A method for optimizing traditional Chinese medicine preparations based on hemorheology mechanism and the preparations

Abstract

The present invention belongs to the technical field of optimization algorithms and preparation optimization of compound traditional Chinese medicine preparations, and relates to a method and preparation for optimizing traditional Chinese medicine preparations based on hemorheology mechanism. In particular, it relates to a method and preparation for optimizing Buyang Huanwu Decoction for anti-thrombosis. The preparation optimization method determines a preferred extraction method of Buyang Huanwu Decoction through constructing a substance group of intestinal flora regulators, a substance group of anti-thrombosis components and a corresponding substance cluster of products obtained by different extraction methods for the chemical components of the medicinal materials of Buyang Huanwu Decoction, and then conducts optimization, which can effectively guide the optimization of Buyang Huanwu Decoction preparations. The Buyang Huanwu Decoction preparation of the present invention is prepared from the water decoction and ethanol precipitation extraction solution of Astragalus membranaceus, Paeonia lactiflora and Pheretima, and the 70% ethanol reflux extraction solution of the other 4 medicinal materials. Its extracted components have the most intersection substances with the substance group of intestinal flora regulators and the substance group of anti-thrombosis components, and the content of the intersection substances is high, and it has higher anticoagulant and anti-thrombotic effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of optimization algorithms and preparation optimization of compound traditional Chinese medicine preparations, and particularly relates to a method and preparation for optimizing traditional Chinese medicine preparations based on hemorheological mechanisms. Background Art

[0002] The original formula of Buyang Huanwu Decoction comes from "Corrections of Medical Errors" by Wang Qingren, and it is a representative formula reflecting the theory of qi deficiency and blood stasis created by Wang Qingren. It is mainly used to treat hemiplegia, deviation of the mouth and eyes, speech impediment, salivation at the corners of the mouth, flaccidity of the lower limbs, frequent urination, enuresis, etc. Modern pharmacological studies have shown that this formula also has pharmacological effects such as anti-cerebral ischemia injury and anti-thrombosis. So far, its original water decoction process has been used for the preparation of preparations in clinical practice. However, due to different required active ingredients for different indications, using one formula for multiple purposes leads to the lack of pertinence of the preparation for different indications.

[0003] In terms of the pharmacological analysis and mechanism analysis of traditional Chinese medicine, there are currently two main technical means. One 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-likeness, etc., and components that are considered likely to be inactive are excluded according to experience (for example, substances with OB≥30% and DL≥0.18 are usually selected as candidate substances). Then, target analysis is carried out on the required components, and then analysis is carried out on the targets related to the pathological mechanisms of known diseases to construct an interaction map of active ingredients - drug targets - disease targets, so as to analyze the possible multi-target action mechanisms of compound drugs. Such methods and literature can be found in "Network Pharmacology of Rhubarb Against Renal Fibrosis and the Action Mechanisms of Its Active Ingredients" (Chinese Journal of Experimental Traditional Medical Formulae, Vol. 26, No. 10, 2020, pp. 163 - 172), "Discussion on the Action Mechanisms 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), CN103150490A, CN110648726A, etc. This method is very helpful for explaining the multi-component and multi-target action mechanisms of traditional Chinese medicine and compound preparations. However, since it belongs to an ex-post explanatory method and tool and focuses on the docking of targets - components, it leads to the neglect of the overall treatment concept of traditional Chinese medicine, such as the method of using qi-tonifying and stasis-removing together when treating stasis. This cannot be explained and illustrated by target - docking. And excluding components that are considered likely to be inactive according to experience also leads to the neglect of the problem of the mutual influence of the dissolution behaviors of different components of traditional Chinese medicine. In practical applications, there are often components with OB and DL far less than the thresholds that still enter the blood after oral administration. As a result, its explanatory ability is difficult to meet expectations.

[0004] In addition, another means of pharmacological analysis and mechanism analysis of traditional Chinese medicine is based on the tool of serum pharmacochemistry of traditional Chinese medicine. That is, the migrated components detected in the blood after oral administration of traditional Chinese medicine or compound prescriptions are considered to be the effective substances only when they enter the blood. So far, serum pharmacochemistry of traditional Chinese medicine also has the problem of being unable to reasonably explain traditional Chinese medicine and its mechanism, and there are great differences in the components entering the blood in preparations with different preparation methods. This is also due to the neglect of the overall treatment concept of traditional Chinese medicine and the deviation from the treatment methods and principles. For example, there are great differences in the components entering the blood and brain of Buyang Huanwu Decoction obtained from "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 the Components of Buyang Huanwu Decoction Absorbed into the Brain and Blood" (Chinese Journal of Experimental Traditional Medical Formulae, Vol. 25, No. 2, 2019, pp. 8-13), and "Serum Pharmacochemistry Study on the Substances with Pharmacodynamic Effects of Buyang Huanwu Decoction against Cerebral Ischemia Injury" (New Drugs & Clinical Remedies, Vol. 34, No. 9, September 2023, pp. 1227-1235).

[0005] The inherent defects of the above two theories result in their being able to be used to explain the action mechanisms of traditional Chinese medicine and compound prescriptions to a certain extent after the fact, but it is difficult to be used to guide the modernization 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 to achieve the optimization of targeted preparations of traditional Chinese medicine compounds and others for different indications. Summary of the Invention

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

[0008] In traditional Chinese medicine clinical practice, it is found that the change of gastrointestinal function often appears prior to the symptoms of other organ diseases, while the recovery of gastrointestinal function often lags behind the cure speed of other organ diseases. This is particularly prominent in diseases such as cardiovascular and cerebrovascular diseases and kidney diseases. This coincides with the view of the spleen and stomach school in traditional Chinese medicine. The development of modern medicine also shows that there are a series of disease regulation mechanisms based on the digestive system in the human body, such as the "gut-brain axis", "lung-brain axis", "gut-heart axis", "gut-vascular barrier axis", etc., and it is found that the 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, 2022, pp. 1217-1221).

[0009] Therefore, in view of the deficiencies of traditional network pharmacology (such as 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, which cannot be incorporated into the traditional network pharmacology system) and the traditional theory of serum pharmacochemistry of traditional Chinese medicine (entering the blood is not the only mechanism for drugs or chemical substances to exert regulatory effects on the body, and the traditional theory of serum pharmacochemistry of traditional Chinese medicine cannot incorporate non-blood components into the investigation), and the problem that the two theories cannot guide the modern refinement and optimization of traditional Chinese medicine preparations such as Buyang Huanwu Decoction; combining the holistic concept of traditional Chinese medicine and theories such as the "intestinal-vascular barrier axis", the present invention first provides an optimization method for Buyang Huanwu Decoction preparation based on the complex mechanism of hemorheology. Specifically, the present invention first provides an optimization method for an anti-thrombotic Buyang Huanwu Decoction preparation to overcome the shortcomings that the original methods and theories cannot guide the optimization of pharmaceutics.

[0010] The method comprises the following steps:

[0011] S1 Construction of the 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 of Buyang Huanwu Decoction, literature retrieval analysis and / or experimental analysis are carried out, and the chemical components with intestinal flora regulation and promoting the proliferation of probiotics are screened to establish the intestinal flora regulator substance group;

[0012] 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 included in databases such as the TCMSP database and the TCMID database can be integrated for chemical component integration.

[0013] S2 Construction of the 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 of Buyang Huanwu Decoction, without limiting the screening thresholds of the OB value and the DL value, component screening, target prediction, construction of compound-target-disease networks and protein-protein interaction networks are routinely carried out by means of network pharmacology, and the screened anti-thrombotic components are used as the anti-thrombotic component substance group.

[0014] The chemical components of the medicinal materials in this step can be integrated with the chemical components included in databases such as the TCMSP database and the TCMID database for chemical component integration. Specifically, reference can be made to "Discussion on the Mechanism of Buyang Huanwu Decoction in Preventing and Treating Thrombosis Based on Network Pharmacology" (China Medical Herald, Vol. 17, No. 29, October 2020, pp. 17-28). The difference is that in step S2 of the present invention, the screening thresholds of the OB value and the DL value are not limited.

[0015] Construction of the substance groups of Buyang Huanwu Decoction by different extraction methods: The medicinal materials of Buyang Huanwu Decoction were extracted by different extraction methods, and the substance structures and contents were analyzed to obtain the corresponding substance clusters of Buyang Huanwu Decoction by different extraction methods; and the corresponding substance clusters of Buyang Huanwu Decoction by different extraction methods were sorted according to content indexes (such as peak area and percentage content).

[0016] For the different extraction methods in this step, methods such as water decoction method, ethanol reflux extraction method, water extraction and alcohol precipitation method, alcohol extraction and water precipitation method, steam distillation method, etc. can be selected. Given that the water decoction method contains impurities such as proteins and is not conducive to chromatographic analysis, the ethanol reflux extraction method, water extraction and alcohol precipitation method, alcohol extraction and water precipitation method, steam distillation method, etc. are preferably used. For the extraction methods here, except for the steam distillation extract, the obtained substances are all soluble components in the solvent, rather than precipitates and medicinal residues.

[0017] Among them, when ethanol is precipitated in the water extraction and alcohol precipitation method, three ethanol precipitation methods with concentrations of 50%, 60%, and 75% (both are volume percentages) can be set for method comparison; in the ethanol reflux extraction method and the alcohol extraction and water precipitation method, ethanol extraction stages with concentrations of 40%, 60%, 70%, 80%, 90%, etc. can be set for method comparison. Methods such as HPLC-MS and GC-MS can be used for the analysis of the substance structures and contents of the substance clusters.

[0018] Among them, different extraction methods include co-extraction of the medicinal materials of the prescription by the same method (which can be called co-decoction), separate extraction of different medicinal materials by the same method or different methods and then merging the extraction solutions (which can be called single decoction), extraction of some medicinal materials by the same method and extraction of another part of the medicinal materials by another method and then merging the extraction solutions (which can be called separate decoction), etc.

[0019] In this way, the substance groups of Buyang Huanwu Decoction by different extraction methods under the extraction method are constructed.

[0020] S4 Compare the corresponding substance clusters of Buyang Huanwu Decoction by different extraction methods obtained in step S3 with the substance groups of intestinal flora regulators and anti-thrombotic component substance groups 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 and content ranking of the intersection substances of the corresponding substance clusters of Buyang Huanwu Decoction by different extraction methods with the substance groups of intestinal flora regulators and anti-thrombotic component substance groups obtained in S1 and S2; select the extraction method with the most intersection substances between the substances with the top content ranking and the substance groups of intestinal flora regulators and anti-thrombotic component substance groups obtained in S1 and S2 as the main extraction method, and optimize the extraction method conditions.

[0021] The main extraction method refers to a combination of two extraction methods, but the extraction method in which the substance ranked first in terms of content has the largest intersection with the intestinal flora regulator substance group and the antithrombotic component substance group obtained from S1 and S2 is taken as the main method.

[0022] The optimization of the extraction method conditions includes further optimization of the selected method, such as the optimization of parameters such as the degree of comminution, the solid-liquid ratio, the extraction time, the extraction temperature, and the number of extraction times.

[0023] Preferably, in step S4, the extraction method in which the substances ranked in the top two-thirds in terms of content have the largest intersection with the intestinal flora regulator substance group and the antithrombotic component substance group obtained from S1 and S2 is taken as the main extraction method, and on this basis of optimizing the extraction method of Buyang Huanwu Decoction, the combination of extraction methods and the condition optimization are carried out.

[0024] Preferably, in step S4, the extraction method in which the substances ranked in the top one-third in terms of content have the largest intersection with the intestinal flora regulator substance group and the antithrombotic component substance group obtained from S1 and S2 is taken as the main extraction method, and on this basis of optimizing the extraction method of Buyang Huanwu Decoction, the combination of extraction methods and the condition optimization are carried out.

[0025] For example, when the corresponding substance cluster of Buyang Huanwu Decoction obtained by extraction method A has intersections with both the intestinal flora regulator substance group and the antithrombotic component substance group obtained from steps S1 and S2, and the intersections are relatively large, and the intersection substances are substances with relatively high contents (for example, the substance content ranks within the top 50), then method A is taken as the main extraction method for the preparation of Buyang Huanwu Decoction, and parameters such as the solid-liquid ratio, the extraction time, and the extraction temperature are further optimized.

[0026] When extraction method A has an intersection with the intestinal flora regulator substance group obtained from S1, and the intersection is relatively large, and the intersection substances are substances with relatively high contents (for example, the substance content ranks within the top 50), but has a relatively small intersection with the antithrombotic component substance group obtained from S2, or the intersection substances are substances with relatively low contents (the content ranks outside the top 50), then a combination of multiple extraction methods is used as the extraction method for the preparation of Buyang Huanwu Decoction, and other extraction parameters are optimized, such as parameters such as the solid-liquid ratio and the extraction time.

[0027] For example, when there is an intersection between the corresponding material cluster of Buyang Huanwu Decoction extracted by water decoction and the intestinal flora regulator material group obtained from S1, and the intersection is relatively large, and the intersection substances are substances with relatively high contents, while there is a relatively small intersection with the anti-thrombotic component material group obtained from S2. Generally, the number of intersection substances between the corresponding material cluster of Buyang Huanwu Decoction and the intestinal flora regulator material group obtained from S1 and the anti-thrombotic component material group obtained from S2 is the largest, but there is still the deficiency of a relatively small intersection with the anti-thrombotic component material group obtained from S2. And there is a relatively large intersection between the corresponding material cluster of Buyang Huanwu Decoction extracted by ethanol reflux and the anti-thrombotic component material group obtained from S2, and the intersection substances are substances with relatively high contents. Then, the medicinal materials can be first extracted by water decoction to separate the water extract, and then the medicinal residues can be extracted by ethanol reflux.

[0028] The aforementioned steps S1, S2, and S3 are only codes and are not regarded as the order of the steps; as independent and executable steps, S1, S2, and S3 can be executed independently respectively. However, steps S1, S2, and S3 should obviously be executed prior to step S4.

[0029] Based on this method, the present invention provides an anti-thrombotic Buyang Huanwu Decoction preparation. The Buyang Huanwu Decoction is made from the following medicinal materials: raw Astragalus membranaceus, Angelica sinensis tail, Red Peony Root, Pheretima aspergillum, Ligusticum chuanxiong, Safflower, and Peach Kernel; the Buyang Huanwu Decoction preparation is made from the water decoction and alcohol precipitation extract of raw Astragalus membranaceus, Red Peony Root, and Pheretima aspergillum and the 70% ethanol reflux extract of Angelica sinensis tail, Ligusticum chuanxiong, Safflower, and Peach Kernel.

[0030] Preferably, the weight ratio of raw Astragalus membranaceus, Angelica sinensis tail, Red Peony Root, Pheretima aspergillum, Ligusticum chuanxiong, Peach Kernel, and Safflower is: 40:2:1.5:1:1:1:1.

[0031] Preferably, the medicinal materials are pulverized to 20 mesh to 100 mesh before extraction. Further preferably, the medicinal materials are pulverized to 60 mesh to 80 mesh before extraction.

[0032] Preferably, when preparing the water decoction and alcohol precipitation extract, the material-liquid ratio is 1:(8 - 12), that is, 1 g of medicinal materials is added with 8 - 12 mL of water; the number of decoction times is 2 - 4 times, and each decoction is 1 - 4 h; when preparing the water decoction and alcohol precipitation extract, ethanol is added in the alcohol precipitation step until the volume fraction of ethanol reaches 70% - 85%.

[0033] Further preferably, when preparing the water decoction and alcohol precipitation extract, the material-liquid ratio is 1:(10 - 12), that is, 1 g of medicinal materials is added with 10 - 12 mL of water; the number of decoction times is 2 times, and each decoction is 2 h; when preparing the water decoction and alcohol precipitation extract, ethanol is added in the alcohol precipitation step until the volume fraction of ethanol reaches 75%.

[0034] Preferably, when preparing the ethanol reflux extract, the material-liquid ratio is 1:(12 - 30), that is, 1 g of medicinal materials is added to 12 - 30 mL of 70% ethanol; the extraction temperature is 60°C - 80°C, the extraction times are 1 - 3 times, and each extraction lasts for 1 - 3 h.

[0035] More preferably, when preparing the ethanol reflux extract, the material-liquid ratio is 1:(15 - 30), that is, 1 g of medicinal materials is added to 15 - 30 mL of 70% ethanol; the extraction temperature is 60°C, the extraction times are 2 times, and each extraction lasts for 1.5 h.

[0036] 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 solid or semi-solid materials for preparing Buyang Huanwu Decoction preparations.

[0037] Therefore, the Buyang Huanwu Decoction preparation can be an oral liquid preparation or an oral solid preparation.

[0038] The Buyang Huanwu Decoction preparation can 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, sodium carboxymethylcellulose.

[0039] Advantages of the present invention:

[0040] (1) The present invention provides an optimization method for Buyang Huanwu Decoction preparations for anti-thrombosis (preventing or treating thrombotic diseases by anticoagulation or improving hemorheological characteristics, etc.). This method overcomes the defects that traditional network pharmacology or serum pharmacochemistry methods are difficult to guide the optimization of traditional Chinese medicine preparations, as well as the problem of inability to adapt to the overall treatment methods and principles of traditional Chinese medicine. It can effectively reflect the overall treatment methods and principles of traditional Chinese medicine and is used to guide the optimization of the extraction method of Buyang Huanwu Decoction preparations.

[0041] (2) The present invention provides a Buyang Huanwu Decoction preparation for anti-thrombosis (preventing or treating thrombotic diseases by anticoagulation or improving hemorheological characteristics, etc.). Brief Description of the Drawings

[0042] Figure 1 It is a logical schematic diagram of the optimization method for the anti-thrombotic Buyang Huanwu Decoction preparation of the present invention. Detailed Embodiments

[0043] The following further describes the present application with reference to the drawings. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and cannot be used to limit the protection scope of the present application.

[0044] Example 1 Optimization Method for Anti-thrombotic Buyang Huanwu Decoction Preparation

[0045] Please refer to Figure 1Logical schematic diagram. The basic principle of the method for optimizing the Buyang Huanwu Decoction preparation in this embodiment is based on the material intersection of three major material groups, and the number and content ranking of the intersection materials are used as a reference to optimize the preparation extraction method.

[0046] These three major material groups include the intestinal flora regulator material group screened from the chemical components of each medicinal material in the Buyang Huanwu Decoction, the anti-thrombotic component material group screened from the chemical components of each medicinal material in the Buyang Huanwu Decoction, and the corresponding material cluster of the Buyang Huanwu Decoction formed by different extraction methods. The material intersection of the three major material groups includes the material intersection 1 between the corresponding material cluster of the Buyang Huanwu Decoction formed by a specific extraction method and the intestinal flora regulator material group, and the material intersection 2 between the corresponding material cluster of the Buyang Huanwu Decoction formed by a specific extraction method and the anti-thrombotic component material group.

[0047] Among them, the construction of the intestinal flora regulator material group can be completed by literature retrieval and analysis of the activities of the chemical components in each medicinal material of the Buyang Huanwu Decoction, so as to screen out the chemical components with the function of regulating intestinal flora and promoting the proliferation of probiotics, thus completing the construction of the intestinal flora regulator material group.

[0048] The chemical components in each medicinal material of the 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), the TCMID database (Traditional Chinese Medicine Information Database), etc., which is a well-known technical means in this field.

[0049] Among them, the anti-thrombotic component material group is screened for the anti-thrombotic components in the chemical components of each medicinal material of the Buyang Huanwu Decoction by means of network pharmacology. For example, the chemical component information of the 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 component screening, target prediction, and compound-target-disease network construction are carried out by means of network pharmacology, so as to screen out the anti-thrombotic components as the anti-thrombotic component material group based on the mechanism of multi-pathway regulation. For example, the literature method (Discussion on the Mechanism of Buyang Huanwu Decoction in Preventing and Treating Thrombosis Based on Network Pharmacology (China Medical Herald, Vol. 17, No. 29, October 2020, pp. 17-28)) is used to screen and construct the anti-thrombotic component material group. The difference is that the screening thresholds of the OB value and the DL value of the chemical components are not limited.

[0050] 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, for the medicinal materials of Buyang Huanwu Decoction, co-decoction, single decoction, separate decoction, etc. are used, and extraction methods such as water decoction method, ethanol reflux extraction method, water extraction and ethanol precipitation method, ethanol extraction and water precipitation method, steam distillation method, etc. are used to extract the medicinal materials used in the preparation. Taking the products obtained by extracting the medicinal materials of a unit of Buyang Huanwu Decoction as the analysis target, methods such as HPLC-MS and GC-MS are used to analyze the substance composition and content of the products, so as to construct the corresponding substance clusters of Buyang Huanwu Decoction corresponding to different extraction methods.

[0051] Among them, when ethanol precipitation is carried out in the water extraction and ethanol precipitation method, three ethanol precipitation methods with concentrations of 50%, 60%, and 75% (both are volume percentages) can be set for method comparison; in the ethanol reflux extraction method and the ethanol extraction and water precipitation method, ethanol extraction methods with concentrations of 40%, 60%, 70%, 80%, 90%, etc. can be set for method comparison.

[0052] The corresponding substance clusters of Buyang Huanwu Decoction corresponding to different extraction methods can be sorted according to the content index, so as to take the substance content as the priority consideration factor.

[0053] After the construction of the three major substance groups is completed, intersection analysis of the three major substance groups can be carried out, as Figure 1 shown. Compare the corresponding substance clusters of Buyang Huanwu Decoction by different extraction methods with the substance group of intestinal flora regulators and the substance group of antithrombotic components, and take the intersection of the three; optimize the extraction method of the antithrombotic Buyang Huanwu Decoction preparation according to the number of intersection substances and the sorting of the content of the intersection substances of the corresponding substance clusters of Buyang Huanwu Decoction by different extraction methods with the substance group of intestinal flora regulators and the substance group of antithrombotic components obtained from S1 and S2; select the extraction method with the most intersection substances of the substances ranked in the front in terms of content with the substance group of intestinal flora regulators and the substance group of antithrombotic components as the main extraction method, and optimize the extraction method conditions.

[0054] In a specific implementation example, select the substances ranked in the top 1 / 3 in terms of content in the corresponding substance cluster of Buyang Huanwu Decoction (for example, if the corresponding substance cluster of Buyang Huanwu Decoction contains 90 substances, among which the top 30 substances with higher content) to be included in the intersection analysis and comparison, and analyze the number of intersection substances with the substance group of intestinal flora regulators and the substance group of antithrombotic components.

[0055] Example 2 Optimization and comparison of antithrombotic Buyang Huanwu Decoction preparation

[0056] As an exemplary demonstration. In this example, different anti-thrombotic Buyang Huanwu Decoction preparations were prepared, and the intersection comparison of the intestinal flora regulator substance group, anti-thrombotic component substance group, and the corresponding substance clusters of Buyang Huanwu Decoction formed by different extraction methods was carried out according to the method of Example 1. When preparing different anti-thrombotic Buyang Huanwu Decoction preparations, the weight ratio of Astragalus membranaceus, Angelica sinensis tail, Paeonia lactiflora, Pheretima aspergillum, Ligusticum wallichii, Prunus persica, and Carthamus tinctorius was: 40:2:1.5:1:1:1:1. Among them, different extraction methods represent common extraction methods without using toxic solvents such as water extraction method, ethanol extraction method, and steam distillation method, 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.). Among them, the materials were soaked for 40 minutes before the first extraction by different extraction methods to promote solvent penetration (for example, when extracted twice, soaked for 40 minutes before the first extraction; when extracted once, soaked for 40 minutes before extraction).

[0057] Preparation of water extraction preparation A of Buyang Huanwu Decoction: Take the medicinal materials, crush them through a 60-mesh sieve, extract by boiling with water twice. For the first time, the material-liquid ratio is 1:12, boil for 2 hours and collect the filtrate; for the second time, the material-liquid ratio is 1:8, boil for 3 hours and collect the filtrate; combine the filtrates, add ethanol to make the ethanol volume fraction 80%, let it stand and precipitate overnight, collect the supernatant and freeze-dry it to make a freeze-dried powder.

[0058] Preparation of ethanol extraction preparation B of Buyang Huanwu Decoction: Take the medicinal materials, crush them through a 60-mesh sieve, extract with 70% ethanol (volume fraction) at 60°C twice. For the first time, the material-liquid ratio is 1:30, extract for 2 hours, and collect the filtrate; for the second time, the material-liquid ratio is 1:15, extract for 2 hours, and collect the filtrate; combine the filtrates, concentrate under reduced pressure to remove ethanol, and freeze-dry it to make a freeze-dried powder.

[0059] Preparation of steam distillation extraction preparation C of Buyang Huanwu Decoction: Take the medicinal materials, crush them through a 60-mesh sieve, add water according to the material-liquid ratio of 1:6 each time, extract with a volatile oil extractor for 8 hours, collect the volatile oil, and dry it with anhydrous sodium sulfate.

[0060] Preparation of Separately Decocted Preparation D of Buyang Huanwu Decoction: (1) Take raw Astragalus membranaceus, Paeonia lactiflora, and Pheretima aspergillum, crush them through a 60-mesh sieve; decoct with water twice. For the first time, the ratio of material to liquid is 1:12, decoct for 2 hours and collect the filtrate; for the second time, the ratio of material to liquid is 1:10, decoct for 2 hours and collect the filtrate; combine the filtrates, add ethanol to make the ethanol volume fraction reach 75%, let it stand and precipitate overnight, collect the supernatant and freeze-dry it to make freeze-dried powder D1. (2) Take Radix Angelicae Sinensis (Tail), Ligusticum chuanxiong, Flos Carthami, and Semen Persicae, crush them through a 60-mesh sieve; extract with 70% ethanol (volume fraction) at 60 °C twice. For the first time, the ratio of material to liquid is 1:30, extract for 1.5 hours and collect the filtrate; for the second time, the ratio of material to liquid is 1:15, extract for 1.5 hours and collect the filtrate; combine the filtrates, concentrate under reduced pressure to remove ethanol, and freeze-dry to make freeze-dried powder D2; (3) Combine freeze-dried powder D1 and D2 as the separately decocted preparation D of Buyang Huanwu Decoction.

[0061] Conduct component analysis and content analysis on the above preparations:

[0062] Among them, the preparation C of Buyang Huanwu Decoction extracted by steam distillation is detected and analyzed by GC-MS method. The detection method can be referred to "Study on the Chemical Constituents of Chinese Medicine Drug Pairs - GC-MS Analysis of the Volatile Oil of Ligusticum chuanxiong - Paeonia lactiflora" (Acta Chimica Sinica, Vol. 27, No. 3, 2006, pp. 443 - 448).

[0063] Other preparations of Buyang Huanwu Decoction are detected and analyzed by liquid chromatography - mass spectrometry.

[0064] Take 200 mg of the sample, add 10 mL of 50% methanol aqueous solution (V:V, methanol:water = 50:50), sonicate for 30 minutes (200 W, 50 Hz), take 1 mL of the supernatant, centrifuge at 14000 rpm for 5 minutes, and inject the supernatant through a 0.22 μm filter membrane for determination.

[0065] Instrument: Q Exactive Plus Orbitrap high-resolution liquid chromatography - mass spectrometry instrument (Thermo Fisher);

[0066] Liquid chromatography: U3000 ultra-high performance liquid chromatography; the chromatographic column is ACQUITY UPLC HSS T3 (2.1×100 mm, 1.8 μm); the column temperature is 35 °C; the injection volume is 10 μL, and the flow rate is 0.3 mL / min; mobile phase A (deionized water, containing 0.1% formic acid); mobile phase B (acetonitrile, containing 0.1% formic acid), and gradient elution is adopted. The elution program is shown in Table 1.

[0067] Table 1 Liquid chromatography elution program

[0068]

[0069] Mass spectrometry conditions: The Q Exactive Plus Orbitrap high-resolution liquid chromatography-mass spectrometry instrument was used for mass spectrometry data acquisition, and the detection mode was Full MS-ddMS 2 . Scans were performed separately in positive and negative ion modes, and the scan range m / z was 100 - 1200. The MS 1 resolution was set to 70000, the ion source voltage was 3.2 kV, the capillary ion transfer tube temperature (Capillary temperature) was 320 °C, the auxiliary gas heating temperature (Aux 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 triggering MS 2 scan collision energy used the stepped fragmentation voltage NCE, which was set to 30, 40, 50.

[0070] Comparisons of the intersections of the corresponding substance clusters of Buyang Huanwu Decoction formed by different extraction methods with the intestinal flora regulator substance group and the antithrombotic component substance group found that the decocted preparation D of Buyang Huanwu Decoction had the most intersection substances with both the intestinal flora regulator substance group and the antithrombotic component substance group, and the substances within the top 1 / 3 of the content also had the most intersection substances with both the intestinal flora regulator substance group and the antithrombotic component substance group. That is, among the top 33 substances (a total of 98 substances were analyzed), the intersections with the intestinal flora regulator substance group and the antithrombotic component substance group reached 10 and 12 respectively (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 the water extraction preparations A and B of Buyang Huanwu Decoction, and more than 3 times that of the steam distillation extraction preparation C of Buyang Huanwu Decoction. The component analysis results of the decocted preparation D of Buyang Huanwu Decoction are shown in Table 2, where the content indicators include peak area and relative content (%, content in the extract). Since under the given extraction conditions, the product obtained from a unit dosage of the medicinal materials is a fixed value, it can also be converted to absolute content.

[0071] Table 2 Composition list of the decocted preparation D of Buyang Huanwu Decoction

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] Example 3 Comparison of the Antithrombotic Effects of Different Buyang Huanwu Decoction Preparations

[0078] As an exemplary demonstration, in this example, the anticoagulant and antithrombotic effects of different Buyang Huanwu Decoction preparations prepared in Example 2 were investigated according to the same dosing standard. Among them, the gavage doses of different preparations were converted into the doses of crude drugs. Different Buyang Huanwu Decoction preparations were made into gastric lavage solutions by adding water at a concentration of 4 g of crude drug / mL, and were administered by gavage at a dosing dose of 20 g of crude drug / kg body weight / day.

[0079] SPF-grade male SD rats, weighing 200 - 220 g, were randomly divided into a sham operation group (the sham operation group performed the operation but did not undergo modeling), a model group, a water extraction preparation A group of Buyang Huanwu Decoction, an ethanol extraction preparation B group of Buyang Huanwu Decoction, a steam distillation extraction preparation C group of Buyang Huanwu Decoction, and a decocted separately preparation D group of Buyang Huanwu Decoction, with 8 rats in each group. The sham operation group and the model group were gavaged with distilled water; the dosing groups were gavaged with the corresponding gastric lavage solutions, and the dosing duration was 28 d. 0.5 h after the last dose, a 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 Active Parts on TXA 2 、PGI 2 and Antiplatelet Research". Hunan University of Chinese Medicine, 2006), the thrombus weight was measured, and blood was collected. The plasma TXB 2 content was measured by radioimmunoassay; the plasma antithrombin III (AT-III) activity was measured by chromogenic substrate method.

[0080] The data were expressed as mean ± standard deviation, and the comparison between two groups was performed using t test. The results are shown in Table 3.

[0081] Table 3 Determination Results of Thrombus Indexes and Coagulation Parameters in Each Group

[0082]

[0083] The experimental results showed that thrombosis occurred in the model group, and TXB 2 increased, 6-keto-PGF 1α and AT-III activity decreased. The thrombus mass and TXB 2 in preparation D group were significantly lower than those in the model group and other preparation groups ( p <0.01); the 6-keto-PGF 1α and AT-III activity in preparation D group were significantly higher than those in 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 substance clusters corresponding to Buyang Huanwu Decoction with different extraction methods were sorted according to the content index; 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 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, the anti-thrombotic Chinese medicine preparation is a Buyang Huanwu Decoction preparation, the Buyang Huanwu Decoction preparation 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 a water-decoction 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; when preparing the water-decoction alcohol precipitation extract, the solid-liquid ratio is 1: (8-12); the decoction times are 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%; 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 is 1-3 hours; 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.

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

6. 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.

7. 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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