Traditional Chinese medicine preparation optimization method based on hemorheology mechanism and preparation

Abstract

The invention belongs to the technical field of compound traditional Chinese medicine preparation optimization algorithms and preparation optimization, and relates to a traditional Chinese medicine preparation optimization method based on a hemorheology mechanism and a preparation. Particularly relates to an optimization method of an antithrombotic yang-tonifying and recuperating decoction preparation and the preparation. According to the preparation optimization method, an intestinal flora regulator substance group and an anti-thrombus component substance group of medicinal material chemical components of the Yang tonifying and also five decoction and corresponding substance clusters of products obtained by different extraction methods are constructed, a better Yang tonifying and also five decoction extraction method is determined on the basis of intersection comparison of different substance groups, and then optimization is performed. The method can effectively guide the optimization of the Yang-tonifying and Yang-restoring decoction preparation. The Yang-tonifying Huanwu decoction preparation is prepared from a water decoction and alcohol precipitation extracting solution of raw radix astragali, radix paeoniae rubra and lumbricus and a 70% ethanol reflux extracting solution of the other four medicinal materials. The extracted components have the most intersection substances with the intestinal flora regulator substance group and the antithrombotic component substance group and the most intersection substances with high content, and have higher anticoagulant and antithrombotic effects.

Description

Technical Field

[0001] The invention belongs to the technical field of compound Chinese medicine preparation optimization algorithm and preparation optimization, and specifically relates to a Chinese medicine preparation optimization method based on blood rheology mechanism and a preparation. Background Art

[0002] The original prescription of Buyang Huanwu Decoction comes from Wang Qingren's "Yi Lin Gaicuo". It is a representative prescription that embodies Wang Qingren's theory of qi deficiency and blood stasis. It is mainly used to treat hemiplegia, facial paralysis, slurred speech, drooling, lower limb paralysis, frequent urination, and incontinence. Modern pharmacological studies have shown that this prescription also has pharmacological effects such as anti-cerebral ischemic injury and anti-thrombotic effects. So far, the original water decoction process has been used clinically for preparation. However, due to the different required active ingredients for different indications, the use of one prescription for multiple purposes leads to the lack of specificity of the preparation for different indications.

[0003] In terms of pharmacological analysis and mechanism analysis of traditional Chinese medicine, there are currently two main technical means, one of which is called network pharmacology. That is, for all known components of the medicinal materials used in the prescription, thresholds are set according to their oral bioavailability, drug-like properties, etc., and components that may not be active are eliminated based on experience (for example, substances with OB ≥ 30% and DL ≥ 0.18 are usually selected as candidate substances), and then the required components are subjected to target analysis, and then the targets related to the pathological mechanisms of known diseases are analyzed, and the interaction diagram of active ingredients-drug targets-disease targets is constructed, so as to analyze the possible multi-target mechanism of action of compound drugs. Such methods and literature can be found in "Network Pharmacology of Rhubarb in Anti-renal Fibrosis and the Mechanism of Action of Its Active Ingredients" (Chinese Journal of Experimental Traditional Chinese Medicine, Vol. 26, No. 10, pp. 163-172, 2020), "Exploration of the Mechanism of Action of Buyang Huanwu Decoction in Preventing and Treating Thrombosis Based on Network Pharmacology" (China Medical Herald, Vol. 17, No. 29, pp. 17-28, October 2020), CN103150490A, CN110648726A, etc. This method is helpful to a great extent in explaining the multi-component and multi-target mechanism of action of traditional Chinese medicine and compound prescriptions. However, because it is a post hoc explanatory method and tool, and focuses on the docking of target-component, it ignores the overall treatment concept of traditional Chinese medicine, such as the use of a combined method of invigorating qi and removing blood stasis when treating blood stasis. This cannot be explained and illustrated by target-docking. Moreover, the elimination of ingredients that may not be active based on experience also leads to the neglect of the mutual influence of dissolution behavior between different components of traditional Chinese medicine. In real applications, it is often seen that OB and DL are far less than the threshold, but the components still enter the blood after oral administration, which makes its explanatory ability difficult to meet expectations.

[0004] In addition, another means of pharmacological analysis and mechanism analysis of traditional Chinese medicine is based on tools of traditional Chinese medicine serum pharmacology. That is, after taking traditional Chinese medicine or compound medicine orally, the components that enter the blood are detected, and it is believed that only the components that enter the blood are the effective substances. So far, traditional Chinese medicine serum pharmacology also has the problem of being unable to reasonably explain traditional Chinese medicine and its mechanism, and the preparations prepared by different methods have very different components entering the blood. This is also due to ignoring the overall treatment concept of traditional Chinese medicine and deviating from the treatment methods and principles. For example, there are great differences in the blood and brain components of Buyang Huanwu Decoction obtained in "HPLC–MS / MS Analysis of a Traditional Chinese Medical Formulation of Bu-Yang-Huan-Wu-Tang and Its Pharmacokinetics after Oral Administration to Rats" (PLOS ONE, Vol. 7, No. 8, 2012, e43848), "UPLC-Q-TOF-MS / MS Analysis of Components of Buyang Huanwu Decoction Absorbed into the Brain and Blood" (Chinese Journal of Experimental Traditional Chinese Medicine, Vol. 25, No. 2, pp. 8-13, 2019), and "Serum Pharmacochemical Study on the Material Basis of the Efficacy of Buyang Huanwu Decoction against Cerebral Ischemia" (New Chinese Medicine and Clinical Pharmacology, Vol. 34, No. 9, September 2023, pp. 1227-1235).

[0005] The inherent defects of the above two theories mean that although they can be used to explain the mechanism of action of traditional Chinese medicine and compound prescriptions to a certain extent, they are difficult to be used to guide the modernization and improvement of traditional Chinese medicine preparations.

[0006] Therefore, there is an urgent need for a method that can be used to guide the optimization of traditional Chinese medicine preparations, so as to achieve the optimization of targeted preparations for different indications such as Chinese medicine compound prescriptions. Summary of the invention

[0007] In view of the above problems, the present invention provides a method and preparation for optimizing a traditional Chinese medicine preparation based on the blood rheology mechanism.

[0008] In clinical TCM, it is found that changes in gastrointestinal function often precede the symptoms of other organ diseases, and the recovery of gastrointestinal function is often slower than the speed of recovery of other organ diseases. This is particularly prominent in cardiovascular and renal diseases. This coincides with the view of the spleen and stomach school of traditional Chinese medicine. The development of modern medicine also shows that the human body has a series of disease regulation mechanisms based on the digestive system, such as the "gut-brain axis", "lung-brain axis", "gut-heart axis", and "gut-vascular barrier axis", and it has been found that intestinal flora is also related to the occurrence and development of chronic diseases such as thrombosis (Qian Ying et al. Research progress on the relationship between intestinal flora and chronic diseases. Chinese Journal of Microecology, Vol. 34, No. 10, pp. 1217-1221, 2022).

[0009] Therefore, in view of the defects of traditional network pharmacology (the mutual interference of complex components of traditional Chinese medicine in oral bioavailability and metabolism and the influence of intestinal flora on the metabolism of active substances of traditional Chinese medicine, etc., cannot be included in the traditional network pharmacology system) and traditional Chinese medicine serum drug chemistry theory (entering the blood is not the only mechanism for drugs or chemical substances to exert their regulatory effects on the body, and traditional Chinese medicine serum drug chemistry theory cannot include non-entering blood components in the study) and the problem that the two theories cannot guide the modernization of Buyang Huanwu Decoction and other Chinese medicine preparations; combined with the holistic concept of traditional Chinese medicine and the theory of "intestinal-vascular barrier axis", the present invention first provides a method for optimizing Buyang Huanwu Decoction preparations based on the complex mechanism of blood rheology, and specifically, the present invention first provides a method for optimizing anti-thrombotic Buyang Huanwu Decoction preparations. To overcome the shortcomings of the original methods and theories that cannot guide the optimization of pharmaceutics.

[0010] The method comprises the following steps: S1 Construction of intestinal flora regulator substance group based on the chemical components of the medicinal materials of Buyang Huanwu Decoction: Based on the chemical components contained in the medicinal materials used in Buyang Huanwu Decoction, literature search analysis and / or experimental analysis are carried out to screen the chemical components that have the function of regulating intestinal flora and promoting the proliferation of probiotics, and establish an intestinal flora regulator substance group; The chemical components of the medicinal materials in this step can be obtained based on the HPLC-MS analysis results of the medicinal materials, or the chemical components of the medicinal materials included in the TCMSP database, TCMID database, etc. can be integrated for chemical component integration.

[0011] S2 Construction of network pharmacology anti-thrombotic component substance group based on the chemical components of the medicinal materials of Buyang Huanwu Decoction: According to the chemical components contained in the medicinal materials used in Buyang Huanwu Decoction, the screening thresholds of OB value and DL value are not limited. The network pharmacology method is used to perform routine component screening, target prediction, construction of compound-target-disease network and protein interaction network, and the screened anti-thrombotic components are used as the anti-thrombotic component substance group.

[0012] The chemical components of the medicinal materials in this step can be integrated with the chemical components of the medicinal materials included in the TCMSP database, TCMID database, etc. For details, please refer to "Exploring the Mechanism of Buyang Huanwu Decoction in Preventing and Treating Thrombosis Based on Network Pharmacology" (China Medical Herald, October 2020, Vol. 17, No. 29, pp. 17-28). The difference is that step S2 of the present invention does not limit the screening thresholds of OB values ​​and DL values.

[0013] S3 Construction of substance clusters of Buyang Huanwu Decoction with different extraction methods: Different extraction methods were used to extract the medicinal materials of Buyang Huanwu Decoction and analyze the material structure and content, so as to obtain the substance clusters corresponding to Buyang Huanwu Decoction with different extraction methods; and the substance clusters corresponding to Buyang Huanwu Decoction with different extraction methods were sorted according to content indicators (such as peak area, percentage content).

[0014] Different extraction methods for this step can be selected from water decoction, ethanol reflux extraction, water extraction alcohol precipitation, alcohol extraction water precipitation, steam distillation, etc. In view of the fact that water decoction contains impurities such as protein, which is not conducive to chromatographic analysis, ethanol reflux extraction, water extraction alcohol precipitation, alcohol extraction water precipitation, steam distillation, etc. are preferred. In the extraction methods here, except for the steam distillation extract, the substances obtained are all soluble components in the solvent, rather than precipitates and drug residues.

[0015] Among them, the ethanol precipitation of the water extraction and alcohol precipitation method can be set to 50%, 60%, and 75% (all volume percentages) for method comparison; the ethanol extraction stage of the ethanol reflux extraction method and the alcohol extraction and water precipitation method can be set to 40%, 60%, 70%, 80%, 90% and other concentrations of ethanol extraction methods for method comparison. The material structure and content analysis of the material cluster can be carried out using methods such as HPLC-MS and GC-MS.

[0016] The different extraction methods include extracting the medicinal materials in the prescription together using the same method (which can be called combined decoction), extracting different medicinal materials using the same method or different methods separately and then combining the extracts (which can be called single decoction), extracting some medicinal materials using the same method and other medicinal materials using another method and then combining the extracts (which can be called separate decoctions), and other different extraction modes.

[0017] In this way, the substance groups of Buyang Huanwu Decoction with different extraction methods were constructed.

[0018] S4 Compare the substance clusters corresponding to the Buyang Huanwu Decoction obtained by different extraction methods in step S3 with the intestinal flora regulator substance group and the anti-thrombotic component substance group obtained in steps S1 and S2, and take the intersection of the three; optimize the extraction method of the anti-thrombotic Buyang Huanwu Decoction preparation according to the number of intersection substances and the content ranking of the intersection substances between the substance clusters corresponding to the Buyang Huanwu Decoction obtained by different extraction methods and the intestinal flora regulator substance group and the anti-thrombotic component substance group obtained by S1 and S2; select the extraction method with the most intersection substances with the substance ranked first in content and the intestinal flora regulator substance group and the anti-thrombotic component substance group obtained by S1 and S2 as the main extraction method, and optimize the extraction method conditions.

[0019] The main extraction method refers to a combination of two extraction methods, but the extraction method with the most intersection substances of the substance ranked first in content and the intestinal flora regulator substance group and the anti-thrombotic component substance group obtained by S1 and S2 is used as the main one.

[0020] The extraction method condition optimization includes further optimization of the screened method, such as optimization of parameters such as pulverization degree, material-liquid ratio, extraction time, extraction temperature, and extraction times.

[0021] Preferably, in step S4, the extraction method with the most intersection substances between the substances ranked in the top two-thirds in content and the intestinal flora regulator substance group and the anti-thrombotic component substance group obtained in S1 and S2 is selected as the main extraction method to form the basis for optimizing the extraction method of Buyang Huanwu Decoction, and to combine the extraction methods and optimize the conditions.

[0022] Preferably, in step S4, the extraction method with the most intersection substances between the substances ranked in the top one-third in content and the intestinal flora regulator substance group and the anti-thrombotic component substance group obtained in S1 and S2 is selected as the main extraction method to form the basis for optimizing the extraction method of Buyang Huanwu Decoction, and to combine the extraction methods and optimize the conditions.

[0023] For example, when the substance cluster corresponding to the Buyang Huanwu Decoction obtained by extraction method A has intersections with the intestinal flora regulator substance cluster and the anti-thrombotic component substance cluster obtained in steps S1 and S2, and the intersections are large, and the intersection substances are substances with higher content (for example, the substance content ranks within the top 50), method A is used as the main extraction method for the Buyang Huanwu Decoction preparation, and parameters such as the solid-liquid ratio, extraction time, and extraction temperature are further optimized.

[0024] When there is an intersection between the intestinal flora regulator substance group obtained by extraction method A and S1, and the intersection is large, and the intersection substance is a substance with a higher content (for example, the substance content ranks within the top 50), but there is a small intersection with the anti-thrombotic component substance group obtained by S2, or the intersection substance is a substance with a lower content (the content ranks outside the top 50), then a combination of multiple extraction methods is used as the extraction method of the Buyang Huanwu Decoction preparation, and other extraction parameters are optimized, such as the solid-liquid ratio, extraction time and other parameters.

[0025] For example, when the substance cluster corresponding to the Buyang Huanwu Decoction extracted by water decoction has an intersection with the intestinal flora regulator substance group obtained from S1, and the intersection is large, the intersection substance is a substance with a higher content, while there is less intersection with the anti-thrombotic component substance group obtained from S2. Overall, the number of intersection substances with the intestinal flora regulator substance group obtained from S1 and the anti-thrombotic component substance group obtained from S2 is the largest, but there is still a deficiency of less intersection with the anti-thrombotic component substance group obtained from S2. The substance cluster corresponding to the Buyang Huanwu Decoction extracted by ethanol reflux has a large intersection with the anti-thrombotic component substance group obtained from S2, and the intersection substance is a substance with a higher content. The medicinal materials can be first subjected to water decoction extraction to separate the water extract, and then the medicinal residues can be subjected to ethanol reflux extraction.

[0026] The aforementioned steps S1, S2, and S3 are only codes and are not considered as the order of the steps; the steps S1, S2, and S3 that are executed independently can be executed independently. However, the steps S1, S2, and S3 should obviously be executed before the step S4.

[0027] Based on this method, the present invention provides an anti-thrombotic Buyang Huanwu Decoction preparation, which is made from the following medicinal materials: raw astragalus, angelica root, red peony root, earthworm, chuanxiong, safflower and peach kernel; the Buyang Huanwu Decoction preparation is made from a water-decocted alcohol precipitation extract of raw astragalus, red peony root and earthworm and a 70% ethanol reflux extract of angelica root, chuanxiong, safflower and peach kernel.

[0028] Preferably, the weight ratio of raw astragalus, angelica root, red peony root, earthworm, ligusticum chuanxiong, peach kernel and safflower is 40:2:1.5:1:1:1:1.

[0029] Preferably, the medicinal material is crushed to 20-100 mesh before extraction. Further preferably, the medicinal material is crushed to 60-80 mesh before extraction.

[0030] Preferably, when preparing the water decoction alcohol precipitation extract, the solid-liquid ratio is 1: (8-12), that is, 8-12 mL of water is added to 1 g of medicinal material; the number of decoctions is 2-4 times, and each decoction is 1-4 hours; when preparing the water decoction alcohol precipitation extract, ethanol is added in the alcohol precipitation step until the ethanol volume fraction reaches 70%-85%.

[0031] Further preferably, when preparing the water decoction alcohol precipitation extract, the solid-liquid ratio is 1: (10~12), that is, 1g of medicinal material is added with 10~12mL of water; the decoction times are 2 times, and each decoction is 2h; when preparing the water decoction alcohol precipitation extract, ethanol is added in the alcohol precipitation step until the ethanol volume fraction reaches 75%.

[0032] Preferably, when the ethanol reflux extract is prepared, the solid-liquid ratio is 1:(12-30), that is, 1g of medicinal material is added with 12-30mL of 70% ethanol; the extraction temperature is 60°C-80°C, the number of extractions is 1-3 times, and each extraction is 1-3h.

[0033] Further preferably, when the ethanol reflux extract is prepared, the solid-liquid ratio is 1:(15-30), that is, 1 g of medicinal material is added with 15-30 mL of 70% ethanol; the extraction temperature is 60°C, the number of extractions is 2, and each extraction is 1.5 h.

[0034] The water decoction alcohol precipitation extract and the 70% ethanol reflux extract can be concentrated under reduced pressure to recover the solvent or freeze-dried to form a solid or semi-solid material for preparing the Buyang Huanwu Decoction preparation.

[0035] Therefore, the Buyang Huanwu Decoction preparation can be an oral liquid preparation or an oral solid preparation.

[0036] The Buyang Huanwu Decoction preparation may also be added with pharmaceutically acceptable preparation excipients, such as excipients, coating agents, fillers, etc., for example, one or more of starch, dextrin, magnesium stearate, and sodium carboxymethyl cellulose.

[0037] Beneficial effects of the present invention: (1) The present invention provides a method for optimizing a Buyang Huanwu Decoction preparation for anti-thrombosis (preventing or treating thrombotic diseases by anticoagulation or improving blood rheological characteristics). The method overcomes the defects of traditional network pharmacology or serum drug chemistry methods that are difficult to guide the optimization of Chinese medicine preparations, and the problem that they are unable to adapt to the overall treatment methods and principles of traditional Chinese medicine. The method can effectively reflect the overall treatment methods and principles of traditional Chinese medicine, and can be used to guide the optimization of the extraction method of the Buyang Huanwu Decoction preparation.

[0038] (2) The present invention provides a Buyang Huanwu Decoction preparation for antithrombotic purposes (preventing or treating thrombotic diseases by anticoagulation or improving blood rheological characteristics). BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The figure is a logical schematic diagram of the method for optimizing the anti-thrombotic Buyang Huanwu Decoction preparation of the present invention. DETAILED DESCRIPTION

[0040] The present application is further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and cannot be used to limit the protection scope of the present application.

[0041] Example 1 Optimization method of anti-thrombotic Buyang Huanwu Decoction preparation Please combine Figure 1 The basic principle of the preparation optimization method of Buyang Huanwu Decoction in this embodiment is to optimize the preparation extraction method based on the intersection of substances of the three major substance groups and the number and content ranking of the intersection substances as a reference.

[0042] The three major substance groups include the intestinal flora regulator substance group selected from the chemical components of the medicinal materials of Buyang Huanwu Decoction, the antithrombotic component substance group selected from the chemical components of the medicinal materials of Buyang Huanwu Decoction, and the corresponding substance clusters of Buyang Huanwu Decoction formed by different extraction methods. The substance intersection of the three major substance groups includes the substance intersection 1 of the Buyang Huanwu Decoction corresponding substance cluster formed by a specific extraction method and the intestinal flora regulator substance group, and the substance intersection 2 of the Buyang Huanwu Decoction corresponding substance cluster formed by a specific extraction method and the antithrombotic component substance group.

[0043] Among them, the construction of the intestinal flora regulator substance group can be completed by searching the literature and analyzing the activity of the chemical components in the medicinal materials of Buyang Huanwu Decoction, so as to screen out the chemical components that have the function of regulating intestinal flora and promoting the proliferation of probiotics, thereby completing the construction of the intestinal flora regulator substance group.

[0044] The chemical components of the medicinal materials in Buyang Huanwu Decoction can be obtained from the chemical component information of the medicinal materials included in the TCMSP database (TCMSP Traditional Chinese Medicine Systems Pharmacology Platform), TCMID database (Traditional Chinese Medicine Information Database, comprehensive database of traditional Chinese medicine), etc., which is a technical means known in the art.

[0045] Among them, the anti-thrombotic component substance group uses network pharmacology to screen the anti-thrombotic components in the chemical components of each medicinal material in Buyang Huanwu Decoction. For example, the chemical component information of medicinal materials is obtained from the TCMSP database and the TCMID database, and the screening thresholds of the OB value and the DL value are not limited. The network pharmacology method is used to routinely perform component screening, target prediction, and construction of compound-target-disease networks, so as to screen out anti-thrombotic components as anti-thrombotic component substance groups based on the mechanism of multi-pathway regulation. For example, the literature method ("Exploring the Mechanism of Action of Buyang Huanwu Decoction in Preventing and Treating Thrombosis Based on Network Pharmacology" (China Medical Herald, October 2020, Vol. 17, No. 29, pp. 17-28) is used to screen and construct anti-thrombotic component substance groups. The difference is that the screening thresholds of the OB value and DL value of the chemical components are not limited.

[0046] Among them, the corresponding substance clusters of Buyang Huanwu Decoction formed by different extraction methods are obtained based on the chromatographic analysis of the products obtained by different extraction methods, that is, the medicinal materials of Buyang Huanwu Decoction are extracted by combined decoction, single decoction, separate decoction, etc., and the medicinal materials used in the preparation are extracted by water decoction, ethanol reflux extraction, water extraction alcohol precipitation, alcohol extraction water precipitation, steam distillation and other extraction methods. The products extracted from the medicinal materials used in Buyang Huanwu Decoction are taken as the analysis targets, and the material composition and content of the products are analyzed by HPLC-MS, GC-MS and other methods, so as to construct the corresponding substance clusters of Buyang Huanwu Decoction corresponding to different extraction methods.

[0047] Among them, for the ethanol precipitation of the water extraction and alcohol precipitation method, three ethanol precipitation methods with concentrations of 50%, 60%, and 75% (all volume percentages) can be set for method comparison; in the ethanol reflux extraction method and the alcohol extraction and water precipitation method, ethanol extraction methods with concentrations of 40%, 60%, 70%, 80%, and 90% can be set for method comparison.

[0048] The substance clusters corresponding to Buyang Huanwu Decoction with different extraction methods can be sorted according to the content index, so that the substance content is taken as the priority factor.

[0049] When the three major material groups are constructed, the intersection analysis of the three major material groups can be performed, such as Figure 1As shown. The corresponding substance clusters of Buyang Huanwu Decoction with different extraction methods were compared with the intestinal flora regulator substance group and the antithrombotic component substance group, and the intersection of the three was taken; the extraction method of the antithrombotic Buyang Huanwu Decoction preparation was optimized according to the number of intersection substances and the content ranking of the intersection substances of the corresponding substance clusters of Buyang Huanwu Decoction with different extraction methods and the intestinal flora regulator substance group and the antithrombotic component substance group obtained by S1 and S2; the extraction method with the most intersection substances with the intestinal flora regulator substance group and the antithrombotic component substance group was selected as the main extraction method, and the extraction method conditions were optimized.

[0050] In a specific implementation scheme, substances ranked in the top 1 / 3 in content in the substance cluster corresponding to Buyang Huanwu Decoction (e.g., the substance cluster corresponding to Buyang Huanwu Decoction contains 90 substances, of which the top 30 substances are in content) are selected for inclusion in intersection analysis and comparison, and the number of intersection substances with the intestinal flora regulator substance cluster and the anti-thrombotic component substance cluster is analyzed.

[0051] Example 2 Optimization comparison of anti-thrombotic Buyang Huanwu decoction preparations As an exemplary display. This embodiment prepares different anti-thrombotic Buyang Huanwu Decoction preparations, and the intersection comparison of the intestinal flora regulator substance group, the anti-thrombotic component substance group and the Buyang Huanwu Decoction corresponding substance cluster formed by different extraction methods is performed according to the method of Example 1. When preparing different anti-thrombotic Buyang Huanwu Decoction preparations, the weight ratio of raw astragalus, angelica root, red peony root, earthworm, Chuanxiong, peach kernel, and safflower is: 40:2:1.5:1:1:1:1. Among them, the different extraction methods represent the commonly used extraction methods such as water extraction, ethanol extraction and steam distillation that do not use toxic solvents, and are respectively the better extraction methods of the same type of extraction methods after exploration (for example, the extraction effects of different ethanol concentrations, the effects of different alcohol precipitation conditions, etc. have been compared). Among them, different extraction methods are soaked for 40 minutes before the first extraction to promote solvent penetration (for example, when extracting twice, soak for 40 minutes before the first extraction; when extracting once, soak for 40 minutes before extraction).

[0052] Preparation of Buyang Huanwu Decoction Water Extract Preparation A: Take the medicinal materials, crush them through a 60-mesh sieve, add water and boil them for extraction twice. The first time, the solid-liquid ratio was 1:12, and the filtrate was collected after boiling for 2 hours; the second time, the solid-liquid ratio was 1:8, and the filtrate was collected after boiling for 3 hours; the filtrate was combined, ethanol was added to a volume fraction of 80% by volume of ethanol, and the mixture was allowed to stand and precipitate overnight, and the supernatant was collected and freeze-dried to prepare freeze-dried powder.

[0053] Preparation of Buyang Huanwu Decoction ethanol extract preparation B: Take the medicinal material, crush it through a 60-mesh sieve, add 70% ethanol (volume fraction) at 60°C and extract twice, the first solid-liquid ratio is 1:30, extract for 2 hours, and collect the filtrate; the second solid-liquid ratio is 1:15, extract for 2 hours, and collect the filtrate; the combined filtrates are concentrated under reduced pressure to remove ethanol, and freeze-dried to prepare freeze-dried powder.

[0054] Preparation of Buyang Huanwu Decoction steam distillation extract preparation C: Take the medicinal materials, grind them through a 60-mesh sieve, add water at a solid-liquid ratio of 1:6, use a volatile oil extractor to extract for 8 hours, collect the volatile oil, and dry it over anhydrous sodium sulfate.

[0055] Preparation of Buyang Huanwu Decoction Decoction D: (1) Take raw Astragalus, Red Peony Root, and Earthworm, crush and pass through a 60-mesh sieve; add water and decoct twice, the first time the solid-liquid ratio is 1:12, decoct for 2 hours and collect the filtrate; the second time the solid-liquid ratio is 1:10, decoct for 2 hours and collect the filtrate; combine the filtrates, add ethanol to 75% ethanol volume fraction, let stand overnight, collect the supernatant and freeze-dry to make freeze-dried powder D1. (2) Take Angelica root, Chuanxiong, Carthamus tinctorius, and Prunus persica, crush and pass through a 60-mesh sieve; add 70% ethanol (volume fraction) at 60°C and extract twice, the first time the solid-liquid ratio is 1:30, extract for 1.5 hours, collect the filtrate; the second time the solid-liquid ratio is 1:15, extract for 1.5 hours, collect the filtrate; combine the filtrates, decompress and concentrate to remove ethanol, and freeze-dry to make freeze-dried powder D2; (3) Combine freeze-dried powders D1 and D2 as Buyang Huanwu Decoction Decoction D.

[0056] The above preparations were analyzed for composition and content: Among them, the steam distillation extract preparation C of Buyang Huanwu Decoction was detected and analyzed by GC-MS method. The detection method can be found in "Study on Chemical Components of Traditional Chinese Medicine Pairs - GC-MS Analysis of Volatile Oils of Chuanxiong and Red Peony Root" (Journal of Chemistry in Universities, Vol. 27, No. 3, 2006, pp. 443-448).

[0057] Other Buyang Huanwu Decoction preparations were tested and analyzed by LC-MS / MS.

[0058] Take 200 mg of sample, add 10 mL of 50% methanol aqueous solution (V:V, methanol: water = 50:50), ultrasonicate for 30 min (200 W, 50 Hz), take 1 mL of supernatant, centrifuge at 14000 rpm for 5 min, filter the supernatant through 0.22 μm filter membrane and inject for determination.

[0059] Instrument: Q Exactive Plus Orbitrap high-resolution liquid spectrometer (Thermo Fisher); Liquid chromatography: U3000 ultra-high performance liquid chromatography; chromatographic column: ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm); column temperature: 35°C; injection volume: 10 μL, flow rate: 0.3 mL / min; mobile phase A: deionized water, containing 0.1% formic acid; mobile phase B: acetonitrile, containing 0.1% formic acid, gradient elution was used, and the elution program is shown in Table 1.

[0060] Table 1 Liquid chromatography elution program

[0061] Mass spectrometry conditions: Q Exactive Plus Orbitrap high-resolution liquid chromatography-mass spectrometry was used for mass spectrometry data acquisition, and the detection mode was Full MS-ddMS 2 . Scan in positive ion mode and negative ion mode respectively, with a scanning range of m / z 100-1200.MS 1 The resolution was set to 70000, the ion source voltage was 3.2 kV, the capillary temperature was 320 °C, the auxiliary gas heater temperature was 350 °C, the sheath gas flow rate was 40 L / min, the auxiliary gas flow rate was 15 L / min, the AGCTarget was set to 1e6, the TopN was set to 5, and the MS was triggered. 2 The collision energy was scanned using a stepwise crushing voltage NCE, which was set to 30, 40, and 50.

[0062] The corresponding substance clusters of Buyang Huanwu Decoction formed by different extraction methods were compared with the intestinal flora regulator substance group and the antithrombotic component substance group. It was found that Buyang Huanwu Decoction decoction preparation D had the most intersection substances with the intestinal flora regulator substance group and the antithrombotic component substance group, and the substances in the top 1 / 3 of the content had the most intersection substances with the intestinal flora regulator substance group and the antithrombotic component substance group. That is, among the top 33 substances in terms of content (a total of 98 substances were analyzed), the intersection with the intestinal flora regulator substance group and the antithrombotic component substance group reached 10 and 12 respectively (among which, among the top 33 substances, there were only 5 intersection substances between the intestinal flora regulator substance group and the antithrombotic component substance group). The intersection substances were about 1.5~2 times that of Buyang Huanwu Decoction water extract preparations A and B. It was more than 3 times that of Buyang Huanwu Decoction steam distillation extract preparation C. The results of component analysis of Buyang Huanwu Decoction decoction preparation D are shown in Table 2, where the content indicators include peak area and relative content (%, content in the extract). Since the product obtained from a unit amount of medicinal materials under given extraction conditions is a fixed value, it can also be converted into absolute content.

[0063] Table 2 List of ingredients of Buyang Huanwu Decoction decoction D

[0064]

[0065]

[0066]

[0067]

[0068] Example 3 Comparison of antithrombotic effects of different Buyang Huanwu Decoction preparations As an exemplary display. In this example, the anticoagulant and antithrombotic effects of different Buyang Huanwu Decoction preparations prepared in Example 2 were investigated according to the same dosage standard, wherein the oral dosages of different preparations were converted into medicinal material dosages, and different Buyang Huanwu Decoction preparations were added with water to prepare oral administration solutions at a concentration of 4 g crude drug / mL, and the oral administration was performed at a dosage of 20 g crude drug / kg body weight / day.

[0069] SPF male SD rats, weighing 200-220 g, were randomly divided into sham operation group (sham operation group underwent surgery but no modeling), model group, Buyang Huanwu Decoction water extract preparation group A, Buyang Huanwu Decoction ethanol extract preparation group B, Buyang Huanwu Decoction steam distillation extract preparation group C, and Buyang Huanwu Decoction decoction preparation group D, with 8 rats in each group. The sham operation group and the model group were gavaged with distilled water; the drug group was gavaged with the corresponding gavage solution, and the drug administration lasted for 28 days. 0.5 h after the last drug administration, the rat common carotid artery thrombosis model was established using the literature method (Yang Jing. "Study on the Effects of Buyang Huanwu Decoction and Its Main Effective Parts on TXA2, PGI2 and Antiplatelet in Rat Arterial Thrombosis". Hunan University of Traditional Chinese Medicine, 2006), the thrombus weight was measured, and blood was collected. The plasma TXB2 content was determined by radioimmunoassay; the plasma antithrombin III (AT-Ⅲ) activity was determined by chromogenic substrate method.

[0070] The data are averaged ± The standard deviation was expressed as t The results are shown in Table 3.

[0071] Table 3 Results of thrombosis indexes and coagulation parameters in each group

[0072] The experimental results showed that thrombosis occurred in the model group, and TXB2 increased and 6-keto-PGF 1α The thrombus mass and TXB2 in the preparation D group were significantly lower than those in the model group and other preparation groups ( p <0.01); Preparation D group 6-keto-PGF 1α , AT-Ⅲ activity was significantly higher than that of the model group and other preparation groups ( p <0.01).

Claims

1. A method for optimizing an anti-thrombotic Chinese medicine preparation, characterized in that: The Chinese medicine preparation optimization method comprises the following steps: S1 Construction of intestinal flora regulator substance group based on the chemical components of the medicinal materials of Buyang Huanwu Decoction: According to the chemical components contained in the medicinal materials used in Buyang Huanwu Decoction, the chemical components that have the function of regulating intestinal flora and promoting the proliferation of probiotics are screened to establish the intestinal flora regulator substance group; S2 Construction of network pharmacology antithrombotic component substance group based on the chemical components of Buyang Huanwu Decoction: Based on the chemical components of the medicinal materials used in Buyang Huanwu Decoction, without limiting the screening thresholds of OB and DL values, conventional network pharmacology methods are used to screen components, predict targets, and construct compound-target-disease networks, and the screened antithrombotic components are used as antithrombotic component substance groups; S3 Construction of substance clusters of different extraction methods of Buyang Huanwu Decoction: Different extraction methods were used to extract the ingredients of Buyang Huanwu Decoction and analyze the substance structure and content, and the substance clusters corresponding to Buyang Huanwu Decoction with different extraction methods were obtained; and the content indicators of the substance clusters corresponding to Buyang Huanwu Decoction with different extraction methods were ranked respectively; S4 Compare the substance clusters corresponding to the Buyang Huanwu Decoction obtained by different extraction methods in step S3 with the intestinal flora regulator substance group and the anti-thrombotic component substance group obtained in steps S1 and S2, and take the intersection of the three; optimize the extraction method of the anti-thrombotic Buyang Huanwu Decoction preparation according to the number of intersection substances and the content ranking of the intersection substances between the substance clusters corresponding to the Buyang Huanwu Decoction obtained by different extraction methods and the intestinal flora regulator substance group and the anti-thrombotic component substance group obtained by S1 and S2; select the extraction method with the most intersection substances with the substance ranked first in content and the intestinal flora regulator substance group and the anti-thrombotic component substance group obtained by S1 and S2 as the main extraction method, and optimize the extraction method conditions.

2. The method for optimizing an anti-thrombotic Chinese medicine preparation according to claim 1, characterized in that: In step S4, the extraction method with the most intersection substances of the substances ranked in the top two thirds in content and the intestinal flora regulator substance group and the anti-thrombotic component substance group obtained in S1 and S2 is selected as the main extraction method to optimize the extraction method conditions.

3. The method for optimizing an anti-thrombotic Chinese medicine preparation according to claim 1, characterized in that: In step S4, the extraction method with the most substances in the first third of the content ranking and the intestinal flora regulator substance group and anti-thrombotic component substance group obtained in S1 and S2 is selected as the main extraction method to optimize the extraction method conditions.

4. An anti-thrombotic Chinese medicine preparation, wherein the Buyang Huanwu Decoction is made from the following medicinal materials: raw astragalus, angelica root, red peony root, earthworm, chuanxiong, safflower and peach kernel; characterized in that: The Buyang Huanwu Decoction preparation is prepared from the water-decocted alcohol precipitation extract of raw astragalus, red peony root and earthworm and the 70% ethanol reflux extract of angelica root, chuanxiong, safflower and peach kernel.

5. The anti-thrombotic Chinese medicine preparation according to claim 4, characterized in that: The weight ratio of the raw astragalus, angelica root, red peony root, earthworm, ligusticum chuanxiong, peach kernel and safflower is 40:2:1.5:1:1:1:

1.

6. The anti-thrombotic Chinese medicine preparation according to claim 4, characterized in that: The medicinal materials are crushed to 20-100 meshes before extraction.

7. The anti-thrombotic Chinese medicine preparation according to claim 4, characterized in that: When the water decoction alcohol precipitation extract is prepared, the solid-liquid ratio is 1: (8-12); the decoction times are 2-4 times, and each decoction is 1-4 hours; when the water decoction alcohol precipitation extract is prepared, ethanol is added in the alcohol precipitation step until the ethanol volume fraction reaches 70%-85%.

8. The anti-thrombotic Chinese medicine preparation according to claim 4, characterized in that: When the ethanol reflux extract is prepared, the solid-liquid ratio is 1:(12-30); the extraction temperature is 60°C-80°C, the number of extractions is 1-3 times, and each extraction takes 1-3 hours.

9. The anti-thrombotic Chinese medicine preparation according to claim 4, characterized in that: The Buyang Huanwu Decoction preparation is an oral solid preparation or an oral liquid preparation.

10. The anti-thrombotic Chinese medicine preparation according to claim 4, characterized in that: The Buyang Huanwu Decoction preparation also contains pharmaceutically acceptable preparation excipients.

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