 μg of echinacoside, 0.2 mg of peoniflorin, 5 μg of verbascoside, 10 μg of liquiritin, 40 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, 30 μg of verbascoside and 50 μg of β-asarone per 1 ml, respectively, as the reference substance solution;

[0009] Chromatographic tests were performed on different batches of test solutions and reference solutions to obtain UPLC chromatograms of different batches and reference solutions. The chromatographic conditions were as follows: an Agilent InfinityLab Poroshell 120 EC-C18 column (3.0 mm×150 mm, 2.7 μm) was used; the mobile phase was acetonitrile (A)-phosphoric acid solution (B) with a mass fraction of 0.1%, gradient elution (0-15 min, 15%-25% A; 15-30 min, 25%-55% A; 30-40 min, 55%-95% A; 40-45 min, 95% A); the detection wavelength was 220 nm, and the flow rate was 0.4 mL·min-1 ; Injection volume 2 μL, column temperature 30 °C;

[0010] The UPLC chromatograms of different batches were imported into the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint. The UPLC chromatogram of the first batch was used as the reference chromatogram. The average method was used for multi-point correction and Mark peak matching. A reference chromatogram was generated and the common peaks were calibrated. The UPLC chromatogram of the reference substance was compared with the reference chromatogram to obtain the fingerprint.

[0011] As a further solution of the present invention: the mass fraction of the methanol solution is 70-90%.

[0012] As a further solution of the present invention: the weight is accurately determined using an analytical balance.

[0013] As a further solution of the present invention: the power of ultrasonic treatment is 400-600W, and the frequency is 36-43KHz.

[0014] As a further embodiment of the present invention: the ultrasonic treatment time is 25-38 minutes.

[0015] As a further solution of the present invention: the chromatographic test adopts an ultra-high performance liquid chromatograph, and the ultra-high performance liquid chromatograph is at least one of Thermo U-3000, ACCHROM S6000 and Shimadzu LC-20AT.

[0016] A fingerprint spectrum obtained by the method for establishing a fingerprint spectrum of granules for treating asthenia syndrome.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention established the HPLC fingerprint of granules for treating asthenia syndrome (i.e., Compound Qirong Granules), and preliminarily revealed the effective components and action mechanisms of Compound Qirong Granules by combining the network pharmacology method, providing a reference for the quality control, in-depth development and clinical application of Compound Qirong Granules. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is the UPLC fingerprint and reference chromatogram of the test sample in the embodiment of the present invention.

[0020] Figure 2 UPLC chromatogram and fingerprint of Fufang Qirong Granules of the reference substance in the examples of the present invention. DETAILED DESCRIPTION

[0021] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] The pharmacodynamic components and mechanism of action of Compound Qirong Granules were predicted and analyzed based on fingerprint and network pharmacology. Methods: The UPLC fingerprint of Compound Qirong Granules was established, and the similarity of 15 batches of samples was evaluated using the Chinese medicine chromatographic fingerprint similarity evaluation system software (2012 edition); the pharmacodynamic components of Compound Qirong Granules were predicted based on literature, and the core targets and pathways for the treatment of frailty were analyzed based on network pharmacology, and the "pharmaceutical component-target-pathway" network was established, and the key targets and components were verified using AutoDockVina. Results: UPLC fingerprints of 15 batches of Fufang Qirong granules were established, with similarities ranging from 0.914 to 0.997. Twelve common peaks were calibrated, and 7 common peaks were identified by reference substances, namely echinacoside, paeoniflorin, verbascoside, liquiritin, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, verbascoside, and β-asarone. Combined with network pharmacology analysis, 7 key components, 64 potential targets, and 123 key signaling pathways in Fufang Qirong granules were screened out. Molecular docking results showed that the docking binding energies of key components and corresponding targets were less than -5 kcal·mol -1 . Conclusion: The results of fingerprint combined with network pharmacology show that the key ingredients in Fufang Qirong Granules for the treatment of frailty include calycosin glucoside, β-asarone, chrysanthemum glycoside, paeoniflorin, calycosin glucoside, etc., which can regulate relaxin, dopaminergic synapses, tryptophan metabolism and other signaling pathways through targets such as EGFR, TNF, HSP90AA1 and ALB to exert therapeutic effects. This study established the HPLC fingerprint of granules (i.e., Fufang Qirong Granules) for the treatment of frailty syndrome, and combined with the network pharmacology method, preliminarily revealed the effective components and mechanism of action of Fufang Qirong Granules, providing a reference for the quality control, in-depth development and clinical application of Fufang Qirong Granules.

[0023] Instruments: Ultra-high performance liquid chromatograph, Thermo U-3000 (Thermo Fisher, USA), ACCHROMS6000 (Waters, USA), Shimadzu LC-20AT (Shimadzu, Japan); CNC ultrasonic cleaner, KQ-500DE (Kunshan Ultrasonic Instrument Co., Ltd., power 500W, frequency 40kHz); water bath, EYELA SB-2000 (Shanghai Airo Instrument Co., Ltd.); analytical balance, BT25S, BS2202S and BS124S (Beijing Sartorius Balance Co., Ltd.); ultrapure water integrated system instrument, Millipore Mingche-D (Millipore, USA).

[0024] Reagents, drugs and reference substances: Test drug: Compound Qirong Granules, batch numbers 2210001, 2210002, 2210003, 2218015, 2218016, 2218017, 22219229, 2219230, 2219231, 2219232, 2315310, 2315311, 2315312, 2315313, 2315314 (S1~S15), 15 batches in total, all provided by the General Hospital of PLA.

[0025] Reagents: methanol (chromatographic grade, Lot216571, Fisher Scientific); acetonitrile (chromatographic grade, batch number 211030426002, Tianjin Biaoshiqi Technology Development Co., Ltd.); ultrapure water (prepared by laboratory Millipore Mingche-D, ultrapure water integrated system); phosphoric acid (super pure, batch number 20210831, Beijing Tongguang Fine Chemical Company).

[0026] Reference substances: echinacoside (batch number 111670-201907, purity 91.8%); verbascoside (batch number 111530-201914, purity 95.2%); verbascoside (batch number 111920-201907, purity 96.8%); paeoniflorin (batch number 110736-202145, purity 94.6%); β-asarone (batch number 112018-201802, purity 99.3%); liquiritigenin (batch number 111610-201908, purity 95.0%); 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (110844-202116, purity 94.8%); all were purchased from China Food and Drug Inspection Institutes.

[0027] Preparation of reference solution: Accurately weigh appropriate amount of each reference substance, add 80% methanol by mass to prepare solutions containing 20 μg echinacoside, 0.2 mg paeoniflorin, 5 μg verbascoside, 10 μg licorice glycoside, 40 μg 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, 30 μg verbascoside, and 50 μg β-asarone per 1 mL as reference solution.

[0028] Preparation of test solution: Take an appropriate amount of Fufang Qirong Granules, grind it into powder, take 1.0 g, weigh it accurately, put it in a stoppered conical flask, accurately add 20 mL of 80% methanol by mass, weigh it, ultrasonically treat it (power is 500 W, frequency is 40 kHz) for 30 minutes, let it cool, weigh it again, make up the lost weight with 80% methanol by mass, shake it well, filter it, and take the filtrate to obtain the test solution.

[0029] Chromatographic conditions: Agilent InfinityLab Poroshell 120 EC-C18 column (3.0 mm ×150 mm, 2.7 μm) was used; the mobile phase was acetonitrile (A)-0.1% phosphoric acid solution (B), gradient elution (0-15 min, 15%-25% A; 15-30 min, 25%-55% A; 30-40 min, 55%-95% A; 40-45 min, 95% A); the detection wavelength was 220 nm, the flow rate was 0.4 mL·min-1; the injection volume was 2 μL, and the column temperature was 30°C.

[0030] Precision test: Take an appropriate amount of Fufang Qirong Granules (batch number 2210001), prepare the test solution according to the above method, inject 6 times continuously according to the above chromatographic conditions, and record the UPLC chromatogram. Take 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (peak 6) as the reference peak (S), calculate the relative retention time and relative peak area of ​​each common peak, and find that their RSDs are all less than 2%, and the similarity of each spectrum is greater than 0.95, indicating that the instrument has good precision.

[0031] Stability test: Take an appropriate amount of Fufang Qirong Granules (batch number 2210001) and prepare the test solution according to the above method. Determine under the above chromatographic conditions at 0, 2, 4, 8, 12, and 24 hours, and record the UPLC chromatogram. Take 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (peak 6) as the reference peak (S), calculate the relative retention time and relative peak area of ​​each common peak, and the RSD is less than 2%, and the similarity of each spectrum is greater than 0.95, indicating that the test solution has good stability within 24 hours.

[0032] Repeatability test: Take an appropriate amount of Fufang Qirong Granules (batch number 2210001), prepare the test solution according to the above method, measure according to the above chromatographic conditions, and record the UPLC chromatogram. Take 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (peak 6) as the reference peak (S), calculate the relative retention time and relative peak area of ​​each common peak, and find that their RSDs are all less than 3%, and the similarity of each spectrum is greater than 0.95, indicating that the repeatability of this method is good.

[0033] Fingerprint establishment and similarity evaluation: Take appropriate amount of 15 batches of Fufang Qirong Granules (S1-S15), prepare test solution according to the above method, measure according to the above chromatographic conditions, record UPLC chromatograms, import UPLC chromatograms of 15 batches of samples into "Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System (2012 Edition)", take S1 as reference spectrum, use average method, perform multi-point correction and Mark peak matching, and generate control chromatogram (R), see Figure 1 A total of 12 common peaks were calibrated, and 7 chromatographic peaks were identified by reference substances, including peak 1 for echinacoside, peak 3 for paeoniflorin, peak 4 for verbascoside, peak 5 for licorice glycoside, peak 6 for 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (S, reference peak), peak 7 for verbascoside, and peak 10 for β-asarone, see Figure 2 The 15 batches of samples were matched with the reference chromatograms and the similarity evaluation was performed. The results were between 0.914 and 0.997, indicating that the quality of the samples was uniform and stable among the batches.

[0034] Molecular docking verification: Based on the RCSB Protein Data Bank database (https: / / www.rcsb.org / ), the structure of the main active ingredients was retrieved and the PDB structure file was downloaded. The target protein crystal 3D structure file obtained from the PDB database was opened with PyMOL 1.7.2.1 software, water molecules were removed, and the protein and its own ligand were separated and saved in PDB format. Small molecule compounds were retrieved from the Pubchem database (https: / / pubchem.ncbi.nlm.nih.gov / ) to obtain their sdf format structure files. The downloaded sdf format files were imported into OpenBabel 2.4.1 software to convert the 2D structure of the small molecule compounds into a 3D structure in mol2 format. The proteins and small molecule compounds were dehydrated, hydrogenated, charged, and non-polar hydrogens were processed using AutoDock Tools 1.5.6 software, and then saved as pdbqt format files. The small molecule compounds and proteins were molecularly docked using virtual screening scripts. After docking, the free binding energy of each small molecule compound binding to the protein can be obtained. The lower the binding energy, the better the docking effect. The conformation with the best affinity is selected as the final docking conformation for visualization in pyMOL software, and the spatial structure and hydrogen bonding after binding are further analyzed to draw the small molecule compound-protein docking image. Autodock-vina is used for semi-flexible docking, and the scores and interaction energy results are shown in Table 1.

[0035] Table 1 Molecular docking result information table

[0036]

[0037] A score less than -5.0 and greater than -7.0 indicates that the small molecule compound has good binding ability with the target, and a score between -5.0 and -4.25 indicates that the compound has a certain binding ability with the macromolecular target. As can be seen from Table 1, the autodock-vina docking score is less than -7.0, indicating that the target and the compound have a strong binding ability.

[0038] In summary, the present invention predicts and analyzes the pharmacological components and mechanism of action of Compound Qirong Granules based on fingerprints and network pharmacology. The UPLC fingerprint of Compound Qirong Granules was established, and the similarity evaluation system software of Chinese medicine chromatographic fingerprint (2012 edition) was used to evaluate the similarity of 15 batches of samples; the pharmacological components of Compound Qirong Granules were predicted based on literature, and the core targets and pathways for treating frailty were analyzed based on network pharmacology, and the "pharmacological component-target-pathway" network was established, and the key targets and components were verified using AutoDock Vina.

[0039] The UPLC fingerprints of 15 batches of Fufang Qirong granules were established with similarities ranging from 0.914 to 0.997. Twelve common peaks were calibrated, and 7 common peaks were identified by reference substances, namely echinacoside, paeoniflorin, verbascoside, licorice glycoside, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, verbascoside, and β-asarone. Combined with network pharmacology analysis, 7 key components, 64 potential targets and 123 key signal pathways in Fufang Qirong granules were screened out. Molecular docking results showed that the docking binding energies of the key components and the corresponding targets were less than -5 kcal·mol -1 .

[0040] The results of fingerprint analysis combined with network pharmacology showed that the key ingredients for the treatment of frailty in Compound Qirong Granules include calycosin, β-asarone, chrysanthemum indole, paeoniflorin, calycosin, etc., which can regulate relaxin, dopaminergic synapses, tryptophan metabolism and other signaling pathways through targets such as EGFR, TNF, HSP90AA1 and ALB to exert therapeutic effects. This study established the HPLC fingerprint of granules (i.e., Compound Qirong Granules) for the treatment of frailty syndrome, and combined with network pharmacology methods to preliminarily reveal the effective components and mechanism of action of Compound Qirong Granules, providing a reference for the quality control, in-depth development and clinical application of Compound Qirong Granules.

[0041] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for establishing a fingerprint spectrum of Fufang Qirong Granules, characterized in that: The following steps are involved: Grind 1g of compound Qirong granules and place in a conical flask, add 18-23mL of methanol solution, weigh and ultrasonicate, cool and weigh again, make up the reduced weight with methanol solution, shake well and filter, take the filtrate, which is the test solution; Weigh the reference substance and add methanol solution. The reference substance contains echinacoside, peoniflorin, verbascoside, liquiritin, 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, verbascoside and β-asarone. Prepare solutions containing 20 μg of echinacoside, 0.2 mg of peoniflorin, 5 μg of verbascoside, 10 μg of liquiritin, 40 μg of 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside, 30 μg of verbascoside and 50 μg of β-asarone per 1 ml, respectively, as the reference substance solution; Chromatographic tests were performed on different batches of test solutions and reference solutions to obtain UPLC chromatograms of different batches and UPLC chromatograms of reference solutions. The chromatographic conditions were as follows: Agilent InfinityLab Poroshell 120 EC-C18 column 3.0 mm×150 mm, 2.7 μm; mobile phase A was acetonitrile, mobile phase B was 0.1% phosphoric acid solution by mass fraction, gradient elution program was: 0-15 min, 15%-25% A; 15-30 min, 25%-55% A; 30-40 min, 55%-95% A; 40-45 min, 95% A; detection wavelength was 220 nm, flow rate was 0.4 mL·min -1 ; The injection volume was 2 μL, and the column temperature was 30 °C; The UPLC chromatograms of different batches were imported into the similarity evaluation system of traditional Chinese medicine chromatographic fingerprint. The UPLC chromatogram of the first batch was used as the reference chromatogram. The average method was used for multi-point correction and Mark peak matching. A reference chromatogram was generated and the common peaks were calibrated. The UPLC chromatogram of the reference substance was compared with the reference chromatogram to obtain the fingerprint.

2. The method for establishing the fingerprint spectrum of Fufang Qirong Granules according to claim 1, characterized in that: The mass fraction of the methanol solution is 70-90%.

3. The method for establishing the fingerprint spectrum of the compound Qirong granules according to claim 1 or 2, characterized in that: The weight was precisely determined using an analytical balance.

4. The method for establishing the fingerprint spectrum of Fufang Qirong Granules according to claim 1, characterized in that: The power of the ultrasonic treatment is 400-600W, and the frequency is 36-43KHz.

5. The method for establishing the fingerprint spectrum of the compound Qirong granules according to claim 4, characterized in that: The ultrasonic treatment time is 25-38 minutes.

6. The method for establishing the fingerprint spectrum of Fufang Qirong Granules according to claim 1, characterized in that: The chromatographic test uses an ultra-high performance liquid chromatograph, which is any one of Thermo U-3000, ACCHROM S6000 and Shimadzu LC-20AT.

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