Traditional Chinese medicine composition as well as preparation method and application thereof

Abstract

The invention relates to a traditional Chinese medicine composition which comprises active ingredients extracted from astragalus membranaceus, angelica sinensis, codonopsis pilosula, radix scrophulariae, dendrobe, radix achyranthis bidentatae, honeysuckle and liquorice, and the active ingredients comprise the following components in percentage by mass: more than 0.8 permillage of neochlorogenic acid, more than 1.8 permillage of chlorogenic acid, more than 1.0 permillage of cryptochlorogenic acid, more than 0.50 permillage of isochlorogenic acid A, more than 0.60 permillage of isochlorogenic acid B and more than 0.80 permillage of isochlorogenic acid C, the medicine is prepared from the following raw materials in percentage by weight: more than 1.1 per mill of secoxyloganin, more than 3.5 per mill of glycyrrhizic acid, more than 1.3 per mill of liquiritin, more than 0.25 per mill of harpagoside, more than 0.15 per mill of beta-ecdysterone, more than 0.8 per mill of astragaloside, more than 0.14 per mill of ginsenoside RO, more than 0.1 per mill of senkyunolide A and more than 0.1 per mill of lobetyolin. The method can be used for quality control of the traditional Chinese medicine composition, so that stable quality and reliable curative effect of the traditional Chinese medicine preparation are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine, and in particular to a traditional Chinese medicine compound preparation and a preparation method and application thereof. Background Art

[0002] Atherosclerotic thrombotic cerebral infarction is the most common type of cerebral infarction. It refers to the occurrence of lumen stenosis, occlusion or thrombosis on the basis of vascular wall lesions caused by atherosclerosis, resulting in interruption of blood flow in the local blood supply area of ​​the brain, ischemia, hypoxia and softening necrosis of brain tissue, and corresponding nervous system dysfunction. It belongs to the category of "stroke" in traditional Chinese medicine. Because stroke has an acute onset, changes in many ways and is rapid, such as "the speed of a storm in the arrow and stone", ancient doctors named it "stroke". Because of its sudden onset, it is also called "cerebral stroke". It is a type of disease with sudden fainting, unconsciousness, accompanied by crooked corners of the mouth, speech difficulties and hemiplegia as the main symptoms. It is mainly divided into two categories: hemorrhagic stroke (cerebral hemorrhage or subarachnoid hemorrhage) and ischemic stroke (cerebral infarction, cerebral thrombosis). Among them, ischemic stroke accounts for 86% of all strokes. Stroke is one of the major diseases that endanger the health of the middle-aged and elderly people. It is a common and frequently occurring disease in internal medicine. The annual incidence of stroke in my country is 0.219%, and the disability rate is 75.0%.

[0003] Patent application number: 200410064784.0, discloses a method for preparing a Chinese medicine component for treating ischemic stroke, the formula is 10-20g of Astragalus, 10-18g of Angelica, 8-10g of Scrophularia, 8-15g of Honeysuckle, 8-15g of Dendrobium, 6-10g of Licorice. Experiments show that the above-mentioned compound extract is subjected to SP-825 type macroporous adsorption resin, and the dry extract of 60% ethanol eluent has a good therapeutic effect on ischemic stroke in rats.

[0004] However, for Chinese herbal compound prescriptions, most of the raw materials come from natural products, such as plants, animals, minerals, etc. Since the growth environment of each medicinal material is different, their quality will also be affected by many factors, such as soil fertility, climate, water source, etc. In addition, factors such as harvesting season and picking technology will also affect the quality of medicinal materials. Even if the medicinal materials meet the requirements of the pharmacopoeia, the medicinal materials from different origins and batches will still lead to unstable ingredients and poor quality consistency in different batches of preparations due to differences in active ingredients. It can be seen that prescriptions based only on the weight of medicinal materials cannot meet the production requirements of modern Chinese patent medicines, which seriously restricts the stability and controllability of the clinical efficacy of traditional Chinese medicine and the repeatability and recognition of modern research results. When determining the formula of Chinese herbal compound prescriptions, it is necessary to carefully select and proportion the medicinal materials, and adjust the dosage according to the actual situation to ensure that the content of each active ingredient is within a reasonable range. In addition, during the preparation process, it is also necessary to pay attention to controlling the processing method and time of the medicinal materials to avoid affecting the content and activity of the beneficial ingredients in the medicinal materials. At the same time, the active ingredients in the complex system of Chinese medicine compound are unclear. Which main ingredients have a significant impact on the efficacy and what is the reasonable optimal content range? It is necessary to reasonably control the range of different active ingredients to ensure the stability of the quality of Chinese medicine preparations while ensuring their reliable efficacy. This is a key issue restricting the development of modern Chinese medicine. Summary of the invention

[0005] The present invention aims to further study the traditional Chinese medicine compound composed of eight traditional Chinese medicines, namely, astragalus, angelica, codonopsis, scrophularia, dendrobium, achyranthes, honeysuckle and licorice. In the early stage, a large number of experiments were conducted to carry out component analysis and efficacy tests on the traditional Chinese medicine compound composed of medicinal materials from different origins and batches. Attention was paid to the relationship between the main components with large fluctuations in content in the traditional Chinese medicine formula and the efficacy test results, and the specific active ingredients and appropriate contents that can affect the efficacy were found out, and verified through experiments, and the traditional Chinese medicine composition with stronger activity was screened out. The key active ingredient group was used as a control method to ensure the stable quality and reliable efficacy of the traditional Chinese medicine products.

[0006] In view of this, the present invention proposes a traditional Chinese medicine composition, which contains active ingredients extracted from astragalus, angelica, codonopsis, scrophularia, dendrobium, Achyranthes bidentata, honeysuckle and licorice. The active ingredients include, by mass percentage: neochlorogenic acid 0.8‰ or more, chlorogenic acid 1.8‰ or more, cryptochlorogenic acid 1.0‰ or more, isochlorogenic acid A 0.50‰ or more, isochlorogenic acid B 0.60‰ or more, isochlorogenic acid C 0.80‰ or more, strychnine glycoside 1.1‰ or more, glycyrrhizic acid 3.5‰ or more, liquiritin 1.3‰ or more, harpaoside 0.25‰ or more, β-ecdysterone 0.15‰ or more, astragaloside Ⅳ 0.8‰ or more, ginsenoside RO 0.14‰ or more, ligusticum chuanxiong lactone A 0.1‰ or more, and codonopsis pilosula glycoside 0.1‰ or more.

[0007] Preferably, the active ingredients include: neochlorogenic acid 0.81-2.01‰, chlorogenic acid 1.89-3.01‰, cryptochlorogenic acid 1.05-1.33‰, isochlorogenic acid A 0.50-0.79‰, isochlorogenic acid B 0.63-0.88‰, isochlorogenic acid C 0.84-1.25‰, strychnosyl glycoside 1.18-1.45‰, glycyrrhizic acid 3.68-5.74‰, liquiritin 1.35-2.2‰, harpaoside 0.25-0.37‰, β-ecdysterone 0.17-0.29‰, astragaloside IV 0.81-1.27‰, ginsenoside RO 0.14-0.28‰, ligusticum bisporum lactone A 0.10-0.19‰, and codonopsis pilosula glycoside 0.10-0.18‰.

[0008] The ratio of the above active ingredients is: neochlorogenic acid 0.81-2.01, chlorogenic acid 1.89-3.01, cryptochlorogenic acid 1.05-1.33, isochlorogenic acid A 0.50-0.79, isochlorogenic acid B 0.63-0.88, isochlorogenic acid C 0.84-1.25, strychnoside 1.18-1.45, glycyrrhizic acid 3.68-5.74, liquiritin 1.35-2.2, harpaoside 0.25-0.37, β-ecdysterone 0.17-0.29, astragaloside IV 0.81-1.27, ginsenoside RO 0.14-0.28, ligusticum chuanxiong lactone A 0.10-0.19, and codonopsis ginsenoside 0.10-0.18. The Chinese medicine composition can also be in an optional preparation form.

[0009] Specifically, the preparation forms include but are not limited to tablets, capsules, pills, powders, granules, and pellets.

[0010] The preparation method of the composition as described above comprises the following steps: weighing raw materials of eight medicinal materials, namely, astragalus, angelica, codonopsis, scrophularia, honeysuckle, dendrobium, achyranthes, and liquorice, according to the compound weight ratio of 1:1:1:1:1:1:1:1:1; decocting with water twice, the first time for 1.5 hours and the second time for 1 hour, with the amount of water added being 5 times and 3 times respectively, filtering, combining the decoctions and concentrating until there is no alcohol taste, adding ethanol to make the alcohol content reach 60%, stirring evenly, letting stand, recovering ethanol and concentrating to a thick paste, drying under reduced pressure, and crushing to obtain a dry paste powder.

[0011] In the above-mentioned Chinese medicine composition and preparation method thereof,

[0012] The fingerprint of Astragalus membranaceus and the reference fingerprint have a similarity greater than 0.7, preferably greater than 0.9. Specifically, the peak area and retention time of the reference fingerprint are as follows:

[0013] Peak number Retention time [Min] Peak area 1 3.617 3.442 2 12.623 2.63 3 18.3 2.227 4 26.253 4.53 5 35.82 1.775

[0014] Specifically, the fingerprint of the Radix Astragali and the reference fingerprint are compared with no less than 5 Figure 2 The indicated fingerprint peak is a reference calibration peak with a similarity greater than 0.7, preferably greater than 0.9.

[0015] Specifically, the extraction amount of the four components in the honeysuckle: chlorogenic acid should not be less than 2.0%; neochlorogenic acid should not be less than 0.05%; cryptochlorogenic acid should not be less than 0.05%; and broken oxidized strychnine should not be less than 0.2%; and the similarity between it and the reference fingerprint is greater than 0.8, preferably greater than 0.95. Specifically, the peak area and retention time of the reference fingerprint are as follows:

[0016]

[0017]

[0018] The fingerprints are compared with Figure 3 In comparison, the similarity is greater than 0.9, preferably greater than 0.95.

[0019] The similarity between the fingerprint of the dendrobium and the reference fingerprint is greater than 0.7, preferably greater than 0.9. Specifically, the peak area and retention time of the reference fingerprint are as follows:

[0020] Peak number Retention time [Min] Peak area 1 3.723 3.444 2 12.313 0.65 3 14.85 0.623 4 15.58 1.535 5 18.663 0.644 6 36.697 0.988 7 37.387 1.093 8 38.003 0.69 9 38.697 1.157

[0021] Specifically, the fingerprint of the dendrobium and the reference fingerprint, such as Figure 4 The indicated fingerprint peak is a reference calibration peak with a similarity greater than 0.7, preferably greater than 0.9.

[0022] The similarity between the fingerprint of Codonopsis pilosula and the reference fingerprint is greater than 0.8, preferably greater than 0.9. Specifically, the peak area and retention time of the reference fingerprint are as follows:

[0023] Peak number Retention time [Min] Peak area 1 3.723 3.444 2 12.313 0.65 3 14.85 0.623 4 15.58 1.535 5 18.663 0.644

[0024] Specifically, the fingerprint of Codonopsis pilosula and the reference fingerprint, such as Figure 5 The indicated fingerprint peak is a reference calibration peak, and the similarity is greater than 0.8, preferably greater than 0.9.

[0025] The similarity between the fingerprint of Scrophularia ningpoensis and the reference fingerprint is greater than 0.7, preferably greater than 0.9. Specifically, the peak area and retention time of the reference fingerprint are as follows:

[0026]

[0027]

[0028] Specifically, the fingerprint of the Scrophularia ningpoensis and the control fingerprint, such as Figure 6 The indicated fingerprint peak is a reference calibration peak with a similarity greater than 0.7, preferably greater than 0.9.

[0029] The similarity between the fingerprint of Achyranthes bidentata and the reference fingerprint is greater than 0.7, preferably greater than 0.9. Specifically, the peak area and retention time of the reference fingerprint are as follows:

[0030] Peak number Retention time [Min] Peak area 1 6.337 5.141 2 10.83 3.962 3 25.91 3.451 4 26.717 2.01

[0031] Specifically, the fingerprint of Achyranthes bidentata and the control fingerprint are as follows: Figure 7 The indicated fingerprint peak is a reference calibration peak with a similarity greater than 0.7, preferably greater than 0.9.

[0032] The similarity between the fingerprint of Angelica sinensis and the reference fingerprint is greater than 0.7, preferably greater than 0.9. Specifically, the peak area and retention time of the reference fingerprint are as follows:

[0033] Peak number Retention time [Min] Peak area 1 3.603 8.125 2 13.223 10.227 3 17.927 2.817 4 39.393 2.009 5 40.043 2.3 6 40.743 29.978 7 41.1 9.03 8 47.193 2.076

[0034] Specifically, the fingerprint of the angelica sinensis and the reference fingerprint, such as Figure 8 The indicated fingerprint peak is a reference calibration peak with a similarity greater than 0.7, preferably greater than 0.9.

[0035] The similarity between the fingerprint of licorice and the reference fingerprint is greater than 0.7, preferably greater than 0.9. Specifically, the peak area and retention time of the reference fingerprint are as follows:

[0036] Peak number Retention time [Min] Peak area 1 15.52 5.776 2 15.987 7.782 3 21.25 2.021 4 23.11 2.29 5 26.31 3.405 6 28.35 2.119 7 30.613 3.196 8 33.617 4.638 9 35.877 33.337 10 38.093 6.082 11 43.817 2.346 12 45.06 2.85 13 47.027 5.108

[0037] Specifically, the fingerprint of the licorice and the reference fingerprint, such as Fig. 9 The indicated fingerprint peak is a reference calibration peak with a similarity greater than 0.7, preferably greater than 0.9.

[0038] Furthermore, when the oral preparation is a tablet, the preparation method also includes: adding 2.3% starch, 1% silicon dioxide, 1% talcum powder, 0.2% magnesium stearate and 2.5% ethanol to the dry paste powder, granulating, mixing, tableting, and coating with a gastric soluble film premix w / w 3% to obtain.

[0039] The present invention also proposes the use of any of the above-mentioned Chinese medicine compositions in the preparation of drugs for improving ischemic stroke, ischemic cardiomyopathy, and acute myocardial infarction. The "use" refers to administering the above-mentioned extract to a subject with the corresponding disease or the tendency of the disease, and its purpose is to impart a therapeutic effect, such as curing, alleviating, changing, affecting, improving or preventing the above-mentioned disease, its symptoms or its tendency, and possible sequelae associated with the disease. Those skilled in the art can easily determine the specific effective dose according to the type of disease to be treated, the route of administration, and the use of excipients, and the dose may vary due to the simultaneous use of other drugs.

[0040] The above-mentioned component content relationship also provides a basis for the quality control of the Chinese medicine composition. The present invention also includes a method for detecting the fingerprint of the Chinese medicine composition as described above, which comprises: chromatographic conditions Waters Xbridge BEH C 18 Chromatographic column (4.6×250 mm, 5 μm); mobile phase: 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution, (0-20 min, 1-12% B; 20-60 min, 12%-35% B; 60-70 min, 47%-50% B; flow rate: 1.0 mL·min -1 ; Column temperature 35℃; Wavelength 270nm.

[0041] Furthermore, the fingerprint and reference spectrum of the composition Figure 1 In comparison, the similarity is greater than 0.8, preferably 0.85, and more preferably 0.9.

[0042] There are 17 characteristic peaks in the characteristic spectrum of the test sample. The peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak should be within ±5% of the specified value. The specified values ​​of relative retention time are: 0.26 (peak 1), 0.33 (peak 2), 0.36 (peak 3), 0.48 (peak 4), 0.62 (peak 5), 0.67 (peak 6), 0.96 (peak 7), 0.98 (peak 8), 1 (peak 9), 1.11 (peak 10), 1.14 (peak 11), 1.21 (peak 12), 1.26 (peak 13), 1.35 (peak 14), 1.38 (peak 15), 1.50 (peak 16), and 1.95 (peak 17).

[0043] Further, peak 4 is neochlorogenic acid, peak 5 is chlorogenic acid, peak 6 is cryptochlorogenic acid, peak 9 is liquiritigenin, peak 10 isochlorogenic acid B, peak 11 isochlorogenic acid A, peak 12 isochlorogenic acid C, peak 16 isochlorogenic acid and peak 17 isochlorogenic acid.

[0044] The present invention further controls the quality of each raw material, and uses rat cerebral ischemia reperfusion injury and ISO-induced rat myocardial ischemia experiments to study the effect of the Chinese medicine composition on ischemic stroke, ischemic cardiomyopathy, acute myocardial infarction and other diseases or symptoms. The experimental results show that medicinal raw materials from different origins and batches will still lead to unstable ingredients and poor quality consistency in different batches of preparations due to differences in effective ingredients. The present invention screens out Chinese medicine compositions with specific active ingredient content characteristics through the control of key active ingredients, which have stable and excellent therapeutic effects. The above-mentioned active ingredient content characteristics can be used for the quality control of the Chinese medicine composition, thereby ensuring the stable quality and reliable efficacy of the Chinese medicine preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 HPLC fingerprints of 25 batches of Chinese herbal medicine extracts (A) and their reference fingerprints (B)

[0046] Figure 2 It is the reference fingerprint of Astragalus membranaceus;

[0047] Figure 3 It is the reference fingerprint of honeysuckle medicinal material;

[0048] Figure 4 It is the reference fingerprint of Dendrobium officinale;

[0049] Figure 5 It is the reference fingerprint of Codonopsis pilosula;

[0050] Figure 6 It is the reference fingerprint of Radix Scrophulariae Radix;

[0051] Figure 7 It is the reference fingerprint of Achyranthes bidentata medicinal material;

[0052] Figure 8 It is the reference fingerprint of Chinese angelica root;

[0053] Fig. 9 This is the reference fingerprint of licorice herbs. DETAILED DESCRIPTION

[0054] As mentioned above, the present invention aims to provide a Chinese medicine composition, preparation method, application and quality control method. The following will be described in detail in conjunction with the contents of the experimental examples.

[0055] It is particularly important to point out that similar substitutions and modifications made to the present invention are obvious to those skilled in the art, and they are all considered to be included in the present invention. Relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments.

[0056] If no specific conditions are specified in the present invention, all experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without specifying the manufacturer are conventional products that can be obtained commercially.

[0057] Determination of the content of main components in traditional Chinese medicine compositions

[0058] Content Determination Ⅰ Neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, broken oxidized strychnine, liquiritin, glycyrrhizic acid and harpagoside were determined by referring to the high performance liquid chromatography method (Appendix VI D of Part I of the 2015 edition of the Chinese Pharmacopoeia).

[0059] Liquid chromatography conditions and system suitability test Thermo acclaim TM 120C 18 Chromatographic column (4.6×250 mm, 5 μm; column temperature 35°C; flow rate 1 mL / min; injection volume: 10 μL; detection wavelength: 238 nm (liquiritigenin, broken oxidized strychnine), 252 nm (glycyrrhizic acid), 280 nm (harpagoside), 327 nm (neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C); mobile phase: 0.2% phosphoric acid aqueous solution-acetonitrile, gradient elution, the specific gradient is shown in the table below:

[0060]

[0061] Preparation of reference solution: Precisely weigh 10 mg of neochlorogenic acid, 20 mg of chlorogenic acid, 10 mg of cryptochlorogenic acid, 10 mg of isochlorogenic acid A, 10 mg of isochlorogenic acid B, 10 mg of isochlorogenic acid C, 10 mg of liquiritin, 30 mg of glycyrrhizic acid, and 10 mg of broken oxidized strychnine in a 20 mL volumetric flask. Precisely weigh 10 mg of harpagoside in a 10 mL volumetric flask, dissolve in 50% methanol, dilute to scale, shake well, accurately transfer 1 mL from it and place it in the aforementioned 20 mL volumetric flask, dilute to scale with 50% methanol, and obtain the mixed reference stock solution of neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, isochlorogenic acid A, isochlorogenic acid B, isochlorogenic acid C, broken oxidized strychnine, liquiritin, glycyrrhizic acid, and harpagoside. Accurately measure 1 mL from the mixed reference substance stock solution and place it in a 20 mL volumetric flask, add 50% methanol to make up to volume, and shake well to obtain the mixed reference substance solution.

[0062] Preparation of test solution: Take about 1 g of the Chinese medicine composition, weigh it accurately, put it in a stoppered conical flask, accurately add 25 mL of 50% methanol, weigh it, treat it with ultrasound for 30 minutes, cool it, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain the solution.

[0063] Determination method: Accurately pipette 10 μL of reference solution and test solution respectively, inject into liquid chromatograph, measure, record the chromatogram, and calculate with peak area according to external standard method.

[0064] Content determination IIβ-ecdysterone was determined by high performance liquid chromatography (Appendix VI D of Part I of the 2015 edition of the Chinese Pharmacopoeia). Liquid chromatography conditions and system suitability test The chromatographic column was filled with octadecylsilane bonded silica gel (chromatographic column: Kromasil C18, 3.5μm, 4.6×150mm); mobile phase: acetonitrile-0.1% H3PO4 gradient elution; detection wavelength: 250nm flow rate: 0.8mL / min column temperature: 30℃; injection volume: 5μL.

[0065] The elution gradient is shown in the table below:

[0066]

[0067] Preparation of reference solution Take an appropriate amount of β-ecdysterone reference substance, weigh it accurately, place it in a 25mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and prepare a stock solution containing 0.4 mg per 1mL. Accurately pipette 1mL of the stock solution and dilute it with methanol to obtain the reference solution.

[0068] Preparation of test solution: Take about 2 g of the Chinese medicine composition, accurately weigh it, put it in a stoppered conical flask, accurately add 50 mL of methanol, weigh it, ultrasonicate for 40 min, cool it, weigh it again, make up the loss with methanol, shake it well, filter it, accurately measure 25 mL of the filtrate, evaporate it to dryness, add 10 mL of water to dissolve the residue, shake it with water-saturated n-butanol for 5 times, 30 mL each time, combine the n-butanol solution, wash the n-butanol solution with ammonia test solution for 3 times, 20 mL each time, then take 5 ml of methanol to wash the separating funnel, combine it, evaporate it to dryness, transfer it to a 50 mL small flask, concentrate it under reduced pressure to dryness, dissolve it in methanol, transfer it to a 5 mL volumetric flask, dilute it to the scale, shake it well, centrifuge it, and take the supernatant.

[0069] Determination method: Accurately pipette 5 μL of reference solution and test solution respectively, inject into liquid chromatograph, measure, record the chromatogram, and calculate with peak area according to external standard method.

[0070] Content determination Ⅲ Astragaloside IV was determined by high performance liquid chromatography (General Rules 0512 of Part IV of the 2015 edition of the Chinese Pharmacopoeia). Liquid chromatography conditions and system suitability test The chromatographic column was filled with octadecylsilane bonded silica gel; acetonitrile-water (32:68) was used as the mobile phase; evaporative light scattering detector; flow rate was 1 mL / min, column temperature was 30°C; injection volume was 20 μL. The number of theoretical plates calculated based on the astragaloside IV peak should not be less than 4000.

[0071] Preparation of reference solution: Take an appropriate amount of astragaloside IV reference substance, weigh it accurately, and add methanol to make solutions containing 0.1 mg and 0.5 mg per 1 mL respectively.

[0072] Preparation of the test solution Take about 2g of the Chinese medicine composition, accurately weigh it, put it in a stoppered conical bottle, accurately add 50mL of methanol, weigh it, ultrasonicate for 60min, cool it, weigh it again, make up the lost weight with methanol, shake it well, filter it, accurately measure 25mL of the filtrate, evaporate it to dryness, add 25mL of water to the residue and dissolve it, shake and extract it 5 times with water-saturated n-butanol, 30mL each time, combine the n-butanol solution, wash the n-butanol solution 4 times with ammonia test solution, 30mL each time, evaporate the n-butanol solution, dissolve the residue in methanol and transfer it to a 2mL volumetric bottle, dilute it to the scale with methanol, shake it well, centrifuge it, and take the supernatant to obtain it. Determination method Accurately take 20μL of the reference solution and the test solution, inject them into the liquid chromatograph, determine, and calculate with the external standard two-point method logarithmic equation to obtain it.

[0073] Content Determination Ⅳ Determination of ligusticum chuanxiong lactone A, ginsenoside RO and codonopsis pilosula by ultra performance liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry

[0074] Chromatographic conditions: Chromatographic column: waters BEH C18 (2.1ⅹ100mm, 1.7μm); Column temperature: 35°C; Injection volume: 2μL Flow rate: 0.4mL / min; Mobile phase: Gradient elution, specific gradient see the table below:

[0075]

[0076] Then run for 3 minutes;

[0077] Mass spectrometry conditions: electrospray ion source (ESI); positive and negative ion mode acquisition; mass scan range m / z 100-1500; capillary voltage 3500 V; drying gas nitrogen temperature: 350°C; drying gas flow rate: 10 L·min-1; nebulizer gas nitrogen pressure 50 psi; fragmentation voltage 135 V; cone voltage 65 V.

[0078] Preparation of reference solution: Take appropriate amount of ligusticum chuanxiong lactone A, codonopsis pilosula and ginsenoside RO reference substances respectively, weigh them accurately, add methanol to make a mixed reference solution containing 15-30 mg·L-1 of each component, and centrifuge at 14000 r·min-1 for 10 min.

[0079] Preparation of test solution: Take the Chinese medicine composition, grind it into powder, take about 1g, weigh it accurately, and use 25mL 50% methanol to ultrasonically extract for 45min. Let it cool, weigh it again, make up the lost weight with 50% methanol, shake it well, filter it, and take the filtrate to obtain it. Centrifuge it at 14000r·min-1 for 10min to obtain it.

[0080] The determination method is to accurately pipette 2 μL of the reference solution and the test solution respectively, inject them into ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry, determine, record the total ion current, and calculate the peak area according to the external standard method to obtain the result.

[0081] Establishment of internal control standards

[0082] Based on the selected samples, by tracing back the component content characteristics of the medicinal raw materials, the relevant medicinal material fingerprint map is established as the internal control standard, and the preparation process is improved to ensure the improvement of product quality and uniformity and stability.

[0083] 1. Establish internal control standards for Astragalus membranaceus: Add liquid fingerprint control items based on the pharmacopoeia

[0084] Liquid fingerprint detection method: The fingerprint of the test sample and the reference fingerprint 1, with no less than 5 fingerprint peaks as marked in the reference fingerprint spectrum as reference calibration peaks, calculated by the calculation software, the similarity should be greater than 0.7.

[0085] The peak areas and retention times of the control fingerprints in this example are as follows:

[0086] Mark Peak Number Retention time [Min] Peak area 1 3.617 3.442 2 12.623 2.63 3 18.3 2.227 4 26.253 4.53 5 35.82 1.775

[0087] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as filler: methanol-0.3% phosphoric acid aqueous solution system was used as mobile phase; elution procedure was:

[0088]

[0089]

[0090] The flow rate was 1 ml / min; the detection wavelength was 254 nm and the column temperature was 30°C.

[0091] Preparation of reference solution: Take an appropriate amount of calycosin glucoside reference substance, accurately weigh it, and add 50% methanol to make a solution containing 50 μg per 1 ml.

[0092] Preparation of the test solution: Take the Astragalus root material, crush it, take about 1.0g of powder, accurately weigh it, put it in a 50ml conical flask, add 25ml of 50% methanol, ultrasonically extract for 0.5 hours, filter, and take the filtrate to obtain the solution.

[0093] Determination method: Accurately pipette 10 μl of each test solution and inject it into the liquid chromatograph for determination.

[0094] 2. Establish internal control standards for honeysuckle medicinal materials: based on the pharmacopoeia standards, add chlorogenic acid, neochlorogenic acid, cryptochlorogenic acid, broken-oxidized strychnine glycoside extraction and medicinal liquid fingerprint.

[0095] (1) The extraction amount of the four components in honeysuckle: chlorogenic acid should not be less than 2.0%; neochlorogenic acid should not be less than 0.05%; cryptochlorogenic acid should not be less than 0.05%; and steric acid should not be less than 0.2%. That is, in this embodiment, taking chlorogenic acid as an example, the chlorogenic acid obtained by water extraction of 100g honeysuckle is not less than 2g.

[0096] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler, methanol-0.1% phosphoric acid aqueous solution system was used as mobile phase, and gradient elution was performed according to the table below; the flow rate was 0.8 ml / min; the detection wavelength for chlorogenic acid, neochlorogenic acid and cryptochlorogenic acid was 324 nm, and the detection wavelength for gardenia glycoside and broken-oxidized strychnoside was 237 nm; the theoretical plate number was calculated based on the chlorogenic acid peak and should not be less than 10,000.

[0097]

[0098] Preparation of reference solution: Take appropriate amount of chlorogenic acid reference substance and gardenia glycoside reference substance, add 50% methanol to make a solution containing about 0.3 mg of chlorogenic acid and 0.5 mg of gardenia glycoside per 1 ml, shake well, and obtain.

[0099] Preparation of test solution: Measure 1800ml of water, place in a flask, boil, add 100g of this product, make the water liquid cover the medicinal materials, reflux extraction for 1 hour, filter, add 1800ml of water to the residue, boil, reflux extraction for 1 hour, filter while hot, combine the filtrate, cool, accurately measure 1-10ml of the filtrate, place in a 50ml volumetric bottle, add 50% methanol to the scale. Shake well, and you are done.

[0100] Determination method: Accurately measure 10 μl of the reference solution and the test solution, respectively, and inject them into the liquid chromatograph for determination. Using the chlorogenic acid reference as a reference, calculate the content of chlorogenic acid in this product by the external standard method; using the chlorogenic acid in this product as an internal reference, calculate the content of neochlorogenic acid and cryptochlorogenic acid in this product according to the relative correction factor; using the gardenia glycoside in the reference solution as an internal reference, calculate the concentration of the interrupted oxidized strychnoside in this product according to the relative correction factor; determine the peak position of each component by the relative retention time.

[0101] The relative correction factors and relative retention times of neochlorogenic acid, cryptochlorogenic acid and strychnine are shown in the following table:

[0102]

[0103]

[0104] Note: Relative retention time is the ratio of the retention time of the component to be measured to that of its internal reference.

[0105] Technical parameters (reference): Chromatographic column phenomenex Luna C18 (4.6×250mm, 5μm) or KromasilC18 (4.6×250mm, 5μm).

[0106] Calculate as follows:

[0107]

[0108] Where: f 平均 - average value of correction factors;

[0109] A 样 ——Peak area of ​​chlorogenic acid in the test solution (average of 2 injections);

[0110] - dilution volume of the test article;

[0111] 10 -3 - unit conversion factor;

[0112] m 样 ——Sampling amount of test product (g)

[0113] (2) Liquid fingerprint: The fingerprint of the test sample should be similar to that of the control. The 12 marked chromatographic peaks should be displayed. The similarity calculated by the calculation software should be greater than 0.9.

[0114] The peak areas and retention times of the control fingerprints in this example are as follows:

[0115] Peak number Retention time [Min] Peak area 1 8.47 5.507 2 11.103 2.044 3 13.077 12.133 4 14.397 91.272 5 14.9 37.905 6 17.29 3.353 7 19.153 3.053 8 22.113 7.654 9 25.063 6.939 10 25.983 11.825 11 27.833 2.886 12 28.697 2.287 13 30.447 2.879 14 30.647 50.522 15 31.637 9.526

[0116] Chromatographic conditions and system suitability test: octadecylsilane bonded silica gel was used as filler (chromatographic column: phenomenex luna C18, 4.6×250mm, 5μm); acetonitrile-0.2% phosphoric acid aqueous solution system was used as mobile phase; detection wavelength was 254nm, column temperature was 30℃. The theoretical plate number was calculated based on the reference substance (chlorogenic acid) peak and should be no less than 6000. The elution procedure was:

[0117]

[0118] Preparation of reference solution: Take an appropriate amount of chlorogenic acid reference substance, weigh it accurately, and add 50% methanol to make a solution containing 50 μg per 1 ml.

[0119] Preparation of test solution: Take the solution under the chlorogenic acid extraction amount, filter it, and take the filtrate to obtain the solution.

[0120] Determination method: Accurately pipette 5 μl of reference solution and test solution respectively, inject into liquid chromatograph and determine.

[0121] 3. Establish internal control standards for Dendrobium officinale: Add liquid fingerprint control items based on the pharmacopoeia

[0122] Liquid fingerprint detection method: The fingerprint of the test sample should be similar to that of the control, the 9 marked chromatographic peaks should be displayed, and the similarity calculated by the calculation software should be greater than 0.7.

[0123] The peak areas and retention times of the control fingerprints in this example are as follows:

[0124] Peak number Retention time [Min] Peak area 1 3.723 3.444 2 12.313 0.65 3 14.85 0.623 4 15.58 1.535 5 18.663 0.644 6 36.697 0.988 7 37.387 1.093 8 38.003 0.69 9 38.697 1.157

[0125] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler (chromatographic column: Agilent C18, 4.6×250 mm, 5 μm); methanol-0.2% phosphoric acid aqueous solution system was used as the mobile phase, and the elution procedure was as follows:

[0126]

[0127] The flow rate was 1 ml / min; the detection wavelength was 254 nm; and the column temperature was 25°C.

[0128] Preparation of the test solution: Take the Dendrobium officinale material, crush it, take about 1g of powder, accurately weigh it, put it in a 50ml conical flask, accurately add 25ml of 60% methanol, ultrasonicate for 45 minutes, filter, and take the filtrate to obtain the solution.

[0129] Determination method: Accurately pipette 10 μl of the test solution, inject into liquid chromatograph, and determine.

[0130] 4. Establish internal control standards for Codonopsis pilosula: Add liquid fingerprint control items based on the pharmacopoeia

[0131] Liquid fingerprint detection method: The fingerprint of the test sample should be similar to that of the control, the 5 marked chromatographic peaks should be displayed, and the similarity should be greater than 0.8 calculated by the calculation software.

[0132] The peak areas and retention times of the control fingerprints in this example are as follows:

[0133] Peak number Retention time [Min] Peak area 1 3.723 3.444 2 12.313 0.65 3 14.85 0.623 4 15.58 1.535 5 18.663 0.644

[0134] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler (chromatographic column: Agilent extend C18, 4.6×250mm, 5μm); methanol-0.2% phosphoric acid aqueous solution system was used as the mobile phase, and the elution procedure was as follows:

[0135]

[0136] The flow rate was 1 ml / min; the detection wavelength was 254 nm; and the column temperature was 35°C.

[0137] Preparation of test solution: Take Codonopsis pilosula, crush it, take about 1g of powder, accurately weigh it, put it in a 50ml conical flask, accurately add 25ml of methanol, reflux extraction for 30 minutes, filter, and take the filtrate to obtain the test solution.

[0138] Determination method: Accurately pipette 10 μl of the test solution, inject into liquid chromatograph, and determine.

[0139] 5. Establish internal control standards for Radix Scrophulariae: Add liquid fingerprint control items based on the pharmacopoeia

[0140] Liquid fingerprint detection method: The fingerprint of the test sample should be similar to that of the control, the 7 marked chromatographic peaks should be displayed, and the similarity calculated by the calculation software should be greater than 0.7.

[0141] The peak areas and retention times of the control fingerprints in this example are as follows:

[0142] Peak number Retention time [Min] Peak area 1 6.337 5.141 2 10.83 3.962 3 25.91 3.451 4 26.717 2.01 5 29.42 9.002 6 36.813 6.028 7 38.9 8.159

[0143] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler (chromatographic column: Agilent extend C18, 4.6×250 mm, 5 μm); acetonitrile-0.2% phosphoric acid aqueous solution system was used as the mobile phase, and the elution procedure was as follows:

[0144]

[0145]

[0146] The flow rate was 1 ml / min; the detection wavelength was 238 nm; and the column temperature was 35°C.

[0147] Preparation of reference solution: Take an appropriate amount of harpagoside reference substance, weigh accurately, and add 50% methanol to make a solution containing 30 μg per 1 ml.

[0148] Preparation of test solution: Take Scrophularia ningpoensis, crush it, take about 1g of powder, accurately weigh it, put it in a 50ml conical flask, accurately add 25ml of 50% methanol, ultrasonically extract for 45 minutes, filter, and take the filtrate to obtain the test solution.

[0149] Determination method: Accurately pipette 10 μl of the test solution, inject into liquid chromatograph, and determine.

[0150] 6. Establish internal control standards for Achyranthes bidentata: Add liquid fingerprint control items based on the pharmacopoeia

[0151] Liquid fingerprint detection method: The fingerprint of the test sample should be similar to that of the control, the 7 marked chromatographic peaks should be displayed, and the similarity calculated by the calculation software should be greater than 0.7.

[0152] The peak areas and retention times of the control fingerprints in this example are as follows:

[0153] Peak number Retention time [Min] Peak area 1 6.337 5.141 2 10.83 3.962 3 25.91 3.451 4 26.717 2.01

[0154] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler (chromatographic column: Agilent extend C18, 4.6×250 mm, 5 μm); acetonitrile-0.2% phosphoric acid aqueous solution system was used as the mobile phase, and the elution procedure was as follows:

[0155]

[0156] The flow rate was 1 ml / min; the detection wavelength was 254 nm; and the column temperature was 30°C.

[0157] Preparation of test solution: Take Achyranthes bidentata medicinal material, crush it, take about 1g of powder, accurately weigh it, put it in a 50ml conical flask, accurately add 25ml of 50% methanol, ultrasonically extract for 45 minutes, filter, and take the filtrate to obtain the solution.

[0158] Determination method: Accurately pipette 10 μl of the test solution, inject into liquid chromatograph, and determine.

[0159] 7. Establish internal control standards for Chinese angelica: Add liquid fingerprint control items based on the pharmacopoeia

[0160] Liquid fingerprint detection method: The fingerprint of the test sample should be similar to that of the control, the 8 marked chromatographic peaks should be displayed, and the similarity should be greater than 0.7 calculated by the calculation software.

[0161] The peak areas and retention times of the control fingerprints in this example are as follows:

[0162]

[0163]

[0164] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler (chromatographic column: Agilent extendC18, 4.6×250mm, 5μm); acetonitrile-0.3% phosphoric acid aqueous solution system was used as the mobile phase, and the elution procedure was as follows:

[0165]

[0166] The flow rate was 1 ml / min; the detection wavelength was 238 nm; and the column temperature was 30°C.

[0167] Preparation of test solution: Take Chinese angelica root, crush it, take about 1g of powder, accurately weigh it, put it in a 50ml conical flask, accurately add 25ml of 50% methanol, ultrasonically extract for 45 minutes, filter, and take the filtrate to obtain the solution.

[0168] Determination method: Accurately pipette 10 μl of the test solution, inject into liquid chromatograph, and determine.

[0169] 8. Establish internal control standards for licorice herbs: Add liquid fingerprint control items based on the pharmacopoeia

[0170] Liquid fingerprint detection method: The fingerprint of the test sample should be similar to that of the control, the 15 marked chromatographic peaks should be displayed, and the similarity calculated by the calculation software should be greater than 0.7.

[0171] The peak areas and retention times of the control fingerprints in this example are as follows:

[0172] Peak number Retention time [Min] Peak area 1 15.52 5.776 2 15.987 7.782 3 21.25 2.021 4 23.11 2.29 5 26.31 3.405 6 28.35 2.119 7 30.613 3.196 8 33.617 4.638 9 35.877 33.337 10 38.093 6.082 11 43.817 2.346 12 45.06 2.85 13 47.027 5.108

[0173] Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as the filler (chromatographic column: Agilent extend C18, 4.6×250 mm, 5 μm); acetonitrile-0.3% phosphoric acid aqueous solution system was used as the mobile phase, and the elution procedure was as follows:

[0174]

[0175] The flow rate was 1 ml / min; the detection wavelength was 252 nm; and the column temperature was 35°C.

[0176] Preparation of reference solution: Take an appropriate amount of glycyrrhizic acid reference substance, weigh it accurately, and add 50% methanol to make a solution containing 100 μg per 1 ml.

[0177] Preparation of test solution: Take licorice root, crush it, take about 1g of powder, accurately weigh it, put it in a 50ml conical flask, accurately add 25ml of 50% methanol, ultrasonically extract for 45 minutes, filter, and take the filtrate to obtain the solution.

[0178] Determination method: Accurately pipette 10 μl of the test solution, inject into liquid chromatograph, and determine.

[0179] Preparation Example 1: Preparation of Chinese medicine composition

[0180] Weigh 240g of each of the eight medicinal materials of Astragalus, Angelica, Codonopsis, Scrophularia, Honeysuckle, Dendrobium, Achyranthes, and Licorice respectively; decoct twice with water, the first time for 1.5 hours and the second time for 1 hour, add 5 times and 3 times the amount of water respectively, filter, combine the decoction and concentrate to an extract without alcohol taste and with a relative density of 1.15-1.25 (45-55°C), add ethanol to make the alcohol content reach 60%, stir evenly, stand for 24 hours, recover ethanol and concentrate to a thick paste with a relative density of 1.3-1.35 (50-60°C), dry under reduced pressure, crush, and obtain an extract dry paste. Slowly add an appropriate amount of ethanol to the dry paste powder, mix, first add 2.3% starch, then add 1% silicon dioxide, 1% talc, 0.2% magnesium stearate, add 2.5% ethanol in total, granulate, mix, press into 500 tablets, and coat to obtain 0.35g / tablet.

[0181] A total of 15 batches of Chinese medicine compositions were prepared by selecting medicinal materials of different origins and batches that met the requirements of the pharmacopoeia. The content of the test sample in each composition was determined according to the aforementioned method.

[0182]

[0183] Experimental Example 1: Effect of 15 batches of Chinese herbal medicine compositions on rat cerebral ischemia-reperfusion injury (Middle Cerebral

[0184] Experimental study on artery occlusion (MCAO)

[0185] 1. Experimental Materials

[0186] 1.1 Drugs and reagents

[0187] A total of 15 batches of the Chinese medicine composition AO of the present invention were prepared by Jiangsu Kangyuan Pharmaceutical Co., Ltd.; Shutai 50, VIRBAC, France, specification: 250 mg / 5 mL, batch number: 889EA; Beitsu Mianxin (xylazine hydrochloride injection), Changsha Beit Biotechnology Research Institute, specification: 2 mL: 0.2 g, batch number: 20210102; sodium carboxymethyl cellulose (CMC-Na), Sinopharm Chemical Reagent Co., Ltd., specification: 500 g, batch number: 20220104; sodium chloride injection, Henan Kelun Pharmaceutical Co., Ltd., specification: 500mL: 4.5g, batch number: A21022302B; interleukin-1β (IL-1β) test kit, Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., item number: H002-1-2, specification: 96T; tumor necrosis factor-α (TNF-α) test kit, Nanjing Jiancheng Bioengineering Research Institute Co., Ltd., item number: H052-1-2, specification: 96T.

[0188] 1.2 Main instruments and consumables

[0189] Electronic balance (d = 0.1 g), Changzhou Lucky Electronic Equipment Co., Ltd., model: XY1000-1B; electronic balance (d = 0.0001 g), Mettler-Toledo Instruments (Shanghai) Co., Ltd., model: AL204; PE microplate reader, PerkinElmer, model: EnSpire; microplate reader, MD, model: M2e; low-temperature incubator, I-CVBE, model: FCI-280; cryo-grinder, Shanghai Jingxin Industrial Development Co., Ltd., model: TISSUELYSER-24L; refrigerated centrifuge, Beijing Baiyang Medical Instrument Co., Ltd., model: BY-R20; MCAO thread plug: Changsha Maiyue Biotechnology Co., Ltd., model: 250-280 g.

[0190] 1.3 Experimental animals

[0191] 102 male SD rats, weighing 170-190 g, were purchased from Nantong University, license number: SCXK (Su) 2019-0001. They were kept in a barrier environment of Jiangsu Kangyuan Pharmaceutical Co., Ltd.: room temperature 22±2℃, humidity 50-60%. Experimental animal use license number: SYXK (Su) 2018-0026. Rats were kept in separate cages, 2 rats / cage.

[0192] 2. Methods

[0193] 2.1 Animal grouping and drug administration

[0194] 102 rats were divided into 17 groups (sham operation control group: 6 rats; MCAO model group: 6 rats; drug administration group: Chinese medicine composition AO of the present invention, a total of 15 groups, 90 rats). The dosage of the Chinese medicine composition of the present invention was 31.74 g / kg of the raw drug. The rats in the drug administration group were gavaged with 10 mL / kg (diluted with 0.5% CMC-Na) 14 days before modeling, once a day, for 14 consecutive days. The MCAO model group and the sham operation control group were given 0.5% CMC-Na.

[0195] 2.2 Model preparation

[0196] After fasting for 12 hours and anesthesia, the rats were fixed on the operating table in the supine position. The midline incision of the neck was made to separate the common carotid artery. The external carotid artery and its branches were separated and ligated downwards. The vagus nerve was gently peeled off and the internal carotid artery was separated. The proximal end of the external carotid artery was prepared. The internal carotid artery and the common carotid artery were clamped with an artery clamp. A small cut was made in the external carotid artery 2 mm away from the internal carotid artery. The thread plug was inserted into the external carotid artery and entered the common carotid artery. The prepared thread was lightly tied to prevent bleeding. The external carotid artery was cut, the artery clamp of the internal carotid artery was loosened, and the thread plug was gently withdrawn to enter the internal carotid artery and continued to push downward until there was slight resistance. At this time, the internal carotid artery was stretched and the thread plug was inserted to a depth of about 18 mm, indicating that the thread plug had passed through the starting segment of the middle cerebral artery and reached the proximal end of the anterior cerebral artery, blocking the blood supply of the middle cerebral artery and the posterior cerebral artery. The artery clamp of the common carotid artery was loosened, the prepared thread was tightened, and a 1 cm long thread head was left outside. The skin was sutured and the rats were returned to the cage for feeding. After 2 hours, the suture head was pulled out to the bifurcation of the internal carotid artery and the external carotid artery to cause blood reperfusion. The sham operation group underwent the same surgical method as before, and the common carotid artery and the internal and external carotid arteries were isolated, but no ligation or suture insertion was performed. After blood flow was restored for 24 hours, biological samples were collected.

[0197] 3. Detection indicators

[0198] 3.1 Behavioral scoring of rats

[0199] According to the mNSS scoring method, the rats were scored for neurological deficits, including the tail lifting test, walking test, sensory test, balance beam test, reflexes and abnormal activities. The evaluation criteria were: 1-6 points were considered mild impairment; 7-12 points were considered moderate impairment; 13-18 points were considered severe impairment.

[0200]

[0201] 3.2 Determination of TNF-α and IL-1β Contents in Rat Brain Tissue

[0202] The brain tissue on the ischemic side was obtained and homogenized in pre-cooled physiological saline to prepare brain homogenate. After centrifugation, the supernatant was aspirated and the contents of inflammatory factors IL-1β and TNF-α in the sample were determined according to the Elisa method in the kit instructions.

[0203] 3.3 Statistical analysis

[0204] Data are presented as mean ± standard deviation GraphPad prism 7 statistical software was used to perform data statistical analysis using one-way ANOVA combined with Dunnett's multiple comparison test. P < 0.05 indicated that the data were statistically significant.

[0205] 4. Experimental results

[0206] 4.1 Effects on behavioral scores of MCAO model rats

[0207] The mNSS scores of rats in each group showed that the mNSS scores of rats in the MCAO model group were significantly higher than those in the sham operation group (P<0.001), while the mNSS scores of rats in groups B, C, E, F, I, J, K, L and N were significantly improved compared with the MCAO model group (P<0.05, P<0.01, P<0.001), as shown in Table 2.

[0208] Table 2 Comparison of mNSS scores of rats in each group ( n=6)

[0209]

[0210]

[0211] Note: Compared with the sham operation control group, ### P<0.001; compared with the MCAO model group, * P<0.05, ** P<0.01, *** P<0.001.

[0212] 4.2 Effects on TNF-α and IL-1β levels in brain tissue of MCAO model rats

[0213] Compared with the sham operation control group, the levels of TNF-α and IL-1β in the brain tissue homogenate of rats in the MCAO model group were increased (P<0.001), while those in groups B, C, E, F, I, J, K, L and N were significantly lower than those in the MCAO model group as a whole (P<0.05, P<0.01, P<0.001), as shown in Table 3.

[0214] Table 3 Comparison of TNF-α and IL-1β content in brain tissue of rats in each group ( n=6)

[0215]

[0216] Note: Compared with the sham operation control group, ### P<0.001; compared with the MCAO model group, * P<0.05, ** P<0.01, *** P<0.001. Experimental Example 2: Experimental study on the effect of 15 batches of Chinese herbal medicine composition on ISO-induced myocardial ischemia in rats

[0217] 1. Experimental Materials

[0218] 1.1 Drugs and reagents

[0219] Shutai 50, VIRBAC, France, specification: 250mg / 5mL, batch number: 889EA; Beitsu Mianxin (xylazine hydrochloride injection), Changsha Beit Biotechnology Research Institute Co., Ltd., specification: 2mL: 0.2g, batch number: 20210102; Isoprenaline hydrochloride (Isoprenaline HCl), Shanghai Aladdin Biochemical Technology Co., Ltd., specification: 25g, Lot#H2124300; sodium chloride injection, Henan Kelun Pharmaceutical Co., Ltd., specification: 500mL: 4.5g, batch number: A21022302B; sodium carboxymethyl cellulose (CMC-Na), Sinopharm Chemical Reagent Co., Ltd., specification: 500g, batch number: 20220104; AST kit, Nanjing Jiancheng Bioengineering Institute, item number: C010-2-1 batch number: 20220509, specification 96T; LDH lactate dehydrogenase kit, Nanjing Jiancheng Bioengineering Institute, item number: A020-2-2 batch number: 20220621 specification: 96T.

[0220] 1.2 Main instruments and consumables

[0221] Electronic balance (d=0.1g), Changzhou Lucky Electronic Equipment Co., Ltd., model: XY1000-1B; electronic balance (d=0.0001g), Mettler-Toledo Instruments (Shanghai) Co., Ltd., model: AL204; PE microplate reader, Perkin Elmer, model: EnSpire; low-temperature incubator, I-CVBE, model: FCI-280; refrigerated centrifuge, Beijing Baiyang Medical Instrument Co., Ltd., model: BY-R20; disposable intravenous infusion needle, Shandong Aosate Medical Instrument Co., Ltd., batch number: 20200623; disposable vacuum blood collection tube, Shandong Chengwu County Huabo Medical Instrument Co., Ltd., batch number: 20201103.

[0222] 1.3 Experimental animals

[0223] 102 Wistar rats, male, weighing 180-220 g, were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd., license number: SCXK (Beijing) 2019-0010. They were kept in a barrier environment of Jiangsu Kangyuan Pharmaceutical Co., Ltd.: room temperature 22±2℃, humidity 50-60%. Experimental animal use license number: SYXK (Su) 2018-0026. Rats were kept in separate cages, 2 rats / cage.

[0224] 2. Methods

[0225] 2.1 Animal grouping

[0226] 102 rats were divided into 17 groups (blank control group: 6 rats; ISO model group: 6 rats; drug administration group: the Chinese medicine composition AO of the present invention, a total of 15 groups, 90 rats).

[0227] 2.2 Medication and modeling

[0228] The dosage of the Chinese medicine composition of the present invention is 31.74 g / kg of the raw drug. The rats in the drug administration group were gavaged with 10 mL / kg (diluted with 0.5% CMC-Na) 14 days before modeling, once a day, for 14 consecutive days. The ISO model group and the blank control group were given 0.5% CMC-Na. One hour after gavage on the 13th and 14th days, isoproterenol (150 mg / kg) was subcutaneously injected in each group except the normal control group, and the subcutaneous injection volume was 2 mL / kg.

[0229] 3. Detection indicators

[0230] 48 hours after the first subcutaneous injection of isoproterenol in rats, after anesthesia, fix the rats in the supine position, cut the abdominal skin, separate the abdominal aorta, collect blood, and place in a 4°C refrigerator overnight. Centrifuge at 3000rpm for 15 minutes on the second day. After obtaining the serum, the serum was divided and stored in a -80°C refrigerator for subsequent kit testing. After blood collection, the chest cavity was opened, the heart was quickly removed, washed with saline, and the residual liquid on the surface of the heart was absorbed with absorbent paper and weighed.

[0231] 3.1 Cardiac Index

[0232] The cardiac index was calculated according to the following formula and the differences among the groups were compared.

[0233]

[0234] 3.2 Detection of AST and LDH in serum

[0235] The method refers to the kit instructions.

[0236] 3.3 Statistical analysis

[0237] Data are presented as mean ± standard deviation GraphPad prism 7 statistical software was used to perform data statistical analysis using one-way ANOVA combined with Dunnett's multiple comparison test. P < 0.05 indicated that the data were statistically significant.

[0238] 4. Experimental results

[0239] 4.1 Effects on rat cardiac index

[0240] The results of the cardiac index of rats in each group showed that the cardiac index of rats in the model control group was significantly higher than that in the blank control group (P<0.001), while the cardiac index of rats in groups B, C, E, F, I, J, K, L and N was significantly improved compared with the model control group (P<0.05, P<0.01, P<0.001), as shown in Table 4.

[0241] Table 4 Comparison of cardiac index of rats in each group ( n=6)

[0242]

[0243] Note: Compared with the blank control group, ### P<0.001; compared with the model control group, * P<0.05, ** P<0.01, *** P<0.001.

[0244] 4.2 Effects on AST and LDH in rat serum

[0245] Compared with the blank control group, the serum AST and LDH levels of rats in the model control group were increased (P<0.001), while those in groups B, C, E, F, I, J, K, L and N were significantly decreased compared with the model control group (P<0.05, P<0.01, P<0.001), as shown in Table 5.

[0246] Table 5 Comparison of AST and LDH in serum of rats in each group ( n=6)

[0247]

[0248]

[0249] Note: Compared with the blank control group, ### P<0.001; compared with the model control group, * P<0.05, ** P<0.01, *** P<0.001.

[0250] The preferred contents of the above active ingredients are: neochlorogenic acid 0.81-2.01‰, chlorogenic acid 1.89-3.01‰, cryptochlorogenic acid 1.05-1.33‰, isochlorogenic acid A 0.50-0.79‰, isochlorogenic acid B 0.63-0.88‰, isochlorogenic acid C 0.84-1.25‰, strychnoside 1.18-1.45‰, glycyrrhizic acid 3.68-5.74‰, liquiritin 1.35-2.2‰, harpaoside 0.25-0.37‰, β-ecdysterone 0.17-0.29‰, astragaloside IV 0.81-1.27‰, ginsenoside RO 0.14-0.28‰, ligusticum bisporum lactone A 0.10-0.19‰, and codonopsis pilosula glycoside 0.10-0.18‰. The proportions of the above active ingredients are: neochlorogenic acid 0.81-2.01, chlorogenic acid 1.89-3.01, cryptochlorogenic acid 1.05-1.33, isochlorogenic acid A 0.50-0.79, isochlorogenic acid B 0.63-0.88, isochlorogenic acid C 0.84-1.25, strychnoside 1.18-1.45, glycyrrhizic acid 3.68-5.74, liquiritin 1.35-2.2, harpaoside 0.25-0.37, β-ecdysterone 0.17-0.29, astragaloside IV 0.81-1.27, ginsenoside RO 0.14-0.28, ligusticum chuanxiong lactone A 0.10-0.19, and codonopsis pilosula 0.10-0.18. According to animal experiments, the Chinese medicine composition with the above-mentioned active ingredient content characteristics has a stable and excellent therapeutic effect and is a preferred Chinese medicine composition; the above-mentioned active ingredient content characteristics can also be used for quality control of the Chinese medicine composition.

[0251] Fingerprint detection of the composition of the present invention

[0252] 1. Fingerprint chromatographic conditions Waters Xbridge BEH C 18 Chromatographic column (4.6×250 mm, 5 μm); mobile phase 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution, (0-20 min, 1-12% B; 20-60 min, 12%-35% B; 60-70 min, 47%-50% B; flow rate 1.0 mL·min -1 ; Column temperature 35℃; Wavelength 270nm; Injection volume 10μL.

[0253] 2. Preparation of test solution Take about 1g of the Chinese medicine composition after internal control, accurately weigh it, put it in a conical flask, accurately add 25mL of 50% methanol, weigh it, ultrasonically treat it for 30min, let it cool, make up the weight loss with 50% methanol solution, and spin it at 14000r·min -1 Centrifuge for 10 minutes and take the supernatant.

[0254] 3. Preparation of reference solution: Accurately weigh 10 mg of neochlorogenic acid, 25 mg of chlorogenic acid, 10 mg of cryptochlorogenic acid, 10 mg of liquiritin, 10 mg of isochlorogenic acid A, 100 mg of isochlorogenic acid B, 10 mg of isochlorogenic acid C, 4 mg of harpagoside and 25 mg of glycyrrhizic acid into a 50 mL volumetric flask, add 50% methanol and ultrasonically dissolve, let cool, dilute to the mark, shake well, and obtain the reference solution.

[0255] 4. Establishment of HPLC fingerprint

[0256] 4.1 Precision test Take an appropriate amount and prepare the test solution according to the method under "2". Continuously inject and measure 6 times according to the chromatographic conditions under "1". Take Peak 7 (liquiritigenin) as the reference peak. The relative retention time RSD of the remaining 16 common peaks is 0.01% to 0.78%, and the relative peak area RSD is 0.59% to 1.86%.

[0257] 4.2 Repeatability test Take an appropriate amount of batch Q of Chinese medicine composition, prepare 6 test solutions in parallel according to the test solution preparation method, and measure according to the chromatographic conditions under "2.1.1". Take peak 9 (liquiritigenin) as the reference peak, and measure the relative retention time RSD of the remaining 16 common peaks in the range of 0.01% to 1.59%, and the relative peak area RSD is in the range of 0.67% to 2.91%.

[0258] 4.3 Stability test Take the same sample solution and inject it at 0, 2, 4, 6, 8, 12, and 24 hours according to the chromatographic conditions. Taking peak 9 (liquiritigenin) as the reference peak, the relative retention time RSD of the remaining 16 common peaks is 0.06% to 0.34%, and the relative peak area RSD is 0.35% to 2.68%. This shows that the sample solution has good stability within 24 hours.

[0259] 4.4 Establishment of fingerprints and similarity analysis Take 25 batches of Chinese medicine extracts, prepare test solutions, and measure them. Use "Chinese medicine chromatographic fingerprint similarity evaluation system software (2004A version)" for analysis. Through multi-point correction method and full spectrum peak matching, 25 batches of fingerprints are obtained. The chromatograms are shown in Figure 1. A total of 17 common peaks were marked, among which Peak 9 (liquiritigenin) had good stability, high chromatographic response value and moderate retention time, so it was selected as the reference peak (S). Accordingly, the relative retention time between each characteristic peak and the S peak was calculated, which should be within ±5% of the specified value. The specified values ​​of relative retention time are: 0.26 (peak 1), 0.33 (peak 2), 0.36 (peak 3), 0.48 (peak 4), 0.62 (peak 5), 0.67 (peak 6), 0.96 (peak 7), 0.98 (peak 8), 1 (peak 9, 1.11 (peak 10), 1.14 (peak 11), 1.21 (peak 12), 1.26 (peak 13), 1.35 (peak 14), 1.38 (peak 15), 1.50 (peak 16), 1.95 (peak 17)

[0260] The similarity between the fingerprints of 25 batches of Chinese herbal medicine extracts ranged from 0.854 to 0.996. Figure 1 The results of Table 1 and Table 2 indicate that the Chinese herbal medicine extracts obtained by the internal control method have good similarity between batches, small batch differences, and relatively stable quality, and thus also have better stability and excellent therapeutic effects. After comparing the PDA spectrum and retention time with the reference substance, 9 chromatographic peaks were identified, namely, peak 4 for neochlorogenic acid, peak 5 for chlorogenic acid, peak 6 for cryptochlorogenic acid, peak 9 for liquiritin, peak 10 for isochlorogenic acid B, peak 11 for isochlorogenic acid A, peak 12 for isochlorogenic acid C, peak 16 for harpagoside, and peak 17 for glycyrrhizic acid.

[0261] Table 6 Similarity values ​​of 25 batches of Chinese herbal medicine extracts

[0262]

[0263] The above is only an exemplary description of the implementation method of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Chinese medicine composition, characterized in that: The invention comprises active ingredients extracted from astragalus, angelica, codonopsis, scrophularia, dendrobium, achyranthes, honeysuckle and licorice, and the active ingredients include, by mass percentage, more than 0.8‰ neochlorogenic acid, more than 1.8‰ chlorogenic acid, more than 1.0‰ cryptochlorogenic acid, more than 0.50‰ isochlorogenic acid A, more than 0.60‰ isochlorogenic acid B, more than 0.80‰ isochlorogenic acid C, more than 1.1‰ schizonepeta glycoside, more than 3.5‰ glycyrrhizic acid, more than 1.3‰ liquiritin, more than 0.25‰ harpagoside, more than 0.15‰ β-ecdysterone, more than 0.8‰ astragaloside, more than 0.14‰ ginsenoside RO, more than 0.1‰ ligusticum lactone A and more than 0.1‰ codonopsis ginsenoside.

2. The Chinese medicine composition according to claim 1, characterized in that: The active ingredients include: neochlorogenic acid 0.81-2.01‰, chlorogenic acid 1.89-3.01‰, cryptochlorogenic acid 1.05-1.33‰, isochlorogenic acid A 0.50-0.79‰, isochlorogenic acid B 0.63-0.88‰, isochlorogenic acid C 0.84-1.25‰, strychnoside 1.18-1.45‰, glycyrrhizic acid 3.68-5.74‰, liquiritin 1.35-2.2‰, harpaoside 0.25-0.37‰, β-ecdysterone 0.17-0.29‰, astragaloside IV 0.81-1.27‰, ginsenoside RO 0.14-0.28‰, ligusticum chuanxiong lactone A 0.10-0.19‰, and codonopsis pilosula glycoside 0.10-0.18‰.

3. A Chinese medicine composition, characterized in that: The invention comprises active ingredients extracted from astragalus, angelica, codonopsis, scrophularia, dendrobium, achyranthes, honeysuckle and licorice, wherein the proportion of the active ingredients is as follows: neochlorogenic acid 0.81-2.01, chlorogenic acid 1.89-3.01, cryptochlorogenic acid 1.05-1.33, isochlorogenic acid A 0.50-0.79, isochlorogenic acid B 0.63-0.88, isochlorogenic acid C 0.84-1.25, strychnine glycoside 1.18-1.45, glycyrrhizic acid 3.68-5.74, liquiritin 1.35-2.2, harpaoside 0.25-0.37, β-ecdysterone 0.17-0.29, astragaloside I 0.81-1.27, ginsenoside RO 0.14-0.28, ligusticum chuanxiong lactone A 0.10-0.19, codonopsis pilosula glycoside 0.10-0.

18.

4. The Chinese medicine composition according to claims 1-3, characterized in that: The traditional Chinese medicine composition is in an optional preparation form.

5. The Chinese medicine composition according to claim 4, characterized in that: The preparation forms include tablets, capsules, pills, powders, granules and drop pills.

6. A method for preparing the Chinese medicine composition according to any one of claims 1 to 3, characterized in that: The following steps are involved: Weigh raw materials of Astragalus, Angelica, Codonopsis, Scrophularia, Honeysuckle, Dendrobium, Achyranthes and Licorice according to the weight ratio of 1:1:1:1:1:1:1:1; add water and boil twice, the first time for 1.5 hours and the second time for 1 hour, adding 5 times and 3 times the amount of water respectively, filter, combine the decoctions and concentrate until there is no alcohol taste, add ethanol to make the alcohol content reach 60%, stir well, let stand, recover and concentrate the ethanol, dry under reduced pressure to obtain a dry paste.

7. The preparation method according to claim 6, characterized in that: The oral preparation is a tablet, and the preparation method is as follows: adding 2.3% starch, 1% silicon dioxide, 1% talcum powder, 0.2% magnesium stearate and 2.5% ethanol to the dry paste powder, granulating, mixing, tableting, and coating with a gastric soluble film premix w / w 3% to obtain the tablet.

8. Use of the Chinese medicine composition according to any one of claims 1 to 3 in the preparation of a medicament for improving ischemic stroke, ischemic cardiomyopathy, and acute myocardial infarction.

9. A method for detecting the fingerprint of the Chinese medicine composition according to claim 1, characterized in that: The method includes: Chromatographic conditions Waters Xbridge BEH C 18 Chromatographic column (4.6×250 mm, 5 μm); mobile phase: 0.1% phosphoric acid aqueous solution (A)-acetonitrile (B), gradient elution, (0-20 min, 1-12% B; 20-60 min, 12%-35% B; 60-70 min, 47%-50% B; flow rate: 1.0 mL·min -1 ; Column temperature 35℃; Wavelength 270nm.

10. The method according to claim 9, characterized in that In the fingerprint spectrum, there are 17 characteristic peaks in the characteristic spectrum of the test sample, and the peak corresponding to the reference peak is the S peak. The relative retention time of each characteristic peak and the S peak should be within ±10% of the specified value. The specified value of the relative retention time is: 0.26 (peak 1), 0.33 (peak 2), 0.36 (peak 3), 0.48 (peak 4), 0.62 (peak 5), 0.67 (peak 6), 0.96 (peak 7), 0.98 (peak 8), 1 (peak 9), 1.11 (peak 10), 1.14 (peak 11), 1.21 (peak 12), 1.26 (peak 13), 1.35 (peak 14), 1.38 (peak 15), 1.50 (peak 16), 1.95 (peak 17).

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