CN114699437A

CN114699437A - Oral preparation containing herba Erigerontis extract and its preparation method

Info

Publication number: CN114699437A
Application number: CN202111612015.XA
Authority: CN
Inventors: 林艳和; 蒋建东; 王璐璐
Original assignee: Yunnan Biovalley Pharmaceutical Co ltd; Institute of Medicinal Biotechnology of CAMS
Current assignee: Yunnan Biovalley Pharmaceutical Co ltd; Institute of Medicinal Biotechnology of CAMS
Priority date: 2020-12-29
Filing date: 2021-12-27
Publication date: 2022-07-05
Anticipated expiration: 2041-12-27
Also published as: CN114699437B; WO2022143514A1; CN114748518B; CN114748518A; WO2022143513A1

Abstract

The invention relates to an oral preparation containing erigeron breviscapus extract and a preparation method and application thereof, wherein the preparation method comprises common oral dosage forms such as capsules, tablets, oral liquid, dripping pills or granules, and the like.

Description

Oral preparation containing herba Erigerontis extract and its preparation method

Technical Field

In particular to a preparation method and application of breviscapine and caffeic acid ester extracts or salts thereof, in particular to the application in preparing medicaments for treating metabolic related diseases such as hyperlipidemia, hypertension, diabetes, obesity, non-alcoholic fatty liver disease and the like and cardiovascular and cerebrovascular diseases, and the application in preparing medicaments for treating platelet aggregation and blood coagulation dysfunction caused by the metabolic related diseases.

Background

In recent years, the disease spectrum has been changed significantly with the accelerating aging of the population, the increasing urbanization degree, and the gradual change of the lifestyle. Metabolic-related diseases such as hypertension, hyperlipidemia, obesity, diabetes and the like are widely prevalent, and the incidence of cardiovascular and cerebrovascular diseases caused by the diseases is also on an accelerated rising trend.

Erigeron breviscapus (Vant.) Hand-Mazz (dried whole plant of Erigeron breviscapus (Vant.) Hand) of Erigeron of Compositae) has pungent, slightly bitter and warm nature, has the effects of dispelling cold, relieving exterior syndrome, dispelling pathogenic wind, removing dampness, promoting blood circulation, removing blood stasis, dredging collaterals and relieving pain, and can be used for treating common cold, headache, rheumatalgia, paralysis caused by cerebrovascular accident, etc. (the Chinese herbal medicine assembly). Herba Erigerontis can be used as single ingredient, and the existing marketed varieties include herba Erigerontis injection, herba Erigerontis capsule, herba Erigerontis soft capsule, herba Erigerontis dripping pill, YIMAIKANG, herba Erigerontis granule, etc.; the effective components of erigeron breviscapus are flavonoid breviscapine and caffeic acid ester, while the main emphasis of the existing product is scutellarin as the main component.

The breviscapine product includes breviscapine injection, breviscapine for injection, breviscapine tablet, breviscapine dripping pill, etc. Even the erigeron breviscapus extract products such as erigeron breviscapus capsules, Yimaikang and the like do not make provisions on the content of caffeic acid ester and do not refer to enrichment. The erigeron breviscapus injection is the only product for enriching caffeic acid ester components from the technical aspect. But the ratio of the caffeic acid ester content to the flavonoid content is only in the range of 1:1.2-1: 5.5. Through research on caffeic acid ester substances in erigeron breviscapus plants, the caffeic acid ester components in erigeron breviscapus are found to be rich, and comprise mono-caffeic acid ester components and di-caffeic acid ester components. The plant contains caffeic acid ester, such as plants of Compositae, Caprifoliaceae, Eucommiaceae, Dipsacaceae, Rubiaceae, and Convolvulaceae. At present, different from other plants containing caffeoylquinic acid esters, the plants in the Compositae also contain caffeoyloctanoic acid compounds, namely, mono-caffeoyl, di-caffeoyl and tri-caffeoyloctanoic acid compounds. The caffeoyloctanoic acid sugar acid component contained in herba Erigerontis is dicaffeoyloctanoic acid sugar acid material including erigeron ester B and erigeron breviscapus. Erigeron ester B is isolated from erigeron plant of Compositae, and erigeron breviscapus is only obtained from erigeron breviscapus. Jiianping Zhao, et al (J.Nat.Prod.2014,77,509-515) report that six caffeoyloctanoic acids obtained from the chamomile plant of the genus chamomilla have anti-inflammatory and anti-metabolic disorder effects, so that the content range of the dicaffeonate compounds of the special type is separately listed, and the caffeic acid ester components obtained from the erigeron breviscapus can be clearly classified and controlled, so that the content of the active ingredients of the erigeron breviscapus is easier to control, and the quality of the products is more controllable. The erigeron breviscapus also contains a hepatotoxic substance gamma-pyrone component, the representative compound is pyromeconic acid, the method for removing pyromeconic acid in the prior patent adopts a column chromatography method, the utilized property is that pyromeconic acid has higher polarity, and the pyromeconic acid can be removed by a water washing method in the column chromatography, but an extraction solvent is required to be low-concentration alcohol or water extraction, otherwise, impurities such as chlorophyll and the like are easy to solidify on an adsorption column, so that the elution cannot be realized, or the regeneration of the column is difficult. The pH value of the acidified supernatant is adjusted to be neutral, microfiltration is carried out to remove insoluble macromolecular impurities including most chlorophyll, protein, tannin and the like, and then the supernatant is concentrated by a nanofiltration membrane, wherein the nanofiltration membrane can filter small molecular substances and water, pyromeconic acid can be dissolved in water, and the molecular weight is only 112Da, so 90% of pyromeconic acid can be removed by the method. The concentrated solution after pyromeconic acid is purified through column separation to obtain purified liquid. And can also adopt 50-80% ethanol for extraction in the first step, so as to increase the extraction rate. The extracted breviscapine and caffeic acid ester can be directly dried to obtain the medicinal composition, or the breviscapine and caffeic acid ester components can be adjusted to pH neutral, salified and spray dried to increase water solubility and medicinal property of the two components.

Disclosure of Invention

In view of the above technical problems, the present invention provides an oral preparation containing an erigeron breviscapus extract, wherein the oral preparation contains caffeic acid ester or a salt thereof, breviscapine or a salt thereof and pharmaceutically acceptable excipients, and the weight ratio of the caffeic acid ester or the salt thereof to the breviscapine or the salt thereof is 1: 1-30: 1.

The conventional erigeron breviscapus oral preparation is limited by an extraction method, and the preparation is usually a mixture of caffeic acid ester, breviscapine and other substances and cannot be accurately mixed. The invention adopts a unique preparation method to respectively extract the main components, particularly to prepare and accurately match two main effective component salts, thereby effectively improving the drug effect.

Preferably, the invention further provides an oral preparation containing the erigeron breviscapus extract, wherein the caffeic acid ester or the salt thereof comprises dicaffeate or the salt thereof and monocffeate or the salt thereof, the weight ratio of the dicaffeate or the salt thereof to the breviscapine or the salt thereof is 1.25: 1-6: 1, (preferably 1.25: 1-5.5: 1, more preferably 1.5:1-5:1, most preferably 2:1-4.5:1), and the weight ratio of the monocffeate or the salt thereof to the dicaffeate or the salt thereof is 1:1.2-1: 6.2.

Preferably, the present invention further provides an oral formulation comprising an extract of erigeron breviscapus, wherein the dicaffeonate or a salt thereof comprises 1, 3-O-dicaffeoylquinic acid or a salt thereof, 3, 4-O-dicaffeoylquinic acid or a salt thereof, 3, 5-O-dicaffeoylquinic acid or a salt thereof, 4, 5-O-dicaffeoylquinic acid or a salt thereof, erigeron breviscapus or a salt thereof; the monocaffeic acid ester or its salt includes 3-O-caffeoylquinic acid or its salt, 4-O-caffeoylquinic acid or its salt, 5-O-caffeoylquinic acid or its salt, and breviscapine or its salt.

Preferably, the invention further provides an oral preparation containing erigeron breviscapus extract, wherein 1, 3-O-dicaffeoylquinic acid or a salt thereof, 3, 4-O-dicaffeoylquinic acid or a salt thereof, 3, 5-O-dicaffeoylquinic acid or a salt thereof, 4, 5-O-dicaffeoylquinic acid or a salt thereof in the dicaffeoylquinic acid ester or a salt thereof are isomers; the erigeron breviscapus ester B or salt thereof and the erigeron breviscapus or salt thereof are isomers, namely dicaffeoyloctanone sugar acid or the like, wherein the ratio of the simple substituted dicaffeonate or salt thereof to the dicaffeoyloctanone sugar acid or salt thereof is 2: 1-0.9: 1.

The oral preparation is preferably selected from hard capsule, soft capsule, tablet, granule, oral liquid, dripping pill, watered pill, powder, and pill.

The breviscapine or its salt in the oral preparation comprises scutellarin or its salt, scutellarin salt, and flavone compounds or its salt related to scutellarin or its salt separated from erigeron breviscapus.

The caffeic acid ester or its salt and breviscapine or its salt in the preparation account for 80-99% of the preparation by weight, and the balance is adjuvant. The oral preparation is preferably a capsule, tablet, such as hard capsule.

Further, the dicaffeonate and the breviscapine salt are sodium salt or potassium salt or other medicinal salts dissolved in water; preferably sodium salt, and the weight ratio of the sodium salt is 1: 1-6: 1. I.e. the other product should also be a sodium or potassium salt or other pharmaceutically acceptable and water soluble salt, and likewise the other product should preferably be a sodium salt.

The invention further provides a preparation method of the oral preparation, which comprises the following steps:

extracting herba Erigerontis with 10-90% aqueous ethanol, and concentrating the extractive solution under reduced pressure to obtain extract; dissolving the extract in water, adjusting the pH value to 7-9, filtering, adjusting the pH value to 1-3 with acid, standing overnight, filtering, collecting filtrate and precipitate, refining the precipitate with water and ethanol, and drying to obtain breviscapine; adding alkali to adjust the pH value to 7-8 after precipitation and refining, and performing spray drying to obtain breviscapine salt; adjusting the pH value of the acidified filtrate to 7-9, clarifying with a ceramic microfiltration membrane, concentrating the clarified liquid obtained after clarification with an organic nanofiltration membrane, passing the concentrated liquid through a polyamide chromatographic column, eluting with water, eluting with 50-80% ethanol, collecting ethanol eluate, concentrating, and drying to obtain caffeic acid ester; and (3) concentrating the ethanol eluent, adjusting the pH value to 7-9, filtering, and spray-drying the filtrate to obtain the caffeic acid ester salt. The dicaffeonate or a salt thereof and the breviscapine or a salt thereof are mixed according to the proportion of 1: 1-6: 1 (preferably 1.25: 1-5.5: 1, more preferably 1.5:1-5:1, most preferably 2:1-4.5:1), and proper auxiliary materials are added to prepare tablets, capsules, soft capsules, dripping pills, granules and oral liquid preparations.

In the preparation method of the oral preparation, the alkali is used for adjusting the pH value of the solution to be NaOH and Na₂CO₃,NaHCO₃,KOH,K₂CO₃Or KHCO₃Or other alkali solutions useful for adjusting pH; the pH value of the acid adjusting solution is HCl and H₂SO₄Or H₃PO₄Or other acid solutions that may be used to adjust the pH. The pH value of the alkali adjusting solution is preferably NaOH or Na₂CO₃,NaHCO₃、KOH、K₂CO₃Or KHCO₃(ii) a The pH value of the acid adjusting solution is preferably HCl or H₂SO₄Or H₃PO₄。

The concentration of ethanol eluted by the polyamide chromatographic column is 50-95%.

The invention provides the application of the medicinal composition in preparing medicaments for treating metabolic related diseases such as hypertension, hyperlipidemia, diabetes, obesity, non-alcoholic fatty liver disease and the like and cardiovascular and cerebrovascular diseases, and has the more creative effect that the medicinal composition obtained by screening has the functions of regulating metabolic related diseases and platelet aggregation and blood coagulation dysfunction caused by the metabolic related diseases and obtains an unexpected effect more pleasantly, namely, compared with aspirin and clopidogrel, the composition of the invention has the equivalent anti-platelet aggregation effect as the positive drug and also has better anticoagulation effect, the pharmaceutical and aspirin clopidogrel compound has medical and pharmaceutical advantages which are not possessed by aspirin and clopidogrel, and more importantly, the composition does not cause bleeding symptoms when being taken in a range exceeding the medical advice range, and is vital to preventing atherosclerosis and cardiovascular and cerebrovascular diseases caused by atherosclerosis.

The single-component oral preparation of erigeron breviscapus currently available in the market does not have the step of removing hepatotoxic component pyromeconic acid, nor the step or process of refining caffeic acid ester or its salt components, especially dicaffeic acid ester or its salt components. For example, the existing process of erigeron breviscapus capsules comprises the steps of taking 2000g of erigeron breviscapus, heating and refluxing the erigeron breviscapus with 80% ethanol for two times for 1.5 hours and 1 hour for the second time, combining extracting solutions, adding 640g of active carbon, boiling, filtering, concentrating the filtrate under reduced pressure to be thick paste, adding a proper amount of starch, drying under reduced pressure, crushing, sieving, coating with 3% polyvinyl alcohol solution, adding 0.5g of magnesium stearate, mixing uniformly, and filling into capsules to prepare 1000 capsules.

The document reports that the caffeoylquinic acid ester compounds have the effects of resisting oxidation, inflammation, bacteria and virus, reducing blood sugar, blood fat, regulating immunity and the like, and are active ingredients. Therefore, the refining of the active substances and the quantitative regulation of the mixture ratio of the two components are necessary work for better serving the erigeron breviscapus product for clinic. The breviscapine and the caffeic acid ester substances have poor water solubility and fat solubility, so that the bioavailability is low and the drugability is weak. The invention adopts alkali dissolution and acid precipitation from the preparation method, firstly, the flavonoid ingredient breviscapine is precipitated and further refined; and simultaneously, the acidified supernatant part is kept and is another type of active component caffeic acid ester, macromolecular insoluble substances are removed by microfiltration through a membrane separation technology, most pyromeconic acid is removed by concentration of a nanofiltration membrane, column chromatography is carried out, the component of the dicaffeonate is enriched, and the pyromeconic acid with hepatotoxicity is removed again. More than 99 percent of pyromeconic acid can be removed after nanofiltration membrane and column chromatography.

This result was confirmed by experiments:

TABLE 1 table of the content change of the components after adjusting pH of the acidified supernatant of erigeron breviscapus and membrane filtration

As can be seen from Table 1, after adjusting pH of the acidified supernatant of erigeron breviscapus to be close to neutral, the effective substance dicaffeonate is better retained after passing through a clarifying membrane and a nanofiltration concentrating membrane, and the removal rate of the harmful substance pyromeconic acid reaches 84%. After column chromatography, the dicaffeonate content is better retained, further pyromeconic acid is removed.

The microfiltration clarifying membrane is preferably 100nm or 200nm, and the nanofiltration concentrating membrane is preferably 300D-400D.

After the flavonoid component and the caffeic acid ester component are salified, an in vitro dissolution experiment proves that the dissolution is higher than that of a prototype, so that the drug property is better than that of the prototype. The dissolution curves of erigeron breviscapus tablets and erigeron breviscapus sodium salt tablets in different dissolution media are evaluated. Research results show that the dissolution rates of the two tablets in 0.1MHCl are lower, the tablets reach saturation within 5min, and the cumulative dissolution percentage is less than 15%; in an acetate buffer solution dissolution medium with the pH value of 4.5, the cumulative release amount of the two tablets is basically saturated within 30min, and the cumulative dissolution percentage of the erigeron breviscapus sodium salt tablets is higher than that of the original tablets and is less than 60 percent; in a phosphate buffer solution dissolution medium with the pH value of 6.8, when the rotating speed is 50rpm, the sodium salt tablets of the erigeron breviscapus are completely dissolved out in 15min, and the cumulative dissolution percentage of the erigeron breviscapus is more than 85% when the erigeron breviscapus needs 45 min; the cumulative dissolution percentage of the sodium salt tablet of erigeron breviscapus is greater than that of the erigeron breviscapus tablet at 30 min.

A nonalcoholic steatohepatitis (NASH) model is constructed by feeding C57 mice with MCD (Methionine and Choline Deficient L-Amino Acid Diet) Methionine and Choline-Deficient feed, and the treatment effect of the existing erigeron breviscapus capsule and the erigeron breviscapus capsule prepared by the invention on NASH is explored.

1. Test reagent

MCD feed, cat # a02082002BR, manufacturer: research Diets (USA)

2. Procedure of the test

(1) Animal grouping and treatment 20C 57BL/6J male mice were randomized into 4 groups of 5 mice each after 1 week of acclimation. The control group is normal maintenance feed, the model group is fed with MCD feed, the existing herba Erigerontis capsule powder group and the inventive herba Erigerontis capsule powder group (formula shown in example 6), and the oral liquid is administered by intragastric administration (100mg/kg body weight) 1 time per day. The experimental animals had free access to food and water for 4 weeks.

(2) And measuring the weight and the liver weight of the mice of each group, weighing the weight and the liver wet weight of the mice of each group, and calculating the liver index.

3. Test results

(1) Effects on mouse body weight, liver weight and liver index

After the mice are raised for 4 weeks, the body weight, the liver weight and the liver index of the mice in the model group, the existing erigeron breviscapus capsule powder group and the erigeron breviscapus capsule powder group are all obviously reduced compared with those in the control group, which indicates that the tested medicament can not effectively improve the reduction of the MCD diet-induced body weight and the liver weight. In addition, there was no significant difference between the 2 tested drugs and the model group in body weight, liver weight and liver index, as shown in table 2.

TABLE 2 Effect of erigeron breviscapus capsule powder on mouse body weight, liver weight and liver index

(2) Influence on liver function index of mouse

Compared with the control group, the levels of glutamic-pyruvic transaminase (AST) and glutamic-oxalacetic transaminase (ALT) of the mice in the model group are obviously increased (^###P is less than 0.001). The mouse serum AST and ALT levels of the processed erigeron breviscapus capsule powder group are higher than those of the control group, but compared with the model group, the processed erigeron breviscapus capsule powder obviously reduces the mouse serum AST and ALT rise induced by MCD diet; herba Erigerontis group (such as Yunan Hao Pont for pharmaceutical) in the prior art can significantly reduce ALT level, has a tendency of reducing AST, but has no statistical significance, and is shown in Table 3.

TABLE 3 Effect of erigeron breviscapus capsule powder and the existing erigeron breviscapus pulse-activating capsule powder on NASH mouse liver function index

Group of	Glutamic-pyruvic transaminase (U/L)	Glutamic-oxalacetic transaminase (U/L)
			Control group	37.2±6.57	103.2±22.9
Model set	160.8±49.2^###	181.2±34.1^###
			Technological erigeron breviscapus group of the invention	101.6±28.2*^#	132.1±22.1*
Erigeron breviscapus group in prior art	100.1±22.4*^#	162.2±21.6^##

^###P<0.005vs control group;^##P<0.01vs control group;^#P<0.05vs control group;^*P<0.05vs model set

4. Analysis and conclusions

(1) Effect on body weight MCD diet induced weight loss in NASH mice is a disease hallmark of this dietary model. The research finds that the erigeron breviscapus capsule powder prepared by the invention and the erigeron breviscapus capsule powder prepared by the prior art can not inhibit MCD diet-induced weight loss, which is probably related to the MCD diet structure.

(2) And the serum ALT and AST levels of mice fed on MCD diet are obviously increased, which indicates that the establishment of the NASH model induced by MCD diet is successful. The research shows that the erigeron breviscapus capsule powder prepared by the invention process and the erigeron breviscapus capsule powder prepared by the prior art can improve MCD diet-induced liver function injury, the erigeron breviscapus capsule powder prepared by the invention process has a down-regulation effect on ALT and AST, and although the erigeron breviscapus capsule powder prepared by the invention process is found in the prior art and may have a protection effect on the liver, the experiment shows that the erigeron breviscapus prepared by the invention process has a better liver protection effect after oral administration (100mg/Kg) compared with the erigeron breviscapus prepared by the prior art.

The existing erigeron breviscapus capsule powder is prepared by breviscapinum salt and caffeic acid ester salt according to the proportion in the scope of claims.

Description of the drawings:

FIG. 1 weight change of golden hamster;

FIG. 2 shows the total cholesterol level in the plasma of golden hamster;

FIG. 3 shows the plasma low density lipoprotein-cholesterol (LDL-c) content of golden hamster;

FIG. 4 plasma Triglyceride (TG) levels in golden hamster;

FIG. 5 plasma ALT levels in golden hamster;

FIG. 6 shows the plasma aspartate Aminotransferase (AST) content of golden yellow hamster;

FIG. 7 golden mouse liver index (%);

FIG. 8 fat index (%) of golden hamster;

FIG. 9 shows the hepatic triglyceride content of golden hamster;

fig. 10 platelet aggregation rate (%);

FIG. 11 activated partial thromboplastin times;

FIG. 12 prothrombin time;

FIG. 13 plasma thrombin time;

figure 14 plasma fibrinogen levels.

The experiment aims at researching the curative effects of the main components of the asarum herb in different proportions on metabolic related diseases caused by high-fat diet and platelet aggregation and blood coagulation dysfunction, and provides experimental data support for new clinical application of the medicine.

The experiment shows that the novel process erigeron breviscapus capsule can effectively reduce ALT and AST in a non-alcoholic steatohepatitis model, and then the existing process (Yunnan Hao Pont pharmaceutical Co., Ltd.) is further used for comparing with the process example 6 of the product, and the dicaffeonate and the breviscapine salt in the example are calculated and accurately proportioned at the same time of comparison, so that the optimal proportioning of the dicaffeonate and the breviscapine salt is achieved. The study adopts aspirin and clopidogrel as positive control drugs, and the following experiments only show aspirin determination results because the detection results of the aspirin and the clopidogrel have no significant difference in each index.

2) The experimental method comprises the following steps:

2.1) experimental animals: LVG Syrian golden yellow hamster, male, 6 weeks old, weight 90-110g, acclimatized in a breeding environment for 2 weeks. Animals were purchased from Experimental animals technology, Inc. of Weitonglihua, Beijing.

2.2) experimental feed: basal diet (Normal chow diet, NC group); high fat diet (High fat diet model group, HFD group: 1.0% cholesterol, 0.2% sodium cholate, 10.0% lard, 5.0% egg yolk powder and 83.8% basal diet). The feed was purchased from Beijing Olympic feed Co.

2.3) administration mode: administering the drug-doped feed based on high fat feed for 8 weeks, wherein the drug content in the herba Erigerontis-containing feed is 4g/kg, and the administration amount is 200mg/kg body weight/day; the feed containing aspirin contains 0.4g/kg of medicine, and the intake of the feed is equivalent to the administration amount of 20mg/kg of body weight/day. The same lot of golden hamster was fed with basal diet as a normal control group (NC group).

2.4) Experimental grouping and administration

TABLE 4 Experimental groups and dosing

The erigeron breviscapus is prepared by the prior process; erigeron breviscapus group A-F is prepared by adopting the process of the invention.

2.5) detection indexes and methods:

a. body weight determination

Animal body weights were recorded once a week during the experiment.

b. Biochemical detection of blood

After the experiment is finished, the animals are fasted for 12h, orbital venous plexus blood (0.5ml) is collected in an anticoagulation tube, the centrifugation is carried out for 10min at 3500rpm and 4 ℃, and 100 mu L of supernatant is taken and stored in a refrigerator at minus 80 ℃. Then, the levels of total Cholesterol (CHO), low density lipoprotein-cholesterol (LDL-C), Triglyceride (TG), alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in the plasma are detected by a full-automatic biochemical analyzer. The detection kit is purchased from Zhongsheng Beizhong Biotechnology limited.

c. Liver dampness-weight ratio and epididymis fat dampness-weight ratio

After the experiment is finished, taking the liver tissues and the epididymis fat of the animals, weighing, and respectively calculating the weight percentage of the liver wet weight and the epididymis fat weight of each animal to the weight percentage.

d. Liver tissue lipid content determination

After the experiment, 20mg of liver tissue was weighed for each hamster, and the weight (g): adding 20 volumes of T-PER (protease phosphatase inhibitor containing 1/100) at a volume (ml) ratio of 1:20, mechanically homogenizing under ice-water bath conditions (42HZ, 4-6 min), centrifuging at 2500rpm for 10min, taking 2.5 microliter of supernatant liquid, diluting by 5 times to prepare a sample to be detected, and detecting according to TG detection kit instructions. The remaining liver tissue homogenate was centrifuged at 12000rpm at 4 ℃ for 30min, and the supernatant was aspirated for protein quantification by BCA method. The results were further calculated according to the following formula.

e. Platelet aggregation and coagulation index determination

After the experiment is finished, 5% chloral hydrate (1ml/100g) is anesthetized, a syringe is used for sampling blood by heart, anticoagulant is added into the blood sample according to the proportion of 1:9 of sodium citrate (4%) solution and blood, the blood sample is centrifuged for 10min at 200g to obtain platelet-rich plasma (PRP), and the residual plasma is centrifuged for 10min at 800g to obtain platelet-poor plasma (PPP). In the platelet maximum aggregation rate detection experiment, PPP is used for calibration, PRP is pre-warmed at 37 ℃ for 180 seconds and then placed in a detection tank, 10ul adenosine diphosphate (ADP, 10nM) is added, and quick click is started, and the platelet Maximum Aggregation Rate (MAR) value within 300s is read. In the four detection experiments of blood coagulation, PPP37 ℃ is pre-heated for 180s and then placed in a detection tank, quantitative Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) Fibrinogen (FIB) detection reagents are respectively added according to the requirements of the specification for quick click to start, and the blood coagulation time and the fibrinogen content are read.

2.6) data analysis

All data were entered into an Excel document and expressed in Mean ± SEM. Statistical data analysis Graphpad Prism 8.0 software, single or two-factor analysis of variance comparison method was used, with P <0.05 as the criterion for significant difference.

3) Results of in vivo experiments

3.1) weight Change

Animal body weights were measured once a week during dosing and the results are shown in figure 1. The animals in the high fat diet model group (HFD) had significantly increased body weight compared to the normal diet control group (NC). The antiplatelet drug aspirin has no effect of reducing weight gain caused by high-fat diet. The control drug (Yunan Hao bang, the existing technology) and herba Erigerontis (the technology of the invention) can reduce the weight increase caused by high fat diet; the weight reducing effect of each group of erigeron breviscapus (the process of the invention) is better than that of the erigeron breviscapus (Yunnan Hao Bob, the prior process) of a control; wherein the content of dicaffeonate is as follows: the ratio of breviscapine salt to breviscapine salt is 3:1 and 4.5:1, and the effect is optimal.

3.2) Biochemical detection of blood

a. The results of plasma total cholesterol levels are shown in figure 2 and table 5.

TABLE 5 Total Cholesterol level in plasma

The results show that the plasma total cholesterol content of the animals in the high-fat diet model is obviously higher than that of the healthy control group (P < 0.001); aspirin and control erigeron breviscapus (Yunnan Hao Pont, existing technology) have no effect of lowering total cholesterol in blood plasma; the experimental erigeron breviscapus (the process of the invention) has the tendency of reducing the total cholesterol in blood plasma, but has no significant difference compared with the HFD group.

b. The results of plasma low density lipoprotein-cholesterol (LDL-c) content are shown in FIG. 3 and Table 6.

TABLE 6 plasma Low Density lipoprotein-Cholesterol (LDL-c) levels

The results show that the plasma low density lipoprotein-cholesterol content of the animals in the high fat diet model is obviously increased compared with that of the healthy control group (P < 0.001); aspirin and herba Erigerontis (conventional process) have no effect of lowering low density lipoprotein-cholesterol in blood plasma; experimental Breviscapine (inventive process) has effects of reducing low density lipoprotein-cholesterol of blood plasma, wherein dicaffeonate: the two proportioning groups of 4:1 and 4.5:1 of breviscapine salt have obvious LDL-c reducing effect (P is 0.01), and the proportioning groups of 3:1(P is 0.085) and 6:1(P is 0.071) also have certain plasma low density lipoprotein-cholesterol reducing effect.

c. The results of plasma Triglyceride (TG) content are shown in fig. 4 and table 7.

TABLE 7 plasma Triglyceride (TG) content

The results showed that plasma Triglyceride (TG) levels in animals of the high fat diet model were significantly elevated compared to healthy controls (. about.. P < 0.001); aspirin and herba Erigerontis (conventional process) have no effect in lowering triglyceride; experimental erigeron breviscapus (inventive process) dicaffeonate: the breviscapine salt 4.5:1 ratio group has obvious effect of reducing plasma triglyceride (P is less than 0.05), and the 3:1(P is 0.0548) ratio group also has certain effect of reducing plasma triglyceride.

d. The results of plasma glutamic pyruvic transaminase (ALT) levels are shown in figure 5 and table 8.

TABLE 8 plasma alanine Aminotransferase (ALT) levels

The results show that the plasma glutamic pyruvic transaminase (ALT) content of the high-fat diet model animals is obviously increased compared with that of a healthy control group (P < 0.05); aspirin and herba Erigerontis (conventional process) have no effect of lowering blood plasma glutamic-pyruvic transaminase; experimental erigeron breviscapus (inventive process) dicaffeonate: the breviscapine salt with the ratio of 3:1 and the ratio of 4.5:1 has the function of obviously reducing the ALT of the blood plasma (P is less than 0.05); the 4:1(P is 0.0573) proportioning group also has certain function of reducing the blood plasma glutamic pyruvic transaminase.

e. The results of plasma aspartate Aminotransferase (AST) levels are shown in figure 6 and table 9.

TABLE 9 plasma aspartate Aminotransferase (AST) content

The results show that the plasma glutamic-oxaloacetic transaminase (AST) content of the high-fat diet model animal is obviously increased compared with that of a healthy control group (P < 0.05); the effect of reducing plasma glutamic-oxalacetic transaminase of each experimental group is not achieved.

3.3) liver and fat index detection

a. The results of the liver index are shown in fig. 7 and table 10.

TABLE 10 liver index

The liver index refers to the ratio of the wet weight of the liver to the body weight. The results showed a significant increase in liver index in animals with high fat diet model compared to healthy controls (. about.p < 0.001); aspirin and herba Erigerontis (conventional process) have no effect of lowering liver index; experimental erigeron breviscapus (inventive process) dicaffeonate: the breviscapine salt with the proportion of 4.5:1 has certain effect of reducing liver index (P is 0.0529), and is suggested to have certain effect of preventing and treating nonalcoholic fatty liver disease caused by high-fat diet.

b. The results of the fat index are shown in FIG. 8 and Table 11

TABLE 11 fat index

The fat index is the ratio of wet weight to body weight of epididymis fat. The results showed a significant increase in the fat index of animals in the high fat diet model compared to the healthy control group (. P < 0.05); aspirin and herba Erigerontis (conventional process) have no effect of lowering fat index; experimental erigeron breviscapus (inventive process) dicaffeonate: the breviscapine salt with the proportion of 4.5:1 has certain effect of reducing the fat index.

3.4) detection result of lipid content in liver tissue

The liver lipid content is expressed as liver triglycerides, and the results are shown in fig. 9 and table 12.

TABLE 12 hepatic triglyceride content

The results show that the liver tissue TG content of the animals with high fat diet model is obviously increased compared with the healthy control group (P < 0.001); aspirin and erigeron breviscapus (existing process) have no effect of reducing liver tissue TG; the experimental erigeron breviscapus (the process of the invention) can obviously reduce the TG of the liver tissue, wherein the dicaffeonate salt: the action of the breviscapine salt is most obvious in the two groups of 3:1 and 4.5:1(P is less than 0.01), and the action of the breviscapine salt can also be used for remarkably reducing the TG of the liver tissue (P is less than 0.05) in the groups of 1.25:1, 2:1, 4:1 and 6:1, so that the effect of preventing and treating the non-alcoholic fatty liver disease is better.

3.5) results of platelet aggregation and blood coagulation assays

a. The platelet aggregation results are shown in fig. 10 and table 13.

TABLE 13 platelet aggregation Rate

The increase of the platelet aggregation rate is easy to form thrombus and block blood vessels, is also one of the factors of the attack of coronary heart disease, and can cause the formation of atherosclerosis. The results show that the platelet aggregation rate of the animals in the high fat diet model is obviously higher than that of the healthy control group (P < 0.001); aspirin significantly reduced platelet aggregation induced by high-fat diet (× P < 0.001); the platelet aggregation caused by high fat diet can be obviously reduced by erigeron breviscapus (the prior art) of a control group and erigeron breviscapus (the invention process) of each experimental group in each proportion, wherein the dicaffeonate salt: the breviscapine salt 3:1 and 4.5:1 had the most significant effects (P <0.01) in the two groups, and the groups 1.25:1, 2:1, 4:1 and 6:1 also had the significant effect of reducing platelet aggregation (P <0.05) caused by high fat diet, and the effects were all superior to those of control erigeron breviscapus (existing process).

b. The Activated Partial Thromboplastin Time (APTT) results are shown in FIG. 11 and Table 14.

TABLE 14 activated partial thromboplastin time

The Activated Partial Thromboplastin Time (APTT) is a blood coagulation function inspection index reflecting the comprehensive activity of intrinsic blood coagulation pathways, particularly blood coagulation factors in the first stage, and the time shortening can be seen in a hypercoagulable state, thromboembolic diseases and the like. The results show that the APTT of the animals with the high fat diet model is obviously reduced compared with the healthy control group (P < 0.05); aspirin and herba Erigerontis (conventional process) have no effect of increasing APTT; experimental group erigeron breviscapus (inventive process) dicaffeonate: the breviscapine salt 4:1(P ═ 0.052) and 4.5:1(P ═ 0.089) have certain APTT increasing effect, and the possibility of preventing thrombotic diseases caused by high fat diet is suggested.

c. The Prothrombin Time (PT) results are shown in figure 12 and table 15.

TABLE 15 Prothrombin Time (PT)

Prothrombin time is an index reflecting the activity of clotting factors I, II, V, VII, X in plasma. Low prothrombin time is common in hypercoagulable blood conditions, such as in myocardial infarction or cerebral thrombosis. The results showed that animals with high fat diet model had significantly reduced PT compared to healthy control group (. about.p < 0.001); aspirin and herba Erigerontis (in the prior art) have no effect of increasing PT; experimental group erigeron breviscapus (inventive process) dicaffeonate: the breviscapine salt 4.5:1 ratio group can remarkably increase the PT reduction (P <0.05) caused by high-fat diet, and is suggested to be possible to prevent thrombotic diseases caused by high-fat diet.

d. The Thrombin Time (TT) results are shown in fig. 13 and table 16.

TABLE 16 Thrombin time

The plasma thrombin time refers to the time required for the conversion of plasma fibrinogen to fibrin, after the addition of "standardised" thrombin to the plasma being tested, which is low, may be related to hyperlipidemia in the patient's own blood and may also be the result of thromboembolic disease. The results showed that animals with high fat diet model had significantly reduced TT compared to healthy control group (. about.p < 0.001); aspirin and herba Erigerontis (conventional process) have no PT increasing effect; experimental group erigeron breviscapus (inventive process) dicaffeonate: the breviscapine salt 4.5:1 ratio group can remarkably increase TT reduction (P <0.05) caused by high fat diet, and is suggested to be possible to prevent thrombotic diseases caused by high fat diet.

d. The Fibrinogen (FIB) results are shown in fig. 14 and table 17.

TABLE 17 fibrinogen

Fibrinogen (FIB) is a protein having a blood coagulation function, which is synthesized mainly by hepatocytes, and is the blood coagulation factor with the highest content in plasma. Clinical data show that: in patients with elevated fibrinogen levels, myocardial infarction or sudden death can occur in most patients, and the higher the fibrinogen level, the greater the risk; elevated plasma fibrinogen is an important factor in the promotion of the onset of atherosclerosis; the fibrinogen is increased, the blood is in a high coagulation state, the blood flow speed is reduced, the blood viscosity is increased, and the thrombus is easy to generate; hypertension is often accompanied by elevated fibrinogen levels, which is a significant cause of induction and exacerbation of hypertension; fibrinogen has a close relationship with fat metabolism; in addition, aging, smoking, obesity, stress, diabetes, etc. can promote elevated fibrinogen levels in the body. The results showed that the plasma fibrinogen of the high fat diet model animals was significantly elevated compared to the healthy control group (. about.p < 0.001); aspirin and erigeron breviscapus (existing process) have no effect of reducing fibrinogen rise caused by high fat diet; experimental group erigeron breviscapus (inventive process) dicaffeonate: the breviscapine salt 4.5:1 ratio group can obviously reduce the fibrinogen rise (P <0.05) caused by high fat diet, and the dicaffeonate salt: the breviscapine salt 2:1(P ═ 0.064), 3:1(P ═ 0.058) and 6:1(P ═ 0.057) groups also have a certain plasma fibrinogen reducing effect.

4) Conclusion

4.1) the weight, the liver index, the fat index, the liver lipid, the blood fat and the liver enzyme level of golden hamster can be obviously increased after eight weeks of high fat diet molding, and the model is successful.

4.2) the antiplatelet drug aspirin has no weight gain effect caused by high-fat diet (the detection result of another positive drug clopidogrel is the same as that of aspirin); the prepared erigeron breviscapus can reduce the weight gain caused by high fat diet; the weight reducing effect is better than that of a control erigeron breviscapus (Haobang, a traditional process); wherein the content of dicaffeonate is as follows: the ratio of breviscapine salt to breviscapine salt is 3:1 and 4.5:1, and the effect is optimal.

4.3) the anti-platelet drug aspirin and the control erigeron breviscapus (Haobang, the existing technology) have no function of reducing blood fat, including total cholesterol, low-density lipoprotein-cholesterol and triglyceride of blood plasma (the detection result of another positive drug clopidogrel is the same as that of aspirin); the experimental erigeron breviscapus (the invention process) has the function of reducing blood fat, and the dicaffeonate salt: the ratio of the breviscapine salt to the breviscapine salt is 4.5:1, and the preparation is optimal.

4.4) aspirin and herba Erigerontis (Haobang, existing technology) have no effect of reducing liver enzyme in blood plasma (the detection result of another positive drug, clopidogrel, is the same as that of aspirin); the experimental erigeron breviscapus (the process of the invention) has the function of reducing glutamic-pyruvic transaminase, and the dicaffeonate salt: the ratio of the breviscapine salt to the breviscapine salt is 4.5:1, and the preparation is optimal.

4.5) aspirin and herba Erigerontis (Haobang, available in the art) have no effects of reducing liver index and fat index and liver tissue lipid accumulation (the detection result of another positive drug, clopidogrel, is the same as that of aspirin); the erigeron breviscapus (new process) has the function of adjusting the indexes, and the dicaffeonate salt: the ratio of the breviscapine salt to the breviscapine salt is 4.5:1, and the preparation is optimal.

4.6) the erigeron breviscapus (the invention process) can obviously improve platelet aggregation and blood coagulation dysfunction induced by high fat diet, including four indexes of platelet aggregation rate and blood coagulation; the effect is better than that of erigeron breviscapus (the prior art); wherein the content of dicaffeonate is as follows: the comprehensive effect of the breviscapine salt is optimal, wherein the ratio of the breviscapine salt to the breviscapine salt is 4.5: 1; aspirin is effective in reducing platelet aggregation but has no anticoagulant effect (clopidogrel, another positive drug, has the same test result as aspirin).

7. By combining the results, the experimental erigeron breviscapus (the process of the invention) has good effects of preventing and treating metabolic-related diseases such as hyperlipidemia, non-alcoholic fatty liver disease, obesity and the like, can effectively prevent and treat platelet aggregation and blood coagulation dysfunction caused by the metabolic-related diseases, and has the effects of preventing and treating cardiovascular and cerebrovascular diseases; the function is related to the preparation process and the proportion of the main components.

The existing erigeron breviscapus capsule powder is prepared by erigeron breviscapus salt and caffeic acid ester salt according to the proportion in the scope of the claims.

The specific implementation mode is as follows:

example 1: preparation of crude drug extract

Decocting herba Erigerontis 2000g with 50% aqueous ethanol twice, each for 2 hr, filtering, mixing filtrates, and concentrating under reduced pressure to obtain extract (relative density of 1.15-1.25, 75 deg.C); dissolving the extract with 5 times of water, adding 5% sodium hydroxide solution while stirring, adjusting the pH value to 7.5-8.5, filtering, adding 10% sulfuric acid solution to adjust the pH value to 1-3, standing overnight, filtering, collecting filtrate and precipitate, refining the precipitate with ethanol, adding 5% sodium hydroxide to adjust the pH value to 7-8, and spray-drying to obtain powder 1; adjusting the pH value of the acidified filtrate to 7-9, clarifying with a 100nm ceramic membrane, concentrating the clarified liquid with a 350D organic membrane, passing the concentrated liquid through a 30-60-mesh polyamide chromatographic column (the diameter and length ratio is 1:4), eluting with water for 3 column volumes, eluting with 65% ethanol for 4 column volumes, collecting ethanol eluate, adjusting the pH value to 7-9, and spray-drying to obtain powder 2; mixing the powder 1 and the powder 2 in proportion to obtain a mixture of breviscapine sodium salt powder and caffeic acid ester sodium salt powder in a ratio of 1:4, thus obtaining the raw material medicine composition components of the medicinal composition. Wherein the powder 1 contains scutellarin sodium salt 36.3% and 5.4 g. Powder 2 contained 30.9g of dicaffeonate 21.0% and 9.1g of monocffeonate. Wherein the amount of the simple substituted dicaffeonate is 19.0g, and the amount of the dicaffeoyloctanone gluconate is 11.9 g. 162g of the obtained total mixed erigeron breviscapus powder.

Example 2: preparation of crude drug extract

Decocting herba Erigerontis 2000g with 50% aqueous ethanol twice, each for 2 hr, filtering, mixing filtrates, and concentrating under reduced pressure to obtain extract (relative density of 1.15-1.25, 75 deg.C); dissolving the extract with 5 times of water, adding 5% sodium hydroxide solution while stirring, adjusting the pH to 7.5-8.5, filtering, adding 10% sulfuric acid solution to adjust the pH to 1-3, standing overnight, filtering, collecting filtrate and precipitate, refining the precipitate with ethanol, and drying under reduced pressure to obtain powder 1; adjusting the pH value of the acidified filtrate to 7-9, clarifying with a 100nm ceramic membrane, concentrating the clarified liquid with a 350D organic membrane, passing the concentrated liquid through a 30-60-mesh polyamide chromatographic column (the diameter and length ratio is 1:4), eluting with water for 3 column volumes, eluting with 65% ethanol for 4 column volumes, collecting ethanol eluate, concentrating, and drying under reduced pressure to obtain powder 2; mixing the powder 1 and the powder 2 in proportion to obtain a mixture of breviscapine powder and caffeic acid ester powder in a ratio of 1:4, thus obtaining the raw material medicine composition components of the medicinal composition. Wherein the powder 1 contains scutellarin 35.7% and 5.6 g. Powder 2 contained 21.5% dicaffeonate totaling 31.4g and 9.1g of monocaffeate. Wherein the amount of the simple substituted dicaffeonate is 18.5g, and the amount of the dicaffeoyloctanone sugar acid is 12.9 g. 161.7g of herba Erigerontis total mixed powder.

Example 3: preparation of crude drug extract

Decocting herba Erigerontis 3000g with 60% aqueous ethanol twice, each for 2 hr, filtering, mixing filtrates, and concentrating under reduced pressure to obtain extract (relative density of 1.15-1.25, 75 deg.C); dissolving the extract with 5 times of water, adding 5% sodium hydroxide solution while stirring, adjusting the pH value to 7-9, filtering, adding 10% hydrochloric acid solution to adjust the pH value to 1-3, standing overnight, filtering, collecting filtrate and precipitate, refining the precipitate with ethanol, adding 5% sodium hydroxide to adjust the pH value to 7-8, and spray-drying to obtain powder 1;

adjusting the pH value of the acidified filtrate to 7-9, clarifying with a 200nm ceramic membrane, concentrating the clarified liquid with a 400D organic membrane, passing the concentrated liquid through a 30-60-mesh polyamide chromatographic column (the diameter to length ratio is 1:4), eluting with 3 column volumes of water, eluting with 3 column volumes of 70% ethanol, collecting the ethanol eluate, adjusting the pH value to 7-9 after concentration, and spray-drying to obtain powder 2; obtaining about 18.7g of powder 1 and about 208.3g of powder 2, and mixing the powder 1 and the powder 2 according to the proportion to obtain a mixture of 1:3 of breviscapine sodium salt powder and caffeic acid ester sodium salt powder, thus obtaining the raw material medicine composition components of the medicinal composition.

Wherein the powder 1 contains scutellarin sodium salt 45% and 8.4g in total. Powder 2 contained 18.6% dicaffeonate salt and a total of 38.7g and 7.3g of monocffeonate salt. Wherein the amount of the simple substituted dicaffeonate is 18.3g, and the amount of the dicaffeoyloctanone sugar acid salt is 20.4 g.

Example 4: preparation of crude drug extract

Decocting herba Erigerontis 3000g with 65% aqueous ethanol twice, each for 2 hr, filtering, mixing filtrates, and concentrating under reduced pressure to obtain extract (relative density of 1.15-1.25, 75 deg.C); dissolving the extract with 5 times of water, adding 5% sodium hydroxide solution while stirring, adjusting the pH to 7-9, filtering, adding 10% hydrochloric acid solution to adjust the pH to 1-3, standing overnight, filtering, collecting filtrate and precipitate, refining the precipitate with ethanol, and reducing pressure to obtain powder 1; adjusting the pH value of the acidified filtrate to 7-9, clarifying with a 200nm ceramic membrane, concentrating the clarified liquid with a 400D organic membrane, passing the concentrated liquid through a 30-60-mesh polyamide chromatographic column (the diameter to length ratio is 1:4), eluting with 3 column volumes of water, eluting with 3 column volumes of 70% ethanol, collecting the ethanol eluate, adjusting the pH value to 7-9 after concentration, and spray-drying to obtain powder 2; obtaining about 25.3g of powder 1 and about 223g of powder 2, and mixing the powder 1 and the powder 2 according to the proportion to obtain a mixture of 1:3.5 of breviscapine and dicaffeonate sodium salt powder, thus obtaining the raw material medicine composition components of the medicinal composition.

Wherein the powder 1 contains scutellarin 32% and 8.1 g. Powder 2 contained 12.7% dicaffeonate salt (28.4 g in total) and 7.3g monocffeonate salt. Wherein the amount of the simple substituted dicaffeonate is 13.4g, and the amount of the dicaffeoyloctanone gluconate is 15 g.

Example 5: preparation of crude drug extract

Adding 2500g of herba Erigerontis into 75% aqueous ethanol, decocting twice, each for 2 hr, filtering, mixing filtrates, and concentrating under reduced pressure to obtain extract (relative density of 1.15-1.25, 75 deg.C); dissolving the extract with 5 times of water, adding 5% sodium hydroxide solution while stirring, adjusting the pH value to 7.5-9.0, filtering, adding 10% sulfuric acid solution to adjust the pH value to 1-3, standing overnight, filtering, collecting filtrate and precipitate, refining the precipitate with ethanol, adding 5% sodium hydroxide to adjust the pH value to 7-8, and spray-drying to obtain powder 1;

acidifying the filtrate, adjusting the pH value to 7-9, clarifying with a 100nm ceramic membrane, concentrating the clarified liquid with a 300D organic membrane, passing the concentrated liquid through a 30-60-mesh polyamide chromatographic column (the diameter to length ratio is 1:4), eluting with 3.5 column volumes of water, eluting with 3 column volumes of 75% ethanol, collecting the ethanol eluate, adjusting the pH value to 7-9 after concentration, and spray-drying to obtain powder 2; obtaining 13.5g of powder 1 and 164g of powder 2, and mixing the obtained breviscapine sodium salt powder and caffeic acid ester sodium salt powder according to a ratio of 1:2 to obtain the raw material medicine composition components of the medicinal composition. Wherein the powder 1 contains scutellarin sodium salt 51% and 6.9 g. Powder 2 contained 32.0g of dicaffeonate 19.5% and 9.7g of monocffeonate. Wherein the amount of the simple substituted dicaffeonate is 19.6g, and the amount of the dicaffeoyloctanone gluconate is 12.4 g.

The inventor also simultaneously and respectively uses 10%, 30%, 40%, 45%, 50%, 55%, 60%, 75% and 90% of aqueous ethanol to extract erigeron breviscapus, other steps are the same as the example 1, spray dry powder is obtained after acidification and membrane concentration, experiments prove that the erigeron breviscapus extract can be obtained by 10-90% of ethanol extraction, but the extract obtained by ethanol with the concentration of more than 40% is relatively better than the ethanol extract with the concentration of less than 40%, the ethanol extract with the concentration of 40-75% is obviously better and more suitable for the process of the invention, and the erigeron breviscapus is extracted by ethanol with the concentration of 50% most preferably.

Example 6: the invention process is different from the prior art in the content of effective components

The invention process comprises the following steps: decocting herba Erigerontis 2000g with 50% aqueous ethanol twice, each for 2 hr, filtering, mixing filtrates, and concentrating under reduced pressure to obtain extract (relative density of 1.15-1.25, 75 deg.C); dissolving the extract with 5 times of water, adding 5% sodium hydroxide solution while stirring, adjusting the pH value to 7.5-8.5, filtering, adding 10% sulfuric acid solution to adjust the pH value to 1-3, standing overnight, filtering, collecting filtrate and precipitate, refining the precipitate with ethanol, adding 5% sodium hydroxide to adjust the pH value to 7-8, and spray-drying to obtain powder 1; adjusting the pH value of the acidified filtrate to 7-9, clarifying with a 100nm ceramic membrane, concentrating the clarified liquid with a 350D organic membrane, passing the concentrated liquid through a 30-60-mesh polyamide chromatographic column (the diameter and length ratio is 1:4), eluting with water for 3 column volumes, eluting with 65% ethanol for 4 column volumes, collecting ethanol eluate, adjusting the pH value to 7-9, and spray-drying to obtain powder 2; mixing 15g of the powder 1 with 147g of the powder 2 to obtain breviscapine sodium salt powder and caffeic acid ester sodium salt powder.

The prior art comprises the following steps: extracting herba Erigerontis 2000g with 80% ethanol under reflux twice (1.5 hr for the first time and 1 hr for the second time), mixing extractive solutions, adding active carbon 640g, boiling, filtering, concentrating the filtrate under reduced pressure to obtain soft extract, adding appropriate amount of starch, drying under reduced pressure, pulverizing, and sieving. To obtain herba Erigerontis extract powder.

The content of each component is calculated by taking the weight of the total mixed powder as a denominator and the weight of each component as a numerator

TABLE 18 comparison of Components of Prior Process and inventive Process

Breviscapine is extract defined by pharmacopoeia, contains scutellarin as main ingredient, and other substances such as breviscapine, apigenin, scutellarin, etc., and its content measurement is represented by scutellarin.

In view of the high content of the above-mentioned effective substances, the ratio of the experimental groups was based on the test results, using the

powders

1 and 2 for formulation.

Example 5: preparation of capsules

And (3) taking 162g of the extraction mixture prepared in the example 1, adding 18g of starch, uniformly mixing, and filling into capsules to obtain the capsule.

Example 6: preparation of capsules

And (3) taking 151g of the extraction mixture prepared in the example 2, adding 29g of starch, uniformly mixing, and filling into capsules to obtain the capsule.

Example 7: preparation of capsules

And (3) taking 142g of the extraction mixture prepared in the example 1, adding 34g of starch and 4g of magnesium stearate, uniformly mixing, and filling into capsules to obtain the capsule.

Example 8: preparation of tablets

Taking 151g of the extraction mixture prepared in the example 2, 100g of starch and 10g of dextrin, sieving with a 14-mesh sieve, granulating, carrying out ventilation drying at 60-70 ℃, and adding 3g of magnesium stearate. Making into tablet and coating.

Example 9: preparation of dropping pills

Taking 15g of the extraction mixture prepared in the embodiment 3, adding 45g of the extraction mixture and the polyethylene glycol 4000, uniformly mixing, melting, dripping into low-temperature liquid paraffin, selecting pills, and removing the liquid paraffin to obtain the traditional Chinese medicine composition.

Example 10 preparation of oral liquid

Mixing 20g of the extraction mixture prepared in example 1 with 300g of honey, 50g of sucrose, 2g of sodium benzoate and 300ml of distilled water, heating for dissolving, and filtering at constant temperature to obtain the traditional Chinese medicine composition.

Example 11: preparation of granules

And (3) uniformly mixing 9g of the extraction mixture prepared in the example 3 with 40g of microcrystalline cellulose, adding a 3% povidone ethanol solution to prepare a soft material, sieving with a 18-mesh sieve to prepare granules, drying at 600 ℃ for 30-45 minutes, grading, adding 4g of talcum powder, uniformly mixing, grading and bagging to obtain the compound vitamin E tablet.

Example 12: preparation of Soft capsules

Taking 120g of the extraction mixture prepared in the embodiment 3, adding 5% of glycerol, 1% of glycine and 400-400 g of polyethylene glycol, uniformly mixing, and pressing into 1000 soft capsules to obtain the soft capsule.

Claims

1. An oral preparation containing erigeron breviscapus extract is characterized by comprising caffeic acid ester or a salt thereof, breviscapine or a salt thereof and pharmaceutically acceptable auxiliary materials, wherein the weight ratio of the caffeic acid ester or the salt thereof to the breviscapine or the salt thereof is 1: 1-30: 1.

2. The oral preparation according to claim 1, wherein the caffeate or the salt thereof comprises dicaffeate or the salt thereof and monocffeate or the salt thereof, wherein the weight ratio of the dicaffeate or the salt thereof to the breviscapine or the salt thereof is 1: 1-6: 1, and the weight ratio of the monocffeate or the salt thereof to the dicaffeate or the salt thereof is 1:1.2-1: 6.2.

3. The oral formulation of claim 2, wherein said dicaffeonate ester or salt thereof comprises 1, 3-O-dicaffeoylquinic acid or salt thereof, 3, 4-O-dicaffeoylquinic acid or salt thereof, 3, 5-O-dicaffeoylquinic acid or salt thereof, 4, 5-O-dicaffeoylquinic acid or salt thereof, erigeron ester or salt thereof, and erigeron breviscapus or salt thereof; the monocaffeic acid ester or its salt includes 3-O-caffeoylquinic acid or its salt, 4-O-caffeoylquinic acid or its salt, 5-O-caffeoylquinic acid or its salt, and breviscapine or its salt.

4. The oral preparation according to claim 3, wherein 1, 3-O-dicaffeoylquinic acid or a salt thereof, 3, 4-O-dicaffeoylquinic acid or a salt thereof, 3, 5-O-dicaffeoylquinic acid or a salt thereof, 4, 5-O-dicaffeoylquinic acid or a salt thereof, collectively referred to as simply substituted dicaffeoylquinic acid ester or a salt thereof; the erigeron breviscapus ester B or salt thereof and the erigeron breviscapus or salt thereof are isomers and are called dicaffeoyloctanone acid or salt thereof, wherein the ratio of the simple substituted dicaffeoylester or salt thereof to the dicaffeoyloctanone acid or salt thereof is 2: 1-0.9: 1.

5. The oral preparation according to any one of claims 1 to 4, which is a hard capsule, a soft capsule, a tablet, a granule, an oral liquid, a dripping pill, a water-paste pill, a powder, a pill.

6. The oral preparation according to claim 5, wherein the weight ratio of the caffeic acid ester or the salt thereof to the breviscapine or the salt thereof is 1: 1-30: 1, and the caffeic acid ester salt and the breviscapine salt are sodium salt or potassium salt or other pharmaceutically acceptable and water-soluble salts.

7. A method for preparing the oral formulation of claim 1, wherein: extracting herba Erigerontis with 10-90% aqueous ethanol, and concentrating the extractive solution under reduced pressure to obtain extract; dissolving the extract in water, adjusting the pH value to 7-9, filtering, adjusting the pH value to 1-3 with acid, standing overnight, filtering, collecting filtrate and precipitate, refining the precipitate with water and ethanol, and drying to obtain breviscapine; adding alkali to adjust the pH value to 7-8 after precipitation and refining, and performing spray drying to obtain breviscapine salt; adjusting the pH value of the acidified filtrate to 7-9, clarifying with a ceramic microfiltration membrane, concentrating clarified liquid obtained after clarification with an organic nanofiltration membrane, passing the concentrated liquid through a polyamide chromatographic column, eluting with water, eluting with 50-80% ethanol, collecting ethanol eluate, concentrating, and drying to obtain caffeic acid ester; concentrating the ethanol eluent, adjusting the pH value to 7-9, filtering, and spray-drying the filtrate to obtain caffeic acid ester salt; the caffeic acid ester or the salt thereof and the breviscapine or the salt thereof are mixed according to the proportion of 1: 1-30: 1 to obtain an erigeron breviscapus oral preparation, and the erigeron breviscapus oral preparation is added with proper auxiliary materials to be prepared into tablets, capsules, soft capsules, dripping pills, granules and oral liquid preparations.

8. The method of claim 7, wherein: the pH value of the alkali adjusting solution is NaOH and Na₂CO₃,NaHCO₃,KOH,K₂CO₃Or KHCO₃Or other alkali solutions useful for adjusting pH; the pH value of the acid adjusting solution is HCl and H₂SO₄Or H₃PO₄Or other acid solutions that may be used to adjust the pH.

9. The use of the oral preparation of claim 1 in the preparation of medicaments for treating metabolic-related diseases such as hyperlipidemia, hypertension, diabetes, obesity, non-alcoholic fatty liver disease and the like, and cardiovascular and cerebrovascular diseases.

10. The use of the oral formulation of claim 1 for the preparation of a medicament for inhibiting or treating platelet aggregation and coagulation disorders associated with metabolic disorders.