Microecological preparation for improving thrombus as well as preparation method and application of microecological preparation

Abstract

The invention belongs to the technical field of microecological preparations and plant polysaccharide sterilization, and particularly relates to a microecological preparation for improving thrombus and a preparation method and application thereof. Specifically, a trichosanthes kirilowii maxim aqueous extract and a Chinese wolfberry fruit aqueous extract are taken as examples, the trichosanthes kirilowii maxim aqueous extract and the Chinese wolfberry fruit aqueous extract which are effectively sterilized are successfully obtained by adopting a method of combining low-temperature heating sterilization and aqueous membrane filtration, meanwhile, the physicochemical properties and the biological activity of the trichosanthes kirilowii maxim aqueous extract and the Chinese wolfberry fruit aqueous extract are effectively maintained, and particularly, the antithrombotic activity of the trichosanthes kirilowii maxim aqueous extract and the Chinese wolfberry fruit aqueous extract is effectively retained; the sterilization method is simple and easy to operate, short in time consumption, free of chemical waste liquid, free of pollution to the environment, green and safe, and the sterilized trichosanthes kirilowii maxim aqueous extract and the sterilized wolfberry aqueous extract are low in loss rate and high in recovery rate, so that the sterilization method has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of microecological preparations and plant polysaccharide sterilization, and specifically relates to a microecological preparation for improving thrombosis, and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Currently, the incidence of diabetes and its complications is on the rise. There are more than 100 complications of diabetes, including diabetic macroangiopathy (DMD), diabetic cardiomyopathy, diabetic peripheral vascular disease, diabetic neuropathy, etc., and DMD is one of the main and dangerous complications of diabetes. DMD often affects large arteries such as the carotid artery, aorta, coronary artery and lower limb artery. Therefore, the risk of cardiovascular and cerebrovascular diseases in diabetic patients is 1.5-2.5 times that of non-diabetic patients, and the mortality rate is twice that of non-diabetic patients. More than 50% of diabetic patients die from DMD. DMD can be followed by pathological changes such as plaque hemorrhage, plaque rupture, thrombosis, aneurysm rupture and atherosclerotic stenosis, causing critical consequences such as stroke, myocardial infarction, amputation, etc., threatening the life safety of patients.

[0004] Trichosanthes is considered to be a basic drug for the treatment of chest obstruction. The basic clinical symptoms of chest obstruction mainly include chest compression, pain in the chest, back, shoulders and arms, and shortness of breath, and these symptoms are common in cardiovascular diseases. Lycium barbarum L. is a product of both medicinal and edible origin. Lycium barbarum polysaccharide can relieve symptoms such as dizziness, fatigue, cough, back pain, and diabetes. After extracting Trichosanthes and Lycium barbarum, the corresponding extracts are obtained and microecological preparations are prepared by microbial fermentation. There are few studies on the treatment of diabetes and vascular diseases.

[0005] The earliest record of Chinese medicine fermentation technology was in the earliest existing Chinese medicine monograph "Shennong Bencao Jing". At present, many studies have proved that microbial fermentation of Chinese medicine has the advantages of protecting the active ingredients of Chinese medicine from destruction, improving efficacy, reducing toxic side effects, and improving taste. However, during the microbial fermentation process, the raw materials are easily contaminated by microorganisms, resulting in a decline in quality or even a complete loss of biological activity. Therefore, it is particularly important to effectively sterilize plant extracts.

[0006] At present, the sterilization methods for plant extracts include chemical sterilization, such as hydrogen peroxide sterilization, ethanol sterilization, etc.; physical sterilization, such as high-temperature steam sterilization, irradiation sterilization, etc. In recent years, with the continuous deepening of research on plant polysaccharide sterilization methods, some new sterilization technologies have gradually emerged. For example, physical means such as high-voltage electric fields and ultrasound are used for sterilization.

[0007] However, despite certain progress in the sterilization treatment of plant extracts, there are still some drawbacks. Among them, both chemical sterilization and physical sterilization may damage the structure and activity of plant extracts (such as plant polysaccharides). This damage may lead to a reduction or loss of biological activity, thereby affecting its application effect in the fields of medicine and food. Especially when used for the sterilization of fermented liquids, liquid plant polysaccharides cannot be directly sterilized by sterile 0.22μm filtration due to the high viscosity of their aqueous solutions, resulting in large polysaccharide losses. The emerging sterilization technology also has the above-mentioned problems, and the high energy consumption and high sterilization cost also limit its further practical application. Therefore, how to achieve thorough sterilization of plant polysaccharides without affecting the physicochemical properties of natural polysaccharides and their subsequent biological activity has become an urgent problem to be solved in the field of plant polysaccharide sterilization. Summary of the invention

[0008] In view of the deficiencies in the prior art, the present invention provides a microecological preparation for improving thrombosis and its preparation method and application. Specifically, the present invention provides a method for treating Trichosanthes kirilowii water extract and Lycium barbarum water extract by combining low-temperature heating sterilization and water system membrane filtration, and adding lactic acid bacteria for fermentation and culture, and finally obtaining a microecological preparation that can be used to significantly improve thrombosis. Experiments have shown that the method is simple to operate, has a fast sterilization speed, is thoroughly sterilized, does not affect the physicochemical properties and biological activity of the plant extract, and the sterilization process has very little loss to the extract. Based on the above research results, the present invention is completed.

[0009] Specifically, the present invention relates to the following technical solutions:

[0010] The first aspect of the present invention provides a probiotic preparation for improving thrombosis. The probiotic preparation is obtained by adding lactic acid bacteria to a water extract of Trichosanthes kirilowii and a water extract of Lycium barbarum after sterilization for fermentation and culture.

[0011] The sterilization method comprises: filtering the water extract of Trichosanthes kirilowii and the water extract of Lycium barbarum through a microporous filter membrane respectively, then heating the filtrate, cycling 1-3 times, and cooling to obtain a sterilized water extract; wherein the pore size of the microporous filter membrane is 0.22-0.45 μm, and the heating treatment temperature is controlled at 60-80°C.

[0012] The present invention has found through research that since the active structures of the water extracts of Trichosanthes kirilowii and Lycium barbarum will be destroyed at high temperatures, especially continuous high temperatures, the present invention first filters them through a microporous filter membrane and then uses a low-temperature heating treatment technology for cyclic treatment, thereby successfully achieving a thorough sterilization treatment without affecting their physical and chemical properties and biological activities.

[0013] The second aspect of the present invention provides the use of the above-mentioned microecological preparation for improving thrombosis in the preparation of anti-thrombotic products.

[0014] Beneficial technical effects of one or more of the above technical solutions:

[0015] The above technical scheme takes the water extract of Trichosanthes kirilowii and the water extract of Lycium barbarum as examples. When preparing the microecological preparation, the water extract of Trichosanthes kirilowii and the water extract of Lycium barbarum that are effectively sterilized are successfully obtained by combining low-temperature heating sterilization and aqueous membrane filtration. At the same time, their physical and chemical properties and biological activities are effectively maintained, especially their anti-thrombotic activity is effectively retained. The combination of the two produces a synergistic anti-thrombotic effect. At the same time, the above sterilization method is simple and easy to operate, short time consumption, does not produce chemical waste liquid, has no pollution to the environment, is green and safe, and the sterilized water extract of Trichosanthes kirilowii and the water extract of Lycium barbarum have a low loss rate and a high recovery rate, so it has good practical application value. DETAILED DESCRIPTION

[0016] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0017] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not indicated in the following specific embodiments, they are generally in accordance with conventional methods and conditions within the art, and such techniques and conditions are fully explained in the literature.

[0018] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

[0019] In a typical embodiment of the present invention, a probiotic preparation for improving thrombosis is provided. The probiotic preparation is obtained by adding lactic acid bacteria to a water extract of Trichosanthes kirilowii and a water extract of Lycium barbarum after sterilization for fermentation and culture.

[0020] The sterilization method comprises: filtering the water extract of Trichosanthes kirilowii and the water extract of Lycium barbarum through a microporous filter membrane respectively, then heating the filtrate, cycling 1-3 times, and cooling to obtain a sterilized water extract; wherein the pore size of the microporous filter membrane is 0.22-0.45 μm, and the heating treatment temperature is controlled at 60-80°C.

[0021] The concentration of the aqueous solution of the water extract of Trichosanthes kirilowii and the water extract of Lycium barbarum is 0.1-1 mg / mL, further 0.4-0.5 mg / mL.

[0022] The pore size of the microporous membrane is 0.34-0.45 μm, and further 0.34 μm. The present invention has found through research that the use of a water-based microporous membrane is more conducive to the filtration and purification of extracts, especially polysaccharide solutions, so the microporous membrane in the present invention is preferably a water-based microporous membrane.

[0023] The heating treatment can be specifically performed by water bath heating treatment. Furthermore, the specific method of the water bath heating treatment is: water bath heating treatment at 60-80°C, water bath heating treatment time is 10-30min, the above treatment is cycled 1-3 times, the cycle interval time is 5-20min, and natural cooling is adopted during the cycle interval; further, the specific method of the water bath heating treatment is: water bath heating treatment at 80°C for 10min, cooling for 10min, and then water bath heating treatment at 80°C for 10min again, and natural cooling is performed to obtain the product. By controlling the heating treatment temperature and interval time, the growth of miscellaneous bacteria can be effectively reduced, while avoiding the influence on the activity of the natural active ingredients of the plant.

[0024] The present invention has found through research that since the active structures of the water extracts of Trichosanthes kirilowii and Lycium barbarum will be destroyed at high temperatures, especially continuous high temperatures, the present invention first filters them through a microporous filter membrane and then uses a low-temperature heating treatment technology for cyclic treatment, thereby successfully achieving a thorough sterilization treatment without affecting their physical and chemical properties and biological activities.

[0025] In the present invention, the mass ratio of the water extract of Trichosanthes kirilowii to the water extract of Lycium barbarum is 0.5-5:1, further 0.8-2:1, further 1.25:1;

[0026] The lactic acid bacteria are lactobacillus, which can be specifically a combination of plant lactobacillus and casei lactobacillus. The inventors have found through research that fermenting the water extract obtained by the two lactobacilli can further effectively improve the biological activity of the water extract after fermentation, especially the anti-thrombotic effect. Furthermore, the plant lactobacillus is specifically (Lactobacillus plantarum) CGMCC 1.12732, and the casei lactobacillus is specifically Lactobacillus casei (Lactobacillus casei) CGMCC 1.8727. Both of the above strains can be purchased from the China General Microbiological Culture Collection Administration Center.

[0027] Furthermore, the water extract of Trichosanthes kirilowii is prepared by the following method: crushing the Trichosanthes kirilowii into powder, adding water for reflux extraction, and taking the filtrate for vacuum concentration and drying to obtain the water extract of Trichosanthes kirilowii.

[0028] Wherein, the mass ratio of the Trichosanthes powder to water is 1:8-15, preferably 1:10.

[0029] The reflux extraction can be performed 1-3 times, preferably 2 times, each time for 2 hours, and the filtrate is filtered and combined.

[0030] Furthermore, the preparation method of the wolfberry water extract includes: grinding and sieving the wolfberry, adding water and performing ultrasonic treatment, centrifuging and removing the precipitate from the aqueous solution and performing rotary evaporation, adding ethanol solution thereto, heating (such as 40-50° C.) and stirring, and drying the precipitate after centrifugation to obtain the wolfberry water extract.

[0031] Wherein, the sieving mesh number is 180-220 meshes, preferably 200 meshes.

[0032] The specific conditions of the ultrasonic treatment are: ultrasonic frequency is 30-70 Hz (preferably 50 Hz), and the ultrasonic treatment is 10-60 min (preferably 30 min).

[0033] The ethanol solution is high-concentration ethanol, specifically, a 96% ethanol solution. More specifically, the volume ratio of the residue after rotary evaporation to the ethanol solution is 1:5.

[0034] In the above preparation methods, the centrifugal treatment can be carried out at a centrifugal speed of 4000-6000 rpm (preferably 5000 rpm) and a centrifugal treatment time of 1-10 min (preferably 5 min).

[0035] In another specific embodiment of the present invention, there is provided the use of the above-mentioned microecological preparation for improving thrombosis in the preparation of anti-thrombotic products.

[0036] The product can be food or medicine. Further, it can be medicine. The present invention has found through research that the combination of the two exhibits good synergistic antithrombotic activity.

[0037] The present invention is further explained by the following examples, but it is not intended to limit the present invention. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0038] Example 1 Sterilization experiment of water extract of Trichosanthes kirilowii and Lycium barbarum seed raw materials

[0039] 1. Materials and Instruments

[0040] 1.1 Materials

[0041] Trichosanthes kirilowii and wolfberry kernels are from Shandong (origin)

[0042] (1) Trichosanthes kirilowii water extract: The Trichosanthes kirilowii was crushed into powder, and distilled water 10 times its weight was added, and reflux extraction was performed twice by a distillation apparatus, each time for 2 hours, and the filtrate was filtered and combined, concentrated under reduced pressure, and freeze-dried to obtain the Trichosanthes kirilowii water extract.

[0043] (2) Lycium barbarum water extract: After drying, the lycium barbarum is crushed by a wall-breaking machine, and then passed through a 200-mesh sieve. Three times the weight of pure water is added in batches, and ultrasonic treatment is performed at 50 Hz for 30 minutes. The precipitate is removed by centrifugation at 8000 rpm for 5 minutes. Three times the weight of pure water is added to the precipitate, and ultrasonic treatment is performed. This process is repeated three times. The aqueous solution after the precipitate is removed is combined and rotary distilled until it becomes a viscous semi-fluid. Five times the weight of 96% ethanol solution is added, and the mixture is stirred at 45°C for 15 minutes. The supernatant is removed by centrifugation at 5000 rpm for 5 minutes. This process is repeated three times. The precipitate is dried at 25°C for 24 hours to obtain the lycium barbarum water extract.

[0044] 0.45μm water system microporous filter membrane, 0.33μm water system microporous filter membrane, 0.22μm water system microporous filter membrane (Millipore China Co., Ltd.), 10ml syringe, LB culture medium (Qingdao Haibo Biotechnology Co., Ltd.).

[0045] 1.2 Instruments

[0046] Microplate reader (model: 1SPA-0093), Dynex, USA.

[0047] 1.3 Sample preparation

[0048] (1) 5 mg / mL Trichosanthes kirilowii aqueous extract solution: Weigh 50 mg of Trichosanthes kirilowii aqueous extract, add 10 ml of distilled water, and stir at room temperature to dissolve.

[0049] 4 mg / mL wolfberry water extract solution: weigh 40 mg wolfberry water extract, add 10 ml distilled water, and stir at room temperature to dissolve.

[0050] 1.4 Experimental plan:

[0051] Control group:

[0052] (1) 10 ml of the prepared water extract of Trichosanthes kirilowii or Lycium barbarum is set aside without any treatment.

[0053] (2) Take 30 μl of the prepared solution, spread it on an LB plate, and culture it at 37°C for 24 hours to observe whether any bacteria grow.

[0054] 1. (1) 10 ml of the prepared water extract of Trichosanthes or Lycium barbarum was filtered through a 0.45 μm microporous filter membrane, heated at 60°C for 10 min in a water bath, cooled for 10 min, and heated again at 60°C for 10 min for later use.

[0055] (2) The solution obtained through the above steps is poured into a measuring cylinder, and the loss rate of the solution after the above sterilization steps is detected: (original solution - solution after sterilization) / original solution × 100%.

[0056] (3) Take 30 μl of the solution, spread it on an LB plate, and incubate it at 37°C for 24 hours to observe whether any bacteria grow.

[0057] 2. (1) 10 ml of the prepared water extract of Trichosanthes or Lycium barbarum was filtered through a 0.45 μm microporous filter membrane, heated at 70°C for 10 min in a water bath, cooled for 10 min, and heated again at 70°C for 10 min for later use.

[0058] (2) The solution obtained through the above steps is poured into a measuring cylinder, and the loss rate of the solution after the above sterilization steps is detected: (original solution - solution after sterilization) / original solution × 100%.

[0059] (3) Take 30 μl of the solution, spread it on an LB plate, and incubate it at 37°C for 24 hours to observe whether any bacteria grow.

[0060] 3. (1) 10 ml of the prepared water extract of Trichosanthes or Lycium barbarum was filtered through a 0.45 μm microporous filter membrane, heated at 80°C for 10 min in a water bath, cooled for 10 min, and heated again at 80°C for 10 min for later use.

[0061] (2) The solution obtained through the above steps is poured into a measuring cylinder, and the loss rate of the solution after the above sterilization steps is detected: (original solution - solution after sterilization) / original solution × 100%.

[0062] (3) Take 30 μl of the solution, spread it on an LB plate, and incubate it at 37°C for 24 hours to observe whether any bacteria grow.

[0063] 4. (1) 10 ml of the prepared water extract of Trichosanthes or Lycium barbarum was filtered through a 0.45 μm microporous filter membrane, heated at 90°C for 10 min in a water bath, cooled for 10 min, and heated again at 90°C for 10 min for later use.

[0064] (2) The solution obtained through the above steps is poured into a measuring cylinder, and the loss rate of the solution after the above sterilization steps is detected: (original solution - solution after sterilization) / original solution × 100%.

[0065] (3) Take 30 μl of the solution, spread it on an LB plate, and incubate it at 37°C for 24 hours to observe whether any bacteria grow.

[0066] 5. (1) 10 ml of the prepared water extract of Trichosanthes or Lycium barbarum was filtered through a 0.34 μm microporous filter membrane, heated at 60°C for 10 min in a water bath, cooled for 10 min, and heated again at 60°C for 10 min for later use.

[0067] (2) The solution obtained through the above steps is poured into a measuring cylinder, and the loss rate of the solution after the above sterilization steps is detected: (original solution - solution after sterilization) / original solution × 100%.

[0068] (3) Take 30 μl of the solution, spread it on an LB plate, and incubate it at 37°C for 24 hours to observe whether any bacteria grow.

[0069] 6. (1) 10 ml of the prepared water extract of Trichosanthes or Lycium barbarum was filtered through a 0.34 μm microporous filter membrane, heated at 70°C for 10 min in a water bath, cooled for 10 min, and heated again at 70°C for 10 min for later use.

[0070] (2) The solution obtained through the above steps is poured into a measuring cylinder, and the loss rate of the solution after the above sterilization steps is detected: (original solution - solution after sterilization) / original solution × 100%.

[0071] (3) Take 30 μl of the solution, spread it on an LB plate, and incubate it at 37°C for 24 hours to observe whether any bacteria grow.

[0072] 7. (1) 10 ml of the prepared water extract of Trichosanthes or Lycium barbarum was filtered through a 0.34 μm microporous filter membrane, heated at 80°C for 10 min in a water bath, cooled for 10 min, and heated again at 80°C for 10 min for later use.

[0073] (2) The solution obtained through the above steps is poured into a measuring cylinder, and the loss rate of the solution after the above sterilization steps is detected: (original solution - solution after sterilization) / original solution × 100%.

[0074] (3) Take 30 μl of the solution, spread it on an LB plate, and incubate it at 37°C for 24 hours to observe whether any bacteria grow.

[0075] 8. (1) 10 ml of the prepared water extract of Trichosanthes or Lycium barbarum was filtered through a 0.34 μm microporous filter membrane, heated at 90°C for 10 min in a water bath, cooled for 10 min, and heated again at 90°C for 10 min for later use.

[0076] (2) The solution obtained through the above steps is poured into a measuring cylinder, and the loss rate of the solution after the above sterilization steps is detected: (original solution - solution after sterilization) / original solution × 100%.

[0077] (3) Take 30 μl of the solution, spread it on an LB plate, and incubate it at 37°C for 24 hours to observe whether any bacteria grow.

[0078] 9. (1) 10 ml of the prepared water extract of Trichosanthes or Lycium barbarum was filtered through a 0.22 μm microporous filter membrane, heated at 70°C for 10 min in a water bath, cooled for 10 min, and heated again at 70°C for 10 min for later use.

[0079] (2) The solution obtained through the above steps is poured into a measuring cylinder and tested after the above sterilization steps. The loss rate of the solution = (original solution - solution after sterilization) / original solution × 100%.

[0080] (3) Take 30 μl of the solution, spread it on an LB plate, and incubate it at 37°C for 24 hours to observe whether any bacteria grow.

[0081] result:

[0082] 1. Solution loss:

[0083]

[0084]

[0085] 2. Microbial detection in sterilized solution

[0086]

[0087] The above results show that the pore size and heating temperature of the sterilization filter membrane affect the loss of the solution and the microbial colony during the sterilization process. The loss rate of the extract after filtration with a 0.45μm microporous filter membrane is lower than that of 0.34μm and 0.22μm, but it is relatively weak in filtering microorganisms. The 0.22μm filter membrane has a strong ability to filter microorganisms, but the molecular weight of the extract, especially the plant polysaccharides, is large, which makes it difficult to pass through the filter membrane, resulting in high polysaccharide loss, a small amount of final acquisition, and high cost. The 0.34μm filter membrane has a relatively strong ability to filter microorganisms, and the loss of polysaccharides is less when filtering polysaccharides. The preferred 0.34μm filter membrane is used for initial sterilization. After sterilization and heating at 60℃-80℃, the properties of the water extract remain good. Taking all factors into consideration, the sterilization method of Scheme 7 is used to sterilize the water extract solution of Trichosanthes kirilowii and the water extract solution of Lycium barbarum, so as to serve as fermentation raw materials and the preparation of subsequent microecological preparations.

[0088] Example 2 Antithrombotic Experiment of Probiotic Preparations

[0089] 1. Materials and Instruments

[0090] 1.1 Materials

[0091] Trichosanthes kirilowii and wolfberry kernel were from Shandong (origin), Lactobacillus plantarum CGMCC 1.12732, Lactobacillus casei CGMCC1.8727, zebrafish for experiments (Institute of Biology, Shandong Academy of Sciences), arachidonic acid SA9940 (Beijing Solebold Technology Co., Ltd.), aspirin M5426-01 (AbMole Biotechnology (Shanghai) Co., Ltd.), dimethyl sulfoxide (DMSO) (Sinopharm Chemical Reagent Co., Ltd.), ethanol (Sinopharm Chemical Reagent Co., Ltd.), methylene blue (Sinopharm Chemical Reagent Co., Ltd.), o-dianisidine (Sigma, USA), and methylcellulose (Shanghai Yuanye Biotechnology Co., Ltd.).

[0092] 1.2 Instruments

[0093] Refrigerator, light incubator (Donglian Electronic Technology Co., Ltd.), low-temperature centrifuge, vortexer, rotary evaporator, freeze dryer, electric constant temperature blast drying oven, ultrasonic cleaning machine, electronic balance, stereo microscope COIC-ZSA302 (Olympus Company, Japan), zebrafish breeding and reproduction system (Beijing Aisheng Technology Co., Ltd.).

[0094] 1.3 Reagent preparation

[0095] (1) Arachidonic acid (AA) stock solution: Accurately weigh 10 mg of arachidonic acid into a 1.5 mL EP tube, add 410.55 μL of DMSO, vortex to dissolve, and obtain 80 mM AA stock solution, which is then stored in a -20°C refrigerator for later use.

[0096] (2) Aspirin (ASP) stock solution: Accurately weigh 10 mg of aspirin into a 1.5 mL EP tube, add 400 μL of DMSO, vortex to dissolve, and obtain 25 mg / mL ASP stock solution, which is then stored in a -20°C refrigerator for later use.

[0097] (3) o-Dianisidine dye solution: Accurately weigh 20.5 mg of sodium acetate and 25 mg of o-dianisidine into a 25 mL volumetric flask, add 550 μL of 35% hydrogen peroxide, and finally make up to volume with 40% ethanol. Ultrasonicate until the solid is completely dissolved to obtain a 1 mg / mL o-dianisidine dye solution, which is then stored in a 4°C refrigerator for later use.

[0098] (4) Methylcellulose: Accurately weigh 8.4 g of methylcellulose powder and add it to 200 mL of hot purified water. Add the solution in small amounts and multiple times and stir continuously until the liquid cools down. Ultrasonicate at 35°C until there are no bubbles in the colloid to obtain a 1 mg / mL methylcellulose solution. Store in a refrigerator at 4°C until use.

[0099] 1.4 Sample preparation

[0100] (1) Trichosanthes kirilowii water extract: The Trichosanthes kirilowii was crushed into powder, and distilled water 10 times its weight was added, and reflux extraction was performed twice by a distillation apparatus, each time for 2 hours, and the filtrate was filtered and combined, concentrated under reduced pressure, and freeze-dried to obtain the Trichosanthes kirilowii water extract.

[0101] (2) Lycium barbarum water extract: After drying, the wolfberry is crushed by a wall-breaking machine, and then passed through a 200-mesh sieve. Three times its weight of pure water is added in batches, and ultrasonic treatment is performed at 50Hz for 30 minutes. The precipitate is removed by centrifugation at 8000rpm for 5 minutes. Three times of pure water is added to the precipitate and ultrasonic treatment is performed. This process is repeated three times. The aqueous solutions after the precipitate is removed are combined and rotary evaporated until they become a viscous semi-fluid. Five times of 96% ethanol solution is added, and the mixture is heated and stirred at 45°C for 15 minutes. The supernatant is removed by centrifugation at 5000rpm for 5 minutes. This process is repeated three times. The precipitate is dried at 25°C for 24 hours to obtain the wolfberry water extract. (3) Preparation of culture medium:

[0102] 1). 2g beef extract, 2g yeast extract, 2g sodium acetate, 1g diammonium hydrogen citrate, 2g dipotassium hydrogen phosphate, water extract of Trichosanthes kirilowii, add water to 1L, and sterilize with high pressure steam.

[0103] 2). After the above culture materials are sterilized, add the water extract solution of Trichosanthes kirilowii and / or the water extract solution of Lycium barbarum obtained by the sterilization method of Scheme 7 of Example 1, adjust the pH of the culture medium to 7, and then add lactic acid bacteria for fermentation culture at 28°C for 72h; the microecological preparation is obtained. 1.5 Sample types are as follows:

[0104] (1) Trichosanthes kirilowii aqueous extract: weigh 50 mg of Trichosanthes kirilowii aqueous extract into a conical flask, add 10 mL of purified water, vortex to dissolve, obtain 5 mg / mL of Trichosanthes kirilowii aqueous extract, and store in a 4°C refrigerator for later use. (2) Lycium barbarum aqueous extract: weigh 40 mg of Lycium barbarum aqueous extract, add 10 mL of distilled water, vortex to dissolve, obtain 4 mg / mL of Lycium barbarum aqueous extract, and store in a 4°C refrigerator for later use. (3) Mixed solution of Trichosanthes kirilowii aqueous extract and Lycium barbarum aqueous extract-1 (H-1): 100 mg of Trichosanthes kirilowii aqueous extract and 80 mg of Lycium barbarum aqueous extract, use 100 ml of distilled water to completely dissolve, and store in a 4°C refrigerator for later use.

[0105] (4) Mixed solution of Trichosanthes kirilowii water extract and Lycium barbarum water extract-2 (H-2): 50 mg of Trichosanthes kirilowii water extract and 40 mg of Lycium barbarum water extract were completely dissolved in 100 ml of distilled water and stored in a refrigerator at 4°C for later use.

[0106] (5) Probiotic preparation-1: A probiotic preparation was prepared according to the method described in 1.4(3), wherein the water extract of Trichosanthes kirilowii was 5 mg / mL, and the initial cell concentration of Lactobacillus plantarum was 1×10 4 cfu / ml, the initial cell concentration of Lactobacillus casei was 1×10 4 cfu / ml.

[0107] (6) Probiotic preparation-2: A probiotic preparation was prepared according to the method described in 1.4(3), wherein the wolfberry water extract was 4 mg / mL and the initial cell concentration of Lactobacillus plantarum was 1×10 4 cfu / ml, the initial cell concentration of Lactobacillus casei was 1×10 4 cfu / ml.

[0108] (7) Probiotic preparation-3: A probiotic preparation was prepared according to the method described in 1.4(3), wherein the water extract of Trichosanthes kirilowii was 5 mg / mL, the water extract of Lycium barbarum was 4 mg / mL, and the initial cell concentration of Lactobacillus plantarum was 1×10 4 cfu / ml.

[0109] (8) Probiotic preparation-4: A probiotic preparation was prepared according to the method described in 1.4(3), wherein the water extract of Trichosanthes kirilowii was 5 mg / mL, the water extract of Lycium barbarum was 4 mg / mL, and the initial cell concentration of Lactobacillus casei was 1×10 4 cfu / ml.

[0110] (9) Probiotic preparation-5: A probiotic preparation was prepared according to the method described in 1.4(3), wherein the water extract of Trichosanthes kirilowii was 5 mg / mL, the water extract of Lycium barbarum was 4 mg / mL, and the initial cell concentrations of Lactobacillus plantarum and Lactobacillus casei were 1×10 4 cfu / ml.

[0111] 2.1 Effects of probiotics on thrombosis

[0112] Normally developed juveniles were selected from wild zebrafish of the AB line at 72 hours after fertilization (72hpf) and placed in a 24-well plate, with 10 fish per well. A blank group, a model group, a positive control group, aspirin (25μg / mL) and water extract of Trichosanthes kirilowii, water extract of Lycium barbarum, mixed solution of water extract of Trichosanthes kirilowii and Lycium barbarum-1 (H-1), mixed solution of water extract of Trichosanthes kirilowii and Lycium barbarum-2 (H-2), probiotics-1, probiotics-2, probiotics-3, probiotics-4, probiotics-5 were set up. Fish water was added to the model group, the well plate was covered, and it was placed in a constant temperature incubator at 28°C for 6 hours. Arachidonic acid (AA) was added to the model group, the positive control group, and the extract group to a final concentration of 80μM, and then returned to the constant temperature incubator for 1.5 hours. Three replicate wells were set for each group in the above experiment, and the experiment was repeated three times. After the experiment, the solution in the well plate was drained, and o-dianisidine dye solution was added to immerse the fry. The fish were incubated for 15 minutes in a dark environment, and the dye solution was discarded. The fish were washed three times with 100% DMSO. Finally, the zebrafish were fixed on methylcellulose, and the heart of the zebrafish was observed under a stereomicroscope. Images were collected and the red blood cell staining area and staining intensity in the heart region of the zebrafish were used as indicators to evaluate whether the thrombosis in the zebrafish was serious.

[0113]

[0114] The antithrombotic activity of water extract of Trichosanthes kirilowii, water extract of Lycium barbarum and probiotics 1-5 was screened using the zebrafish model. The results in Table 1 show that the red blood cell staining area and staining intensity in the zebrafish heart region of the arachidonic acid model group were significantly reduced compared with the blank control group, indicating that the zebrafish in the model group had severe thrombosis, leading to vascular blockage and insufficient blood supply to the heart; aspirin in the positive control group can significantly alleviate the decrease in the red blood cell staining area and staining intensity induced by AA in the zebrafish heart. When the zebrafish were treated with water extract of Trichosanthes kirilowii, water extract of Lycium barbarum, H-1 group, H-2 group, probiotics-1, probiotics-2, probiotics-3, probiotics-4 and probiotics-5, the red blood cell staining area and staining intensity in the zebrafish heart region increased significantly, and the effect was most significant after treatment with probiotics-5, indicating that probiotics-5 has significant antithrombotic activity.

[0115] 2.2 Effects of Chinese herbal medicine compositions and microecological preparations prepared from mixed strains on platelet clot retraction

[0116] The stepwise screening method starts with the fermentation of one Chinese medicine to determine the optimal dose, adds a second medicine, and determines the dose of the two medicines. The indicator is the drug's ability to inhibit thrombin-induced clot retraction on platelets as the observation point, and calculates the combined index (CI).

[0117] Two kinds of Chinese herbal extracts (Trichosanthes kirilowii and Lycium barbarum) with thrombotic activity were fermented to obtain microecological preparations for platelet clot retraction detection. The standard chessboard titration method was used to evaluate the effect of combined medication using the factional inhibition concentration index (FIC). FIC was calculated as follows: ∑FIC = FICA + FICB, where FICA = MICA (in the presence of B) / MICA (alone), FICB = MICB (in the presence of A) / MICB (alone). The FIC index interpretation criteria are as follows: ≤0.5, synergistic effect; 0.5-1, additive effect; 1-4.0, irrelevant effect; >4.0, antagonistic effect. The experiment was repeated 3 times.

[0118] In the same method as above, strains are selected for fermentation to obtain microecological preparations (Lactobacillus plantarum and Lactobacillus casei) for platelet clot retraction detection.

[0119] 3. The results showed that the ∑FIC of probiotic preparation-5 (W-5) in the platelet clot retraction experiment was 0.4, indicating that the composition obtained by the combined fermentation of Lactobacillus plantarum and Lactobacillus casei has synergistic anti-thrombotic activity.

[0120] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A microecological preparation for improving thrombosis, characterized in that: The microecological preparation is obtained by adding lactic acid bacteria to the water extract of Trichosanthes kirilowii and the water extract of Lycium barbarum after sterilization for fermentation and cultivation.

2. The microecological preparation according to claim 1, characterized in that: The sterilization treatment method comprises: filtering the water extract of Trichosanthes kirilowii and the water extract of Lycium barbarum through a microporous filter membrane respectively, then heating the filtrate, circulating 1-3 times, and cooling to obtain a sterilized water extract; wherein the pore size of the microporous filter membrane is 0.22-0.45 μm, and the heating treatment temperature is controlled at 60-80°C.

3. The microecological preparation according to claim 1, characterized in that: The pore size of the microporous filter membrane is 0.34-0.45 μm, and further 0.34 μm; the microporous filter membrane is a water-based microporous filter membrane.

4. The microecological preparation according to claim 1, characterized in that: The heating treatment specifically adopts water bath heating treatment. Furthermore, the specific method of the water bath heating treatment is: water bath heating treatment at 60-80°C, water bath heating treatment time is 10-30min, the above treatment is cycled 1-3 times, the cycle interval time is 5-20min, and natural cooling is adopted during the cycle interval; further, the specific method of the water bath heating treatment is: water bath heating treatment at 80°C for 10min, cooling for 10min, and then heating at 80°C for 10min again, and naturally cooling to obtain.

5. The microecological preparation according to claim 1, characterized in that: The mass ratio of the water extract of Trichosanthes kirilowii to the water extract of Lycium barbarum is 0.5-5:1, further 0.8-2:1; The lactic acid bacteria are lactobacillus, specifically a combination of Lactobacillus plantarum and Lactobacillus casei. Further, the Lactobacillus plantarum is specifically (Lactobacillus plantarum) CGMCC1.12732, and the Lactobacillus casei is specifically Lactobacillus casei (Lactobacillus casei) CGMCC 1.8727.

6. The microecological preparation according to claim 1, characterized in that: The water extract of Trichosanthes kirilowii is prepared by the following method: crushing the Trichosanthes kirilowii into powder, adding water for reflux extraction, and taking the filtrate for vacuum concentration and drying to obtain the water extract of Trichosanthes kirilowii; Wherein, the mass ratio of the Trichosanthes powder to water is 1:8-15, preferably 1:10; The reflux extraction is performed 1-3 times, preferably 2 times, each time for 2 hours, and the filtrate is filtered and combined.

7. The microecological preparation according to claim 1, characterized in that: The preparation method of the wolfberry water extract comprises: grinding and sieving the wolfberry, adding water and performing ultrasonic treatment, centrifuging and removing the precipitate from the water solution, performing rotary evaporation, adding ethanol solution thereto, heating and stirring, centrifuging and drying the precipitate to obtain the wolfberry water extract; Wherein, the sieve mesh number is 180-220 mesh; The specific conditions of the ultrasonic treatment are: ultrasonic frequency is 30-70 Hz, and the ultrasonic treatment lasts 10-60 min.

8. The microecological preparation according to claim 7, characterized in that: The ethanol solution is high-concentration ethanol, specifically 96% ethanol solution. More specifically, the volume ratio of the residue after rotary evaporation to the ethanol solution is 1:

5.

9. Use of the microecological preparation for improving thrombosis according to any one of claims 1 to 8 in the preparation of anti-thrombotic products.

10. The use according to claim 9, characterized in that: The product is a food or a medicine.

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