Preparation method of safflower seed oil with thrombus removing function and application thereof

Abstract

The present application belongs to the technical field of safflower seed extraction, and particularly relates to a preparation method of safflower seed oil and application thereof. The preparation method comprises the following steps: drying and crushing safflower seeds to obtain safflower seed powder; adding water to the safflower seed powder and adding a compound enzyme to obtain an enzyme solution through enzymolysis; and adding a strain to the enzyme solution for fermentation, centrifugation and collection of an upper oil phase. The present application breaks through the bottleneck of the traditional process, significantly improves the quality of safflower seed oil, increases the stability, and has a stronger inhibitory effect on thrombosis.

Description

Technical Field

[0001] This invention belongs to the field of safflower seed extraction technology, specifically relating to a method for preparing safflower seed oil with thrombolytic function and its application. Background Technology

[0002] Safflower seed oil, a typical representative of traditional Chinese medicinal and edible plant oils, contains core active ingredients including unsaturated fatty acids such as linoleic acid and polyphenolic compounds. Modern medical research has confirmed its multiple physiological functions, including regulating lipid metabolism, improving blood circulation, anti-oxidation, and anti-inflammation. In the current context of high cardiovascular disease prevalence, safflower seed oil can effectively reduce the risk of cardiovascular and cerebrovascular events by lowering blood viscosity, inhibiting platelet aggregation, and protecting vascular endothelial function, making it an important choice for preventative intervention. However, current safflower seed oil preparation technologies still have significant shortcomings, leading to severe product homogenization and insufficient efficacy stability, making it difficult to meet the precise nutritional needs of the health-conscious consumer trend.

[0003] Currently, the main industrial methods for producing safflower seed oil are pressing and solvent extraction. Pressing extracts oil from safflower seeds using external pressure, requiring high-quality raw materials with high oil content. No other chemicals are introduced during processing. However, the oil yield and oilseed meal utilization are relatively low, making it suitable for high-oil-content oilseed crops such as rapeseed, sesame, and sunflower seeds. Solvent extraction, on the other hand, utilizes organic solvents that are similarly miscible with oils to extract oil from the seed. This method results in a low residual oil content in the oilseed meal, indicating thorough oil extraction, but the crude oil may contain some solvent residue. Although both processes have been widely industrialized, they still have certain limitations.

[0004] In recent years, researchers have continuously improved and enhanced the processing methods for safflower seed oil. Due to the low oil content of safflower seeds, research on various auxiliary extraction technologies to increase yield has increased, such as ultrasonic-assisted extraction. With the development of vegetable oil extraction technology, more and more oil preparation methods have emerged, such as microwave-assisted extraction, supercritical CO2 extraction, and subcritical fluid extraction.

[0005] A thrombus is a solid mass formed by the abnormal coagulation of blood within a blood vessel. It can obstruct blood flow, leading to tissue ischemia, organ dysfunction, and in severe cases, death. It does not only occur in the elderly; it can happen at any age and at any time. Moreover, 99% of thrombi have no obvious symptoms in their early stages, earning them the nickname "silent killer."

[0006] The causes of thrombosis include: when the inner wall of a blood vessel is damaged, collagen fibers are exposed, activating platelet aggregation and initiating the coagulation system. Common triggers include trauma, surgery, rupture of atherosclerotic plaques, and chronic diseases such as hypertension and diabetes. Smoking and high blood sugar can also impair vascular endothelial function, increasing the risk of thrombosis. When blood flow slows down, clotting factors and platelets tend to accumulate locally, forming a thrombus. Certain physiological or pathological conditions can make blood more prone to clotting, increasing the risk of thrombosis.

[0007] Safflower seed oil is extremely rich in unsaturated fatty acids, reaching up to 90%, with a high proportion of linoleic acid. Linoleic acid is known as a "vascular cleanser," possessing important benefits such as protecting the cardiovascular system. Furthermore, the phenolic compounds and other active substances in safflower seed oil also have protective effects on the liver, cardiovascular system, and as antioxidants.

[0008] Therefore, there is an urgent need for an innovative preparation method to break through the bottleneck of traditional processes, significantly increase the content of polyphenols and unsaturated fatty acids such as linoleic acid in safflower seed oil, and provide a highly efficient and stable safflower seed oil product with good inhibitory effects on thrombosis, thus filling a market gap. Summary of the Invention

[0009] To overcome the above technical problems, this invention provides a method for preparing safflower seed oil with thrombolytic function and its application.

[0010] To achieve the above objectives, the technical solution provided by this invention is as follows: On one hand, the present invention provides a method for preparing safflower seed oil with thrombolytic function, comprising the following steps: (1) Wash the safflower seeds, dry them at low temperature, and pulverize them to obtain safflower seed powder; (2) Add water to safflower seed powder and add compound enzymes, enzymatically hydrolyze, and then add brewer's yeast and Lactobacillus plantarum to ferment and obtain fermentation liquid; (3) Sterilize the fermentation broth, centrifuge, and collect the upper oil phase.

[0011] As some specific embodiments of the present invention, the low-temperature drying in step (1) is drying at 70-90°C to constant weight; the mass ratio of water to safflower seed powder in step (2) is 10-20:1.

[0012] As some specific embodiments of the present invention, the complex enzymes in step (2) include cellulase, papain and Candida antarcticis lipase B.

[0013] As some specific embodiments of the present invention, the mass ratio of cellulase, papain and Candida antarcticis lipase B in step (2) is 1:1-5:0.5-2.

[0014] As some specific embodiments of the present invention, the total amount of the compound enzyme added in step (2) is 0.5-3% of the mass of water.

[0015] As some specific embodiments of the present invention, the enzymatic hydrolysis in step (2) includes first adding cellulase and hydrolyzing at a temperature of 45-55℃ and a pH of 4.5-5.5 for 1-3 hours, then adding papain and hydrolyzing at a temperature of 50-65℃ and a pH of 6-7 for 1-2 hours, and finally adding Candida antarcticis lipase B and hydrolyzing at a temperature of 60-65℃ and a pH of 8.0-9.0 for 1-2 hours.

[0016] As some specific embodiments of the present invention, the inoculation amounts of Saccharomyces cerevisiae and Lactobacillus plantarum in step (2) are 1-3% and 0.5-2%, respectively.

[0017] As some specific embodiments of the present invention, the fermentation temperature in step (2) is 27-32°C and the fermentation time is 48-96h.

[0018] As some specific embodiments of the present invention, the enzymatic hydrolysate after enzymatic hydrolysis in step (2) needs to be sterilized at a temperature of 118-125°C for 18-35 minutes.

[0019] As some specific embodiments of the present invention, the centrifugation in step (3) is centrifugation at 8000-10000 r / min for 15-35 min.

[0020] On the other hand, the present invention provides the application of safflower seed oil prepared by the above preparation method in the preparation of antithrombotic drugs.

[0021] Explanation of technical features: "Thrombolysis" is a colloquial term for the act of "removing blood clots from the body." In the medical field, the formal term corresponding to "thrombolysis" is thrombolytic therapy, which refers to the active dissolution of existing blood clots through medication or mechanical means to restore vascular patency. Its core mechanism is the activation of the body's own fibrinolytic system, which allows fibrin in the blood clot to be degraded into soluble fragments by plasminogen activators, thereby dissolving the clot. Thrombolytic therapy is mainly applicable to acute thrombotic diseases, such as: acute ischemic stroke (within 4.5 hours of onset), acute myocardial infarction (within 12 hours of onset), acute pulmonary embolism, and large-area deep vein thrombosis.

[0022] Safflower seeds: scientific name: Carthamus tinctorius L., are the dried, mature achenes of safflower, an annual herbaceous plant of the genus Carthamus in the family Asteraceae. They are also known as "white flat seeds" or "grass safflower seeds" and are multifunctional plant seeds with medicinal, edible and industrial value.

[0023] Safflower, belonging to the genus *Carthamus* of the family Asteraceae, is scientifically named *Carthamus tinctorius* L., and is also known as safflower or red and blue safflower. The plant grows to 30–100 cm tall, with an erect stem bearing longitudinal ridges. The leaves are ovate-lanceolate with spines along the margins. The terminal capitula bear tubular florets that are initially yellow, gradually turning orange-red to deep red. The achenes are obovate or elliptical, about 5–8 mm long, quadrangular, with a whitish or grayish-white surface speckled with brown, and lack a pappus. Native to Central Asia and Egypt, it was introduced to China via the Silk Road during the Western Han Dynasty, first appearing in the *Essential Prescriptions of the Golden Chamber*. China is now the world's main producer, with the Tacheng region of Xinjiang accounting for over 70% of the national output. It is also widely cultivated in Yunnan, Henan, and Sichuan. It prefers sunlight, is drought-tolerant, salt-tolerant, and highly adaptable. Flowering occurs from June to July, and fruiting from August to September.

[0024] Microbial fermentation is a green and efficient method for modifying lipids. Specific microbial strains (such as lipase-producing bacteria) can selectively hydrolyze the ester bonds of triglycerides to generate monotriglycerides and diglycerides. Compared with traditional chemical hydrolysis, fermentation has the advantages of mild conditions (ambient temperature and pressure), high selectivity, and diverse products (possibly accompanied by the production of beneficial metabolites such as glycolipids). Small molecule products (especially monotriglycerides and ditriglycerides) have better water dispersibility and antioxidant activity.

[0025] Compared with the prior art, the technical advantages of the present invention are as follows: (1) The present invention uses a complex enzyme system of cellulase, papain and lipase to promote the extraction of safflower seed oil, which has a synergistic effect on cell wall degradation, oil release efficiency and process stability.

[0026] (2) The present invention selects specific lipases to improve the extraction efficiency in the microbial fermentation extraction of oil by catalyzing oil hydrolysis, promoting cell structure destruction and releasing intracellular oil, and at the same time plays a key role in the lipid metabolism regulation of oil-producing microorganisms.

[0027] (3) In the "water-enzyme extraction" process of this invention, the specific lipase, papain and cellulase used work synergistically to not only participate in the early enzymatic hydrolysis process, enabling the enzymatic hydrolysis process to more thoroughly degrade the cell walls and lipoprotein complexes of oilseeds, thus allowing the oil to be fully released, but also selectively hydrolyze triglycerides, transesterification, esterification and other reverse reactions, participating in the regulation of fat synthesis. Studies have found that the specific enzymatic hydrolysis process of this invention significantly improves the quality of safflower seed oil.

[0028] (4) This invention employs specific microbial fermentation, using Saccharomyces cerevisiae and Lactobacillus plantarum to produce safflower seed oil that possesses high oxidative stability, high oil yield, and excellent application performance. This oil not only retains abundant ω-3 polyunsaturated fatty acids but is also rich in bioactive polyphenols and other substances, providing a reference for developing high-stability, high-value-added deep-processed safflower seed oil products and expanding its application in food, functional food, dietary supplements, and other fields. Detailed Implementation

[0029] The present invention will be described below through specific embodiments to make the technical solution of the present invention easier to understand and master, but the present invention is not limited thereto. Unless otherwise specified, the experimental methods described in the following embodiments are conventional methods; the reagents and materials described, unless otherwise specified, can be obtained commercially. Specifically, *Saccharomyces cerevisiae* strain number: CICC1222, purchased from the China Industrial Microbiological Culture Collection Center; *Lactobacillus plantarum* strain number: JYLP-002, supplied by Shandong Zhongke Jiayi. *Saccharomyces cerevisiae* strain number: CGMCC 2.3880, purchased from the China General Microbiological Culture Collection Center. *Lactobacillus plantarum* strain number: CICC 20022, purchased from the China Industrial Microbiological Culture Collection Center. *Candida blakeana* strain number: ATCC 22214, purchased from Ningbo Mingzhou Biotechnology Co., Ltd., product code B80544.

[0030] Example 1 This embodiment provides a method for preparing safflower seed oil, the steps of which are as follows: (1) Wash the safflower seeds, dry them at 80°C to constant weight, pulverize them, and pass them through a 30-mesh sieve to obtain safflower seed powder; (2) Add 15 times the amount of water to the safflower seed powder, add 2.5% of the water mass of the compound enzyme for enzymatic hydrolysis. The compound enzyme is cellulase, papain and Candida antarcticis lipase B in a ratio of 1:1:0.5. The specific operation is as follows: first add cellulase and hydrolyze at 45℃ and pH 5.0 for 2 hours, then add papain and hydrolyze at 55℃ and pH 6 for 1 hour, and finally add Candida antarcticis lipase B and hydrolyze at 60℃ and pH 8.0 for 1 hour to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was sterilized at 121℃ for 20 min, and then the seed liquid of Saccharomyces cerevisiae and seed liquid of Lactobacillus plantarum were added at an inoculation amount of 2% and 0.5% respectively. Fermentation was carried out at 30℃ and pH 5.5 for 48 h, and the fermentation liquid was obtained by filtration. The seed culture was prepared by inoculating Saccharomyces cerevisiae CICC1222 and Lactobacillus plantarum JYLP-002 into MRS liquid medium at an inoculation amount of 1% and fermenting at 37°C for 24 hours to obtain Saccharomyces cerevisiae seed culture and Lactobacillus plantarum seed culture, respectively.

[0031] (3) Sterilize the fermentation broth, centrifuge at 9000 r / min for 20 min, and collect the upper oil phase.

[0032] Example 2 This embodiment provides a method for preparing safflower seed oil, the steps of which are as follows: (1) Wash the safflower seeds, dry them at 90°C to constant weight, pulverize them, and pass them through a 30-mesh sieve to obtain safflower seed powder; (2) Add 15 times the amount of water to the safflower seed powder, and add 0.8% of the water mass of the compound enzyme for enzymatic hydrolysis. The compound enzyme is cellulase, papain and Candida antarcticis lipase B in a 1:5:2 ratio. The specific operation is as follows: first add cellulase and hydrolyze at 45℃ and pH 4.5 for 1 hour, then add papain and hydrolyze at 50℃ and pH 6 for 1 hour, and finally add Candida antarcticis lipase B and hydrolyze at 60℃ and pH 8.0 for 1 hour to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was sterilized at 121℃ for 20 min, and then the seed liquid of Saccharomyces cerevisiae and seed liquid of Lactobacillus plantarum were added at an inoculation amount of 1% and 0.5% respectively. The mixture was fermented at 30℃ for 48 h, filtered, and the fermentation liquid was obtained. The seed culture was prepared by inoculating Saccharomyces cerevisiae CICC1222 and Lactobacillus plantarum JYLP-002 into MRS liquid medium at an inoculation amount of 1% and fermenting at 37°C for 24 hours to obtain Saccharomyces cerevisiae seed culture and Lactobacillus plantarum seed culture, respectively.

[0033] (3) Sterilize the fermentation broth, centrifuge at 8000 r / min for 35 min, and collect the upper oil phase.

[0034] Example 3 This embodiment provides a method for preparing safflower seed oil, the steps of which are as follows: (1) Wash the safflower seeds, dry them at 70°C to constant weight, pulverize them, and pass them through a 30-mesh sieve to obtain safflower seed powder; (2) Add 15 times the amount of water to the safflower seed powder, and add 1.2% of the water mass of a compound enzyme for enzymatic hydrolysis. The compound enzyme is a 1:2:1 mixture of cellulase, papain and Candida antarcticis lipase B. The specific operation is as follows: first add cellulase and hydrolyze at 45-55℃ and pH 5.5 for 2 hours, then add papain and hydrolyze at 65℃ and pH 7 for 1 hour, and finally add Candida antarcticis lipase B and hydrolyze at 65℃ and pH 9.0 for 1 hour to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was sterilized at 121℃ for 20 min, and then the seed liquid of Saccharomyces cerevisiae and seed liquid of Lactobacillus plantarum were added at an inoculation amount of 2% and 1% respectively. The mixture was fermented at 30℃ for 48 h, filtered, and the fermentation liquid was obtained. The seed culture was prepared by inoculating Saccharomyces cerevisiae CICC1222 and Lactobacillus plantarum JYLP-002 into MRS liquid medium at an inoculation amount of 1% and fermenting at 37°C for 24 hours to obtain Saccharomyces cerevisiae seed culture and Lactobacillus plantarum seed culture, respectively.

[0035] (3) Sterilize the fermentation broth, centrifuge at 10000r / min for 15min, and collect the upper oil phase.

[0036] Comparative Example 1 The difference between this comparative example and Example 1 is that *Candida antarcticis* lipase B was not added. Specifically: (1) Wash the safflower seeds, dry them at 80°C to constant weight, pulverize them, and pass them through a 30-mesh sieve to obtain safflower seed powder; (2) Add 15 times the amount of water to the safflower seed powder, add 2.5% of the water mass of the compound enzyme for enzymatic hydrolysis. The compound enzyme is cellulase and papain in a 1:1.5 ratio. The specific operation is to first add cellulase and hydrolyze at 45℃ and pH 5.0 for 2 hours, and then add papain and hydrolyze at 55℃ and pH 6 for 2 hours to obtain the hydrolysate. The enzymatic hydrolysate was sterilized at 121℃ for 20 min, and then the seed liquid of Saccharomyces cerevisiae and seed liquid of Lactobacillus plantarum were added at an inoculation amount of 2% and 0.5% respectively. The mixture was fermented at 30℃ for 48 h, filtered, and the fermentation liquid was obtained. The seed culture was prepared by inoculating Saccharomyces cerevisiae CICC1222 and Lactobacillus plantarum JYLP-002 into MRS liquid medium at an inoculation amount of 1% and fermenting at 37°C for 24 hours to obtain Saccharomyces cerevisiae seed culture and Lactobacillus plantarum seed culture, respectively.

[0037] (3) Sterilize the fermentation broth, centrifuge at 9000 r / min for 20 min, and collect the upper oil phase.

[0038] Comparative Example 2 The difference between this comparative example and Example 1 is that papain was not added. Specifically: (1) Wash the safflower seeds, dry them at 80°C to constant weight, pulverize them, and pass them through a 30-mesh sieve to obtain safflower seed powder; (2) Add 15 times the amount of water to the safflower seed powder, add 2.5% of the water mass of the compound enzyme for enzymatic hydrolysis. The compound enzyme is cellulase and Candida antarcticis lipase B in a 1:1.5 ratio. The specific operation is to first add cellulase and hydrolyze at 45℃ and pH 5.0 for 2 hours, and then add Candida antarcticis lipase B and hydrolyze at 60℃ and pH 8.0 for 2 hours to obtain the hydrolysate. The enzymatic hydrolysate was sterilized at 121℃ for 20 min, and then the seed liquid of Saccharomyces cerevisiae and seed liquid of Lactobacillus plantarum were added at an inoculation amount of 2% and 0.5% respectively. The mixture was fermented at 30℃ for 48 h, filtered, and the fermentation liquid was obtained. The seed culture was prepared by inoculating Saccharomyces cerevisiae CICC1222 and Lactobacillus plantarum JYLP-002 into MRS liquid medium at an inoculation amount of 1% and fermenting at 37°C for 24 hours to obtain Saccharomyces cerevisiae seed culture and Lactobacillus plantarum seed culture, respectively.

[0039] (3) Sterilize the fermentation broth, centrifuge at 9000 r / min for 20 min, and collect the upper oil phase.

[0040] Comparative Example 3 The difference between this comparative example and Example 1 lies in the preparation method. Details are as follows: (1) Wash the safflower seeds, dry them at 80°C to constant weight, pulverize them, and pass them through a 30-mesh sieve to obtain safflower seed powder; (2) Add 15 times the amount of water to the safflower seed powder, add 2.5% of the water mass of the compound enzyme for enzymatic hydrolysis. The compound enzyme is cellulase, papain and Candida antarcticis lipase B in a ratio of 1:1:0.5. The specific operation is as follows: first add cellulase and hydrolyze at 45℃ and pH 5.0 for 2 hours, then add papain and hydrolyze at 55℃ and pH 6 for 1 hour, and finally add Candida antarcticis lipase B and hydrolyze at 60℃ and pH 8.0 for 1 hour to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was sterilized at 121℃ for 20 min, and then the seed liquid of Saccharomyces cerevisiae and seed liquid of Lactobacillus plantarum were added at an inoculation amount of 2% and 0.5% respectively. Fermentation was carried out at 30℃ and pH 5.5 for 48 h, and the fermentation liquid was obtained by filtration. The seed culture was prepared as follows: Saccharomyces cerevisiae CGMCC 2.3880 and Lactobacillus plantarum CICC 20022 were inoculated into MRS liquid medium at an inoculation amount of 1% and fermented at 37°C for 24 hours to obtain Saccharomyces cerevisiae seed culture and Lactobacillus plantarum seed culture, respectively.

[0041] (3) Sterilize the fermentation broth, centrifuge at 9000 r / min for 20 min, and collect the upper oil phase.

[0042] Comparative Example 4 The difference between this comparative example and Example 1 lies in the preparation method. Details are as follows: (1) Wash the safflower seeds, dry them at 80°C to constant weight, pulverize them, and pass them through a 30-mesh sieve to obtain safflower seed powder; (2) Add 15 times the amount of water to the safflower seed powder, add 2.5% of the water mass of the compound enzyme for enzymatic hydrolysis. The compound enzyme is cellulase, papain and Candida antarcticis lipase B in a ratio of 1:1:0.5. The specific operation is as follows: first add cellulase and hydrolyze at 45℃ and pH 5.0 for 2 hours, then add papain and hydrolyze at 55℃ and pH 6 for 1 hour, and finally add Candida antarcticis lipase B and hydrolyze at 60℃ and pH 8.0 for 1 hour to obtain the enzymatic hydrolysate. The enzymatic hydrolysate was sterilized at 121℃ for 20 min, and then 2.5% of the seed culture of *Candida bacillus* ATCC 22214 was added. The mixture was fermented at 30℃ and pH 5.5 for 48 h, filtered, and the fermentation broth was obtained. The seed culture was prepared by inoculating 1% of *Candida baccata* ATCC 22214 into MRS liquid medium and fermenting at 37°C for 24 hours to obtain the seed culture.

[0043] (3) Sterilize the fermentation broth, centrifuge at 9000 r / min for 20 min, and collect the upper oil phase.

[0044] Experiment 1: Determination of Physicochemical Properties of Extracted Safflower Seed Oil (1) Safflower seed oil yield (%) = mass of safflower seed oil / mass of safflower seed raw material × 100%.

[0045] (2) The acid value of safflower seed oil was determined according to GB5009.229-2016.

[0046] (3) The determination of peroxide value of safflower seed oil shall be in accordance with GB5009.227-2016.

[0047] The safflower seed oil extracted in each embodiment and comparative example of the present invention was subjected to physicochemical index determination according to the above-mentioned determination standards. The experiment was conducted in parallel for 3 times, and the average value was taken. The results are shown in Table 1.

[0048] Table 1

[0049] Conclusion: This invention utilizes a synergistic enzymatic hydrolysis-fermentation extraction method to extract safflower seed oil, resulting in low acid value and stable peroxide value. While enzymatic hydrolysis using only a combination of cellulase and lipase yields acceptable acid and peroxide values, it fails to fully release the bound oil, impacting the oil extraction rate. Using only cellulase and protease easily leads to excessive protein hydrolysis and activation of esterase activity, resulting in increased acid value and decreased stability. Fermentation with other microbial strains results in a slightly lower oil yield, but the acid and peroxide values ​​are poorer, inevitably affecting the quality and stability of the oil.

[0050] Experiment 2: Storage Stability Experiment To comprehensively evaluate the stability of the extracted safflower seed oil under different environments, five portions of each oil were placed in capped glass bottles and kept in a constant temperature incubator at (45±1)℃ for 3 months under the required conditions. The odor was observed and recorded, and the acid value and peroxide value were tested. The results are shown in Table 2.

[0051] Table 2

[0052] Conclusion: The oils extracted in Examples 1-3 and Comparative Examples 1-2 of this invention have high stability, with Examples 1-3 being superior.

[0053] Experiment 3: Verification of the efficacy of safflower seed oil (1) Experimental materials SPF-grade SD rats, weighing 300±20g; enteric-coated aspirin tablets (National Drug Approval Number HJ20160685, manufacturer: Bayer Healthcare Co., Ltd., product specification 100mg) 30 tablets; distilled water, chloral hydrate.

[0054] (2) Experimental methods Animal grouping and administration: SD rats were randomly divided into a model group, a positive group, Example 1-3 groups, and Comparative Example 1-4 groups, with 8 rats in each group. Except for the model group, the rats in the other groups were administered safflower seed oil prepared for their respective groups by gavage at a dose of 0.02 mg / g BW once a day for 7 consecutive days. The normal group was given an equal volume of distilled water. One hour after the last administration, the rats were anesthetized with 10% chloral hydrate, fixed in a supine position, and the abdominal wall was opened. The inferior vena cava was separated, and the inferior vena cava was ligated at the intersection of the left renal vein and the inferior vena cava. The abdominal wall was sutured for 6 hours, and the abdominal cavity was reopened. The blood vessel was clamped 2 cm below the ligation, the lumen was opened, the embolus was removed, the residual blood was absorbed with filter paper, and the wet weight was weighed using an electronic balance. The embolus was then placed in an oven at 70°C for 2 hours and weighed dry. The thrombus formation inhibition rate was calculated using the following formula.

[0055] Thrombosis inhibition rate (%) = (Thrombus weight in model group - Thrombus weight in treatment group) / Thrombus weight in model group × 100%.

[0056] (3) Experimental results The results of wet weight, dry weight and thrombus formation inhibition rate of rat thrombi in each group are shown in Table 3.

[0057] Table 3

[0058] Note: Compared with the model group, p < 0.05 p < 0.01; compared with Example 1 group, #p < 0.05, ##p < 0.01.

[0059] The results showed that all examples and comparative examples exhibited significant activity in reducing the wet and dry weight of thrombi. In terms of efficacy, the positive group performed best, with a wet weight inhibition rate of 45.30% and a dry weight inhibition rate of 43.90%. Examples 1-3 also showed inhibitory effects close to those of the positive drug, and were statistically quite effective.

[0060] Comparative Examples 1-2 used other enzymatic hydrolysis processes, and Comparative Examples 3-4 used other microbial fermentation. The safflower seed oil extracted from these methods showed a lower inhibition rate against thrombosis than the extraction process of this invention. This invention uses specific enzyme combinations, which synergistically enhance the enzymatic destruction of cell walls and cell membranes, thereby completely releasing the oil. Simultaneously, the use of specific microbial fermentation increases the polyphenol content in the oil phase and reduces the acid value and peroxide value of the oil, comprehensively improving the quality and functionality of safflower seed oil.

[0061] In summary, safflower seeds not only have significant economic value but also extremely high nutritional and medicinal value. The safflower seed oil obtained by this invention is extremely rich in unsaturated fatty acids, reaching up to 90%, with linoleic acid content as high as 85%. Linoleic acid is known as a "vascular cleanser" and, as an essential fatty acid, has important effects such as protecting the cardiovascular system, promoting blood circulation, and accelerating metabolism. Furthermore, fermentation greatly improves its stability and also makes it rich in phenolic compounds and other active substances. Studies have shown that the safflower seed oil of this invention has good potential for inhibiting thrombus formation and has promising application prospects.

[0062] The above detailed description is a specific description of one of the feasible embodiments of the present invention. This embodiment is not intended to limit the patent scope of the present invention. All equivalent implementations or modifications that do not depart from the present invention should be included within the scope of the technical solution of the present invention.

Claims

1. A method for preparing safflower seed oil with thrombolytic function, characterized in that, Includes the following steps: (1) Wash the safflower seeds, dry them at low temperature, and pulverize them to obtain safflower seed powder; (2) Add water to safflower seed powder and add compound enzymes for enzymatic hydrolysis, then add brewing yeast and Lactobacillus plantarum for fermentation to obtain fermentation liquid; (3) Sterilize the fermentation broth, centrifuge, and collect the upper oil phase.

2. The preparation method according to claim 1, characterized in that, The low-temperature drying in step (1) is drying at 70-90℃ to constant weight; the mass ratio of water to safflower seed powder in step (2) is 10-20:

1.

3. The preparation method according to claim 1, characterized in that, The complex enzymes mentioned in step (2) include cellulase, papain and Candida antarctica lipase B.

4. The preparation method according to claim 3, characterized in that, The mass ratio of cellulase, papain and Candida antarcticis lipase B in step (2) is 1:1-5:0.5-2; And / or, the total amount of the compound enzyme added in step (2) is 0.5-3% of the mass of water.

5. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis in step (2) includes first adding cellulase and hydrolyzing at a temperature of 45-55℃ and a pH of 4.5-5.5 for 1-3 hours, then adding papain and hydrolyzing at a temperature of 50-65℃ and a pH of 6-7 for 1-2 hours, and finally adding Candida antarcticis lipase B and hydrolyzing at a temperature of 60-65℃ and a pH of 8.0-9.0 for 1-2 hours.

6. The preparation method according to claim 1, characterized in that, The inoculation amounts of Saccharomyces cerevisiae and Lactobacillus plantarum in step (2) are 1-3% and 0.5-2%, respectively.

7. The preparation method according to claim 1, characterized in that, The fermentation temperature in step (2) is 27-32℃, the fermentation pH is 5-6, and the fermentation time is 48-96h.

8. The preparation method according to claim 1, characterized in that, In step (2), the enzymatic hydrolysate after enzymatic hydrolysis needs to be sterilized at a temperature of 118-125℃ for 18-35 minutes.

9. The preparation method according to claim 1, characterized in that, The centrifugation mentioned in step (3) is centrifugation at 8000-10000 r / min for 15-35 min.

10. The use of safflower seed oil prepared by the preparation method according to any one of claims 1-9 in the preparation of antithrombotic products.

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