Application of phillyrin, phillygenin, derivatives of phillygenin and composition of phillygenin and phillygenin in preparation of antithrombotic drugs and / or health food

Abstract

The invention provides a novel application of forsythin, forsythiaside, forsythiaside sulfate, forsythiaside glucuronate or a composition thereof in preparation of drugs and / or health-care foods for inhibiting platelet aggregation and resisting thrombus. The phillyrin, the phillygenin, the phillygenin sulfate, the phillygenin glucuronate or the composition of the phillygenin, the phillygenin, the phillygenin sulfate and the phillygenin glucuronate disclosed by the invention can inhibit suppository contraction and platelet aggregation activation in vitro, can prolong mouse tail bleeding time and inhibit tail thrombosis in vivo, and is remarkable in curative effect, quick in effect taking, small in toxic and side effects and free from side effects. The medicine or health-care food for inhibiting platelet aggregation and resisting thrombus is safe, efficient, stable, simple in preparation process, suitable for industrial production and easy to popularize. The invention provides a new medicine source and health food source for preventing and treating platelet aggregation and resisting thrombus.

Description

Technical Field

[0001] The invention relates to forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate and a combination thereof, and their application in inhibiting platelet aggregation and activation and in antithrombotic drugs, belonging to the field of biomedicine. Background Art

[0002] The adhesion between platelets is called platelet aggregation. If the platelet aggregation rate is too high, the risk of thrombosis is high and thromboembolic diseases are likely to occur. High platelet aggregation may lead to atherosclerosis, such as common cerebral embolism and pulmonary embolism.

[0003] Thrombosis is the coagulation of blood due to the activation of platelets and coagulation factors in the flowing state. In the process of thrombosis, platelets first adhere to the exposed collagen surface after the intima is damaged, and swell and deform after being activated by collagen, and then release platelet particles, and then release substances such as ADP, thromboxane A2, 5-HT and platelet factor IV from the particles, so that the platelets in the blood flow continue to adhere locally, forming a reversible platelet pile. With the activation of endogenous and exogenous coagulation pathways, irreversible platelet thrombus is gradually formed, and finally becomes a thrombus. Therefore, whether the activation and aggregation of platelets can be effectively controlled is the key to the prevention and treatment of thrombotic diseases.

[0004] Phillyrin is a lignin compound that can be extracted from the plant Forsythia Suspensa (Thunb.) Vahl. of the Oleaceae family. The structural formula is:

[0005]

[0006] Molecular formula C 27 H 34 O 11 , with a molecular weight of 534, is the main active ingredient of the traditional Chinese medicine Forsythia. Studies have shown that forsythiaside has good pharmacokinetic properties, with a biological oral availability of 36.4%, and can be further metabolized into forsythiaside, forsythiaside glucuronide and forsythiaside sulfate in the body. These metabolites may be the main substances that exert its pharmacological activity. It is reported that forsythiaside has antiviral, antibacterial, antipyretic, analgesic, liver-protecting, antioxidant, anti-inflammatory and antiviral activities, but there has never been a report on its use in inhibiting platelet activation and anticoagulation.

[0007] The inventors have prepared forsythiaside, forsythiaside, forsythiaside sulfate and forsythiaside glucuronide through a large amount of modern scientific research and the use of advanced separation and purification technology or chemical synthesis technology (① a chemical synthesis method of forsythiaside, patent number: ZL201410386621.8; ② a preparation method of forsythiaside, patent number: ZL201410387248.8; ③ forsythiaside sulfate and its derivatives, preparation method and application thereof, patent number: ZL201310580801.5; ④ a preparation method of forsythiaside glucuronic acid derivative and application thereof, patent number: ZL201510319303.4), which can provide patients with a highly effective and low-toxic drug. Summary of the invention

[0008] The primary purpose of the present invention is to address the above-mentioned technical problems of inhibiting platelet aggregation and anti-thrombosis, and to provide new uses of forsythiaside, forsythiaside sulfate, forsythiaside glucuronide and their compositions in the preparation of drugs and health foods for inhibiting platelet aggregation and anti-thrombosis.

[0009] Specifically, on one hand, the present invention provides the use of forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate or a combination thereof in the preparation of a drug or health food for inhibiting platelet aggregation or anti-thrombosis. In addition, the present invention also provides the use of forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate or a combination thereof in the preparation of a drug for treating diseases caused by platelet aggregation or thrombosis.

[0010] The medicine and / or health food is composed of forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate or a combination of two or more thereof (forsythiaside is preferred), and a pharmaceutically and / or food acceptable carrier.

[0011] Particularly, the content of forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate or a combination thereof is ≥ 1%.

[0012] Particularly, the content of forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate or a combination thereof is 1% to 98%.

[0013] The drug may include a pharmaceutically acceptable carrier and be formulated into a pharmaceutical preparation. This is well known to those skilled in the art. Preferably, in the application of the first aspect of the present invention, the drug is in the form of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections (freeze-dried powder injections, needle-free powder injections); or, the health food is in the form of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, or in the form of foods such as dairy products, candies, beverages, biscuits, tea and related products, and wine.

[0014] In particular, the forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate or a combination thereof can be administered orally or by injection. For example, the preferred administration method of forsythiaside is injection.

[0015] Particularly, the in vivo injection dose of the forsythiaside is 0.2 mg / kg to 5 mg / kg, and the in vitro administration dose is 0.01 μM to 100 μM.

[0016] The oral or injectable forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate or the combination thereof of the present invention can be used for the prevention and treatment of thrombosis caused by infection and cardiovascular disease.

[0017] The oral or injectable forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate or the combination thereof of the present invention can be used in single or compound preparations and products of medicine, food, cosmetics and the like.

[0018] Preferably, the medicine and / or health food is a mixture of forsythiaside and forsythiaside with α-, β- or γ-cyclodextrin or its derivatives, or a complex formed by forsythiaside and forsythiaside with α-, β- or γ-cyclodextrin or its derivatives treated by physical or chemical methods.

[0019] In particular, the medicine or health product also includes salvia miltiorrhiza extract, gastrodia elata extract, hawthorn extract, astragalus extract, Panax notoginseng extract, Acanthopanax senticosus extract, honeysuckle extract, wolfberry fruit extract, Ganoderma lucidum extract, grape seed extract, pomegranate seed extract, royal jelly, vitamin C and its derivatives or vitamin E and its derivatives.

[0020] The invention has the advantages that forsythiaside, forsythiaside, forsythiaside glucuronate, forsythiaside sulfate and the combination thereof have an effect on in vitro thrombus contraction, platelet aggregation, platelet spreading, and on tail bleeding time and tail thrombosis of mice, proving that forsythiaside, forsythiaside, forsythiaside glucuronate, forsythiaside sulfate and the combination thereof have the function of inhibiting platelet aggregation and activation, and also have obvious anticoagulation and thrombosis inhibition effects. Therefore, forsythiaside, forsythiaside, forsythiaside glucuronate, forsythiaside sulfate and the combination thereof can be applied to the prevention and treatment of thrombus caused by infection and cardiovascular disease, and can be used in single or compound preparations and products in the pharmaceutical, food and cosmetic industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the test examples are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The effects of forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate on thrombus aggregation; a is the aggregation of thrombi at different time points, and b is the statistical analysis of thrombus shrinkage rate;

[0023] Figure 2 The effects of forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate on platelet aggregation;

[0024] Figure 3 The effects of forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate on platelet adhesion in vitro; a is the result of platelet adhesion on fibrinogen in each group of samples, and b is the statistical result of the area of ​​platelet adhesion in each group;

[0025] Figure 4 The effect of forsythiaside and forsythiaside on lipopolysaccharide-induced disseminated intravascular coagulation in mice;

[0026] Figure 5 To investigate the effects of forsythiaside and forsythiaside on cardiopulmonary embolism in mice induced by collagen combined with adrenaline;

[0027] Figure 6 Effects of forsythiaside on carrageenan-induced tail thrombosis in mice.

[0028] Figure 7 Effects of forsythiaside, forsythiaside sulfate, forsythiaside glucuronide and their combination on APTT, PT and TT in rats DETAILED DESCRIPTION

[0029] Now, various exemplary embodiments of the present invention are described in detail, and this detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the present invention. It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention.

[0030] In addition, for the numerical range in the present invention, it is understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Among them, forsythiaside, forsythiazone, forsythiazone glucuronide, forsythiazone sulfate and their combination include forsythiaside, forsythiazone, forsythiazone glucuronide, forsythiazone sulfate and a combination of two or more thereof.

[0035] Example 1 Forsythiaside Tablets

[0036] 1. Prepare the raw materials according to the following ratio

[0037]

[0038]

[0039] 2. Mix forsythiaside and starch evenly, make granules, add talcum powder and magnesium stearate, mix evenly, and press into 10,000 tablets.

[0040] Example 2 Forsythiaside capsules

[0041] 1. Prepare the raw materials according to the following ratio

[0042] Forsythiaside (purity 60%) 200g

[0043] Starch 1000g

[0044] 2. Mix forsythiaside and starch evenly and put them into capsules to make 10,000 capsules.

[0045] Example 3 Forsythiaside Capsules

[0046] 1. Prepare the raw materials according to the following ratio

[0047] Forsythiaside (purity 98%) 500g

[0048] Starch 1000g

[0049] 2. Mix forsythiaside and starch evenly and put them into capsules to make 10,000 capsules.

[0050] Example 4 Forsythiaside Tablets

[0051] 1. Prepare the raw materials according to the following ratio

[0052]

[0053] 2. Mix forsythiaside, salvia miltiorrhiza extract, vitamin C and starch evenly, make granules, add talcum powder and magnesium stearate, mix evenly and press into 10,000 tablets.

[0054] Example 5 Forsythiaside Capsules

[0055] 1. Prepare the raw materials according to the following ratio

[0056]

[0057]

[0058] 2. Evenly mix the forsythiaside, gastrodia extract, vitamin C and starch, and put them into capsules to make 10,000 capsules.

[0059] Example 6 Forsythiaside Granules

[0060] 1. Prepare the raw materials according to the following ratio

[0061]

[0062] 2. Evenly mix forsythiaside, hawthorn extract, vitamin C and sucrose powder to make granules and pack them into bags, making 10,000 bags.

[0063] Example 7 Forsythiaside oral solution

[0064] 1. Prepare the raw materials according to the following ratio

[0065]

[0066] 2. Take forsythiaside, Panax notoginseng extract and pomegranate seed extract, dissolve them in ethanol, add glucose syrup, add deionized water to 100 ml, mix well, and you have it.

[0067] Example 8 Forsythiaside tablets

[0068] 1. Prepare the raw materials according to the following ratio

[0069]

[0070] 2. Mix forsythiaside and starch evenly, make granules, add talcum powder and magnesium stearate, mix evenly, and press into 10,000 tablets.

[0071] Example 9 Forsythiaside capsules

[0072] 1. Prepare the raw materials according to the following ratio

[0073] Forsythiaside (purity 60%) 200g

[0074] Starch 1000g

[0075] 2. Mix forsythiaside and starch evenly and put them into capsules to make 10,000 capsules.

[0076] Example 10 Forsythiaside capsules

[0077] 1. Prepare the raw materials according to the following ratio

[0078] Forsythiaside (purity 98%) 500g

[0079] Starch 1000g

[0080] 2. Mix forsythiaside and starch evenly and put them into capsules to make 10,000 capsules.

[0081] Example 11 Forsythiaside Tablets

[0082] 1. Prepare the raw materials according to the following ratio

[0083]

[0084] 2. Mix forsythiaside, salvia miltiorrhiza extract, vitamin C and starch evenly, make granules, add talcum powder and magnesium stearate, mix evenly and press into 10,000 tablets.

[0085] Example 12 Forsythiaside capsules

[0086] 1. Prepare the raw materials according to the following ratio

[0087]

[0088] 2. Evenly mix the forsythiaside, astragalus extract, vitamin C and starch, and put them into capsules to make 10,000 capsules.

[0089] Example 13 Forsythiaside Granules

[0090] 1. Prepare the raw materials according to the following ratio

[0091]

[0092] 2. Evenly mix forsythiaside, hawthorn extract, vitamin C and sucrose powder to make granules and pack them into bags, making 10,000 bags.

[0093] Example 14 Forsythiaside oral solution

[0094] 1. Prepare the raw materials according to the following ratio

[0095]

[0096] Deionized water (appropriate amount)

[0097] 2. Take forsythiaside, Acanthopanax senticosus extract and honeysuckle extract, dissolve them in ethanol, add glucose syrup, add deionized water to 100 ml, mix well, and you have it.

[0098] Example 15 Forsythiaside glucuronate tablets

[0099] 1. Prepare the raw materials according to the following ratio

[0100]

[0101] 2. Mix the forsythiaside glucuronate and starch evenly, make granules, add talcum powder and magnesium stearate, mix evenly, and press into 10,000 tablets.

[0102] Example 16 Forsythiaside glucuronate capsules

[0103] 1. Prepare the raw materials according to the following ratio

[0104] Forsythiaside glucuronide (purity 60%) 200g

[0105] Starch 1000g

[0106] 2. Evenly mix the forsythiaside glucuronide and starch and fill them into capsules to make 10,000 capsules.

[0107] Example 17 Forsythiaside glucuronate capsules

[0108] 1. Prepare the raw materials according to the following ratio

[0109] Forsythiaside glucuronide (purity 98%) 500g

[0110] Starch 1000g

[0111] 2. Evenly mix the forsythiaside glucuronide and starch and fill them into capsules to make 10,000 capsules.

[0112] Example 18 Forsythiaside glucuronate tablets

[0113] 1. Prepare the raw materials according to the following ratio

[0114]

[0115] 2. Mix forsythiaside glucuronide, astragalus extract, vitamin C and starch evenly, make granules, add talcum powder and magnesium stearate, mix evenly, and press into 10,000 tablets.

[0116] Example 19 Forsythiaside capsules

[0117] 1. Prepare the raw materials according to the following ratio

[0118]

[0119] 2. Evenly mix the forsythiaside glucuronide, gastrodia extract, vitamin C and starch, and fill them into capsules to make 10,000 capsules.

[0120] Example 20 Forsythiaside glucuronate granules

[0121] 1. Prepare the raw materials according to the following ratio

[0122]

[0123] 2. Evenly mix the forsythiaside glucuronide, hawthorn extract, vitamin C and sucrose powder to make granules and pack them into bags, making 10,000 bags.

[0124] Example 21 Forsythiaside glucuronate oral solution

[0125] 1. Prepare the raw materials according to the following ratio

[0126]

[0127] 2. Take forsythiaside glucuronide, salvia miltiorrhiza extract and wolfberry extract, dissolve them in ethanol, add glucose syrup, add deionized water to 100 ml, mix well, and you have it.

[0128] Example 22 Forsythiaside sulfate tablets

[0129] 1. Prepare the raw materials according to the following ratio

[0130]

[0131] 2. Mix forsythiaside sulfate and starch evenly, make granules, add talcum powder and magnesium stearate, mix evenly, and press into 10,000 tablets.

[0132] Example 23 Forsythiaside Sulfate Capsules

[0133] 1. Prepare the raw materials according to the following ratio

[0134] Forsythiaside sulfate (purity 60%) 200g

[0135] Starch 1000g

[0136] 2. Mix forsythiaside sulfate and starch evenly and put them into capsules to make 10,000 capsules.

[0137] Example 24 Forsythiaside Sulfate Capsules

[0138] 1. Prepare the raw materials according to the following ratio

[0139] Forsythiaside sulfate (purity 98%) 500g

[0140] Starch 1000g

[0141] 2. Mix forsythiaside sulfate and starch evenly and put them into capsules to make 10,000 capsules.

[0142] Example 25 Forsythiaside Sulfate Tablets

[0143] 1. Prepare the raw materials according to the following ratio

[0144]

[0145] 2. Mix forsythiaside sulfate, salvia miltiorrhiza extract, vitamin C and starch evenly, make granules, add talcum powder and magnesium stearate, mix evenly, and press into 10,000 tablets.

[0146] Example 26 Forsythiaside capsules

[0147] 1. Prepare the raw materials according to the following ratio

[0148]

[0149] 2. Evenly mix the forsythiaside sulfate, gastrodia elata extract, vitamin C and starch, and put them into capsules to make 10,000 capsules.

[0150] Example 27 Forsythiaside sulfate granules

[0151] 1. Prepare the raw materials according to the following ratio

[0152]

[0153] 2. Evenly mix forsythiaside sulfate, hawthorn extract, vitamin C and sucrose powder to make granules, and then pack them into bags, making 10,000 bags.

[0154] Example 28 Forsythiaside sulfate oral solution

[0155] 1. Prepare the raw materials according to the following ratio

[0156]

[0157]

[0158] Deionized water (appropriate amount)

[0159] 2. Take forsythiaside sulfate, Panax notoginseng extract and grape seed extract, dissolve them in ethanol, add glucose syrup, add deionized water to 100 ml, mix well, and you have it.

[0160] Example 29-32 Composition tablets of forsythiaside, forsythiaside, forsythiaside glucuronide, and forsythiaside sulfate

[0161] Composition A (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 10:10:20:60);

[0162] Composition B (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 80:10:5:5);

[0163] Composition C (forsythiaside / forsythiaside 90:10);

[0164] Composition D (forsythiaside / forsythiaside 10:90).

[0165] 1. Prepare the raw materials according to the following ratio

[0166]

[0167] 2. Mix the composition A, B, C, and D with starch respectively and make granules, add talcum powder and magnesium stearate, mix evenly, and press into 10,000 tablets.

[0168] Example 33-36 Composition capsule of forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate

[0169] Composition A (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 10:10:20:60);

[0170] Composition B (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 80:10:5:5);

[0171] Composition C (forsythiaside / forsythiaside 90:10);

[0172] Composition D (forsythiaside / forsythiaside 10:90).

[0173] 1. Prepare the raw materials according to the following ratio

[0174] Composition 200g

[0175] Starch 1000g

[0176] 2. Mix the composition A, B, C, D and starch evenly and pack them into capsules to make 10,000 capsules. Example 37-52 Composition capsules of forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate

[0177] Composition A (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 10:10:20:60);

[0178] Composition B (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 80:10:5:5);

[0179] Composition C (forsythiaside / forsythiaside 90:10);

[0180] Composition D (forsythiaside / forsythiaside 10:90).

[0181] In Examples 37-52, compositions A, B, C, and D were mixed evenly with starch according to the weight ratios shown in the table below and then filled into capsules, and 10,000 capsules were prepared for each.

[0182]

[0183] Example 53-68 Combination granules of forsythiaside, forsythiaside, forsythiaside glucuronide, and forsythiaside sulfate

[0184] Composition A (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 10:10:20:60);

[0185] Composition B (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 80:10:5:5);

[0186] Composition C (forsythiaside / forsythiaside 90:10);

[0187] Composition D (forsythiaside / forsythiaside 10:90).

[0188] In Examples 53-68, compositions A, B, C, and D were uniformly mixed with microcrystalline cellulose according to the weight ratios shown in the table below, and then made into granules and packed into bags, so that 10,000 bags were prepared.

[0189]

[0190]

[0191] Examples 69-72 Composition tablets of forsythiaside, forsythiaside, forsythiaside glucuronide, and forsythiaside sulfate

[0192] Composition A (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 10:10:20:60);

[0193] Composition B (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 80:10:5:5);

[0194] Composition C (forsythiaside / forsythiaside 90:10);

[0195] Composition D (forsythiaside / forsythiaside 10:90).

[0196] 1. Prepare the raw materials according to the following ratio

[0197]

[0198] 2. The compositions A, B, C and D were mixed evenly with the salvia miltiorrhiza extract, vitamin C and starch respectively to prepare granules, talcum powder and magnesium stearate were added and mixed evenly, and then pressed into 10,000 tablets.

[0199] Example 73-76 Composition capsule of forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate

[0200] Composition A (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 10:10:20:60);

[0201] Composition B (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 80:10:5:5);

[0202] Composition C (forsythiaside / forsythiaside 90:10);

[0203] Composition D (forsythiaside / forsythiaside 10:90).

[0204] 1. Prepare the raw materials according to the following ratio

[0205]

[0206]

[0207] 2. The compositions A, B, C and D were respectively mixed evenly with Gastrodia elata extract, vitamin C and starch, and then filled into capsules to prepare 10,000 capsules.

[0208] Embodiment 77-80 Composition granules of forsythiaside, forsythiaside, forsythiaside glucuronide, and forsythiaside sulfate

[0209] Composition A (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 10:10:20:60);

[0210] Composition B (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 80:10:5:5);

[0211] Composition C (forsythiaside / forsythiaside 90:10);

[0212] Composition D (forsythiaside / forsythiaside 10:90).

[0213] 1. Prepare the raw materials according to the following ratio

[0214]

[0215] 2. The compositions A, B, C and D were respectively mixed with hawthorn extract, vitamin C and sucrose powder to form granules and then packed into bags, and 10,000 bags were prepared.

[0216] Examples 81-84 Oral liquid of a composition of forsythiaside, forsythiaside, forsythiaside glucuronide, and forsythiaside sulfate

[0217] Composition A (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 10:10:20:60);

[0218] Composition B (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 80:10:5:5);

[0219] Composition C (forsythiaside / forsythiaside 90:10);

[0220] Composition D (forsythiaside / forsythiaside 10:90).

[0221] 1. Prepare the raw materials according to the following ratio

[0222]

[0223] Deionized water (appropriate amount)

[0224] 2. Take the compositions A, B, C, and D, mix them with Panax notoginseng extract and pomegranate seed extract respectively, dissolve them with ethanol, add glucose syrup, add deionized water to 100 ml, mix well, and obtain the product.

[0225] Example 85 Injection of a composition of forsythiaside, forsythiaside, forsythiaside glucuronide, and forsythiaside sulfate

[0226] Composition A (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 10:10:20:60);

[0227] Composition B (forsythiaside / forsythiaside / forsythiaside sulfate / forsythiaside glucuronide 80:10:5:5);

[0228] Composition C (forsythiaside / forsythiaside 90:10);

[0229] Composition D (forsythiaside / forsythiaside 10:90).

[0230] In a specific embodiment of the present invention, the preparation method of the injection solution comprises the following steps:

[0231] (1) Forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate and the combination A, B, C and D thereof dissolved in an organic solvent are mixed with auxiliary materials dissolved in water respectively and then dried under reduced pressure;

[0232] (2) the dried product obtained in step (1) is dissolved in water and then dried under reduced pressure;

[0233] (3) The dried product obtained in step (2) is dissolved in water, and a pH regulator and / or an osmotic pressure regulator is optionally added.

[0234] In another specific embodiment of the present invention, the preparation method of the powder injection comprises the following steps:

[0235] (1) Forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate and the combination A, B, C and D thereof dissolved in an organic solvent are mixed with auxiliary materials dissolved in water respectively and then dried under reduced pressure;

[0236] (2) the dried product obtained in step (1) is dissolved in water and then dried under reduced pressure;

[0237] (3) dissolving the dried product obtained in step (2) in water, and optionally adding a pH regulator and / or an osmotic pressure regulator;

[0238] (4) freeze-drying the injection solution obtained in step (3).

[0239] In a specific embodiment of the present invention, the needle-free powder injection comprises the following steps:

[0240] (1) Forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate and the combination A, B, C and D thereof dissolved in an organic solvent are mixed with auxiliary materials dissolved in water respectively and then dried under reduced pressure;

[0241] (2) the dried product obtained in step (1) is dissolved in water and then dried under reduced pressure;

[0242] (3) dissolving the dried product obtained in step (2) in water, and optionally adding a pH regulator and / or an osmotic pressure regulator;

[0243] (4) freeze-drying the injection solution obtained in step (3);

[0244] (5) Wetting the powder for injection obtained in step (4), granulating, tableting and crushing, and then sieving to collect powder with a particle size between 400 mesh and 120 mesh.

[0245] The forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate used in the following Experimental Examples 1-6 were all from Dalian Fusheng Natural Medicine Development Co., Ltd.; the experimental animals used were wild-type male Kunming mice weighing 18-22 g; SD rats weighing 200-210 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., China; the description is not repeated below.

[0246] Experimental Example 1 Effects of Forsythiaside, Forsythiaside, Forsythiaside Glucuronide and Forsythiaside Sulfate on Thrombus Aggregation

[0247] Prepare washed platelets from rats and adjust the concentration to 3 × 10 8 / mL, 100μL platelets were placed in a siliconized tube, and aspirin (50μM), forsythiaside extract (0.1mg / mL, forsythiaside content of about 0.05mg / mL), forsythiaside (50μM), forsythiaside (50μM), forsythiaside glucuronide (50μM) and forsythiaside sulfate (50μM) were added according to the experimental requirements. The blank group and the model group were controlled with solvent (1‰ DMSO). After incubation at 37℃ for 10min, 1.5mL of fibrinogen (0.5mg / mL) and CaCl were added to the siliconized tube. 2 The platelet diluent (2mmol / L) was mixed evenly, and thrombin (final concentration 1U / mL) was added to all groups except the blank group. The clot retraction experiment was started at 37°C, and pictures of clot retraction were taken every 10 minutes. The two-dimensional area of ​​thrombus formation was analyzed using Image J software, and the proportion of thrombus clot retraction was calculated. The clot retraction rate = 1-(thrombus formation area / thrombus initial area) × 100%. After three independent repeated experiments, Graphpad Prism8.0 software was used for statistical analysis and graphing. The results are shown in Figure 1 As shown in a, after treatment with aspirin, forsythiaside extract, forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate, the thrombus contraction process was significantly delayed, indicating that forsythiaside extract, forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate can inhibit thrombin-induced platelet thrombus aggregation. Statistical analysis of thrombus aggregation contraction rate showed ( Figure 1 b), compared with the model group (47.43%), aspirin (3.37%), forsythiaside extract (14.28%), forsythiaside (7.27%), forsythiaside (4.60%), forsythiaside glucuronide (5.61%), forsythiaside sulfate (19.27%) all showed the effect of inhibiting the contraction of platelet plugs, among which forsythiaside had the most prominent effect. (Compared with the blank group, ### P<0.001; compared with the model group, *** P<0.001; n=3) Experimental Example 2 Effects of forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate on platelet aggregation

[0248] The number of platelets prepared was 2×10 7 / mL rat platelet-rich plasma. Take 100μL and place it in a test tube. According to the experimental requirements, add aspirin (50μM), different concentrations of forsythiaside (0, 0.01, 0.1, 1, 10, 50, 100μM), forsythiaside (0, 0.01, 0.1, 1, 10, 50, 100μM), forsythiaside glucuronide (0, 0.01, 0.1, 1, 10, 50, 100μM) and forsythiaside sulfate (0, 0.01, 0.1, 1, 10, 50, 100μM). The blank group uses 100μL platelet-deficient rat plasma and 1‰ DMSO solvent as a control. After incubation at 37℃ for 5min, take the suspension and add it to a 96-well plate. The absorbance value is measured at 405nm with an enzyme reader and recorded as OD1. Except for the blank group, 10 μL of ADP solution (final concentration of 10 μM) was added to the remaining groups. 10 μL of solvent was added to the blank group. After 200 seconds, the absorbance value at 405 nm was measured by an ELISA instrument and recorded as OD2. The absorbance value of the blank lacking platelet plasma was measured and recorded as OD3. The aggregation rate of platelets in each experimental group was calculated: aggregation rate (%) = [(OD1-OD2) / (OD1-OD3)] × 100%. After five independent repeated experiments, the half-maximal inhibitory concentration IC was calculated using GraphPad Prism8.0 50 The results are as follows Figure 2 As shown in Figure 2, forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate can effectively inhibit ADP-induced platelet aggregation in rats, with IC 50 The values ​​were 9.70 μM forsythiaside, 10.80 μM forsythiaside, 11.70 μM forsythiaside glucuronide and 16.90 μM forsythiaside sulfate. The results of platelet aggregation experiment showed that forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate can effectively inhibit platelet aggregation.

[0249] Experimental Example 3 Effects of Forsythiaside, Forsythiaside, Forsythiaside Glucuronide and Forsythiaside Sulfate on Platelet Adhesion

[0250] The platelet concentration of SD rats was adjusted to 3×10 7 / mL, 100μL was taken and incubated with aspirin (50μM), forsythiaside (50μM), forsythiaside glucuronide (50μM) and forsythiaside sulfate (50μM) at 37℃ for 5min; except for the blank group, the model group and each drug group were stimulated with 10μM ADP for 5min, loaded on a fibrinogen-coated slide, and incubated at 37℃ for 1h. The non-adherent platelets were washed with PBS and The cells were stained with phalloidin labeled with 594 (purchased from Suzhou Youyi Landi Biotechnology Co., Ltd., catalog number YP0052S) at room temperature for 30 min, and then washed and sealed. The cells were observed and photographed using a fully built-in fluorescence reflection microscope (Leica, Germany, model DMi8S). The spreading area of ​​each group was measured using Image J software, and statistically analyzed using GraphPad Prism 8.0 software. The results are shown in Figure 2. Figure 3 As shown in the figure, compared with the platelet adhesion on fibrinogen in the model control group, forsythiaside, forsythiaside, forsythiaside glucuronide and forsythiaside sulfate can significantly inhibit the platelet adhesion on fibrinogen, among which forsythiaside has the most obvious inhibitory effect (compared with the blank group, ### P<0.001; compared with the model group, *** P < 0.001; * P<0.05; n=4).

[0251] Experimental Example 4 Effects of forsythiaside and forsythiaside on lipopolysaccharide-induced disseminated intravascular coagulation in mice

[0252] Kunming mice were randomly divided into blank group (normal saline), model group (normal saline), oral aspirin group (10 mg / kg), oral forsythiaside low-dose group (1 mg / kg), medium-dose group (5 mg / kg) and high-dose group (25 mg / kg), and injection forsythiaside low-dose group (0.2 mg / kg), medium-dose group (1 mg / kg) and high-dose group (5 mg / kg), with 6 mice in each group. Aspirin and forsythiaside were administered orally by gavage, while forsythiaside was administered by intraperitoneal injection, once a day for 3 consecutive days. At 30 minutes after the last administration, 8 mg / kg lipopolysaccharide (purchased from Beijing Solebow Biotechnology Co., Ltd., catalog number L8880) was intraperitoneally injected into the other groups except the blank group to induce the establishment of disseminated intravascular coagulation (DIC) model in mice, and the blank group and model group mice were intraperitoneally injected with the corresponding volume of normal saline. After 5 hours, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). The tail of the mouse was cut 3 mm away from the tip, and the first drop of blood was absorbed with filter paper. The tail of the mouse was immediately placed vertically in 37°C saline and the bleeding time of the tail was recorded. After the bleeding stopped, the observation was continued for 1 minute. If the bleeding stopped within 1 minute, the moment when the blood flow stopped was recorded as the stop time; if the bleeding resumed within 1 minute after the bleeding stopped, the recording was continued until 15 minutes had passed and the bleeding time was recorded as 15 minutes. The results are as follows Figure 4As shown in the results, after lipopolysaccharide induced DIC in mice, the tail bleeding time of mice decreased significantly, while oral administration of forsythiaside (5mg / kg and 25mg / kg) and intraperitoneal injection of forsythiaside (1mg / kg and 5mg / kg) significantly increased the tail bleeding time of DIC model mice. This indicates that oral administration of forsythiaside and injection of forsythiaside have an effect on the coagulation function of mice, and have a preventive and therapeutic effect on thrombosis caused by bacterial lipopolysaccharide (compared with the blank group, ### P<0.001; compared with the model group, *** P<0.001; n=6).

[0253] Experimental Example 5 Effects of forsythiaside and forsythiaside on cardiopulmonary embolism in mice induced by collagen combined with adrenaline

[0254] Kunming mice were randomly divided into a blank group (normal saline), a model group (normal saline), an oral aspirin group (10 mg / kg), an oral forsythiaside low-dose group (1 mg / kg), a medium-dose group (5 mg / kg) and a high-dose group (25 mg / kg), and an injection forsythiaside low-dose group (0.2 mg / kg), a medium-dose group (1 mg / kg) and a high-dose group (5 mg / kg), with 6 mice in each group. Aspirin and forsythiaside were administered orally by gavage, while forsythiaside was administered by intraperitoneal injection, once a day for 3 consecutive days. 30 minutes after the last administration, except for the blank group, the mice in other groups were injected with 0.2 mL of a mixed inducer of collagen and adrenaline (192 μL of 5 mg / mL type I collagen solution + 8 μL of 1 mg / mL adrenaline hydrochloride injection; type I collagen was purchased from Beijing Solebow Biotechnology Co., Ltd., item number C8062, and adrenaline injection was purchased from Huachu Biotechnology Co., Ltd., item number 2204) through the tail vein to induce the establishment of a cardiopulmonary embolism model in mice, and the mice in the blank control group were intraperitoneally injected with a corresponding volume of normal saline. 2 hours after the completion of the injection of all drugs, the mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg), and the tail was cut 3 mm away from the tip of the mouse tail, and the bleeding time of the mouse tail was recorded. For specific operations, see Example 4. The results are as follows Figure 5 As shown in the results, after collagen combined with adrenaline induced a mouse cardiopulmonary embolism model, the tail bleeding time of the mice decreased significantly, while oral administration of forsythiaside (5mg / kg and 25mg / kg) and intraperitoneal injection of forsythiaside (1mg / kg and 5mg / kg) significantly increased the tail bleeding time of the mice in the cardiopulmonary embolism model. This indicates that oral administration of forsythiaside and injection of forsythiaside have a protective effect on the prevention and treatment of thrombosis caused by cardiovascular disease. (Compared with the blank group, ### P<0.001; compared with the model group, *** P<0.001; n=6) Experimental Example 6 Effect of forsythiaside on carrageenan-induced tail thrombosis in mice

[0255] Kunming mice were randomly divided into a blank control group (normal saline), a model group (normal saline), an aspirin group (5 mg / kg) and a forsythiaside group (5 mg / kg), with 3 mice in each group. Aspirin and forsythiaside were administered by intraperitoneal injection, once a day for 3 consecutive days. 30 minutes after the last administration, 20 mg / kg carrageenan (purchased from Shanghai MacLean Biochemical Technology Co., Ltd., catalog number C804872) was intraperitoneally injected into all groups except the blank group to cause thrombosis in the mouse tail. The blank control group mice were intraperitoneally injected with a corresponding volume of normal saline. 36 hours after the injection of all drugs, the thrombosis formation in the mouse tail was observed and photographed. The results are as follows Figure 6 As shown, carrageenan induced the entire tail of mice to be purple-black, which means tail thrombosis, while positive drugs aspirin and forsythiaside can effectively shorten the length of tail thrombosis in mice. This shows that the administration of forsythiaside can effectively control the formation of thrombus in vivo.

[0256] Test Example 7 Anticoagulation in vitro of forsythiaside, forsythiaside, forsythiaside sulfate, forsythiaside glucuronide and their combination

[0257] The thrombin time (TT), prothrombin time (PT) and activated partial thromboplastin time (APTT) were used as detection indicators, heparin sodium was used as a positive control, and normal saline was used as a blank control to investigate the in vitro anticoagulant effect of forsythiaside, forsythiaside, forsythiaside sulfate, forsythiaside glucuronide and their compositions. TT determination method: Take 100 μL of the plasma to be tested, preheat at 37°C for 3 minutes, then add 100 μL of TT reagent preheated at 37°C, and record the instrument measurement results. PT determination method: Take 100 μL of the plasma to be tested, preheat at 37°C for 3 minutes, then add 100 μL of PT reagent preheated at 37°C, and record the instrument measurement results. APTT determination method: Take 100 μL of the plasma to be tested, preheat at 37°C for 3 minutes, then add 100 μL of APTT reagent preheated at 37°C, preheat at 37°C for 5 minutes, and then add 0.025 mol / L CaCl preheated at 37°C 2 The solution was 100 μL, and the instrument measurement results were recorded. The experimental data of each group were expressed as mean ± standard deviation. The difference between the two groups was analyzed by t-test. The results showed that forsythiaside, forsythiaside sulfate, forsythiaside glucuronide and their combination can prolong TT, PT and APTT, indicating that they have good inhibitory effects on both exogenous and intrinsic coagulation pathways.

[0258] The above is only a preferred specific embodiment of the present invention, and those skilled in the art can refer to the content of this article for appropriate improvement. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention, which should be covered within the scope of protection of the present invention.

Claims

1. Use of forsythiaside, forsythiaside, forsythiaside sulfate, forsythiaside glucuronide or a combination thereof in the preparation of drugs and / or health foods for inhibiting platelet aggregation and / or anti-thrombotic effects.

2. The use according to claim 1, characterized in that: The medicine and / or health food is composed of forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate or a combination of two or more thereof, and a pharmaceutically and / or food acceptable carrier.

3. The use according to claim 1 or 2, characterized in that: The medicine is in the form of tablets, capsules, pills, powders, granules, syrups, solutions, injections (lyophilized powder injections, needle-free powder injections); or, the health food is in the form of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, or in the form of dairy products, candies, beverages, biscuits, tea and related products, wine and other foods.

4. The use according to claim 1 or 2, characterized in that: The drug can be administered orally or by injection.

5. The use according to claim 3, characterized in that The in vivo injection dosage of the forsythiaside is 0.2 mg / kg to 5 mg / kg, and the in vitro administration dosage is 0.01 μM to 100 μM.

6. The use according to claim 1 or 2, characterized in that: The content of forsythiaside, forsythiaside, forsythiaside glucuronide, forsythiaside sulfate or the combination thereof in the medicine and / or health food is ≥1%.

7. The use according to claim 6, characterized in that: The content of forsythiaside, forsythiaside, forsythiaside glucuronic acid salt, forsythiaside sulfate or the combination thereof in the medicine and / or health food is 1% to 98%.

8. The use according to claim 1, characterized in that: The antithrombotic agent is thrombosis caused by infection and / or cardiovascular disease.

9. The use according to claim 2, characterized in that: The medicine and / or health food is a mixture of forsythiaside and forsythiaside with α-, β- or γ-cyclodextrin or its derivatives, or a complex formed by forsythiaside and forsythiaside with α-, β- or γ-cyclodextrin or its derivatives through physical and chemical treatment.

10. The use according to claim 1, characterized in that: The medicine and / or health food also includes salvia miltiorrhiza extract, gastrodia elata extract, hawthorn extract, astragalus extract, panax notoginseng extract, eleuthero extract, honeysuckle extract, wolfberry fruit extract, ganoderma lucidum extract, grape seed extract, pomegranate seed extract, royal jelly, vitamin C or its derivatives or vitamin E or its derivatives.

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