A complex with stroke treatment characteristics, its preparation method and application

Abstract

The present invention discloses a complex with stroke treatment characteristics, its preparation method and application. The complex is a complex formed by yeast peptide and hydroxytyrosol; the yeast peptide is a yeast peptide with a molecular weight greater than or equal to 1 kDa and less than 3 kDa produced by papain enzymolysis of yeast protein; when papain enzymolyzes yeast protein, the pH is 6.8 - 7.2, the enzymolysis time is 3.8 - 4.2 h, the enzymolysis temperature is 48 - 52 °C, and the enzyme amount / mass ratio of papain to yeast protein in the enzymolysis system is 8800 - 9200 U / g. The complex is used for manufacturing drugs for treating ischemic stroke. The present invention provides a complex with stroke treatment characteristics that is efficient, safe and economical, has broad application prospects and significant technical advantages, and has a simple preparation method and low cost.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology. Specifically, it is a complex with stroke treatment characteristics, its preparation method and application. Background Art

[0002] Stroke is an acute cerebrovascular disease, characterized by high incidence, high disability rate and high mortality rate, which brings a heavy burden to society and families. At present, the treatment of stroke mainly relies on thrombolytic therapy, antiplatelet therapy, neuroprotective therapy and other means, but these methods all have certain limitations. For example, thrombolytic therapy is limited by problems such as a narrow treatment time window and high bleeding risk; antiplatelet therapy may cause side effects such as bleeding and gastrointestinal discomfort; and the existing neuroprotective drugs have limited efficacy and are difficult to meet clinical needs.

[0003] In recent years, with the continuous development of biotechnology, researchers have begun to explore the use of novel biomolecules such as polypeptides and nanozymes as potential drugs for the treatment of stroke. For example, the research group led by Researcher Ren Lai of the Kunming Institute of Zoology cooperated with Professor Heyu Ni of the University of Toronto and others to identify an active polypeptide with significant therapeutic effects from the forest mountain leech. This polypeptide can specifically inhibit the activities of kallikrein and factor XII, shows good therapeutic effects on both transient ischemic stroke and permanent ischemic stroke, has good biosafety and antithrombotic effects, and also has no bleeding risk, possessing significant characteristics for candidate drug development.

[0004] However, this polypeptide needs to be obtained from the forest mountain leech, and the production is inconvenient and the cost is relatively high. Therefore, it is necessary to further develop novel, efficient, safe and relatively low-cost polypeptides with therapeutic effects on stroke to meet clinical needs. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a complex with stroke treatment characteristics, its preparation method and application, which are efficient, safe and economical. The preparation method of this complex is simple, the cost is low, and it has good therapeutic effects and biosafety.

[0006] To solve the above technical problems, the present invention provides the following technical solutions:

[0007] A complex with stroke treatment properties, the complex containing yeast peptide and hydroxytyrosol; the yeast peptide is a yeast peptide with a molecular weight greater than or equal to 1 kDa and less than 3 kDa produced by papain enzymolysis of yeast protein; when papain enzymolyzes yeast protein, the pH is 6.8 - 7.2, the enzymolysis time is 3.8 - 4.2 h, the enzymolysis temperature is 48 - 52 °C, and the enzyme amount / mass ratio of papain to yeast protein in the enzymolysis system is 8800 - 9200 U / g. Experiments have proved that the yeast peptide prepared by this method has a certain effect of alleviating stroke in mice, and the complex formed by this yeast peptide and hydroxytyrosol also shows an effect of alleviating stroke and reversing oxidative damage during ischemia-reperfusion in mouse experiments.

[0008] The above complex with stroke treatment properties, the complex further containing lecithin and / or a phospholipid membrane prepared from lecithin; the preparation method of the phospholipid membrane is to dissolve lecithin in chloroform and then evaporate the chloroform. Introducing lecithin or a phospholipid membrane into the complex can promote the combination of yeast peptide and hydroxytyrosol.

[0009] The above complex with stroke treatment properties, when lecithin is added to the complex: the molar ratio of yeast peptide, hydroxytyrosol and lecithin is (6.5 - 38.9):(6.5 - 38.9):(0.1 - 0.5); when a phospholipid membrane is added to the complex, the amount of the phospholipid membrane is calculated according to the amount of lecithin used to prepare the phospholipid membrane: the molar ratio of yeast peptide, hydroxytyrosol and the phospholipid membrane is (6.5 - 38.9):(6.5 - 38.9):(0.1 - 0.5).

[0010] The preparation method of the above complex with stroke treatment properties includes the following steps:

[0011] (1) Prepare a yeast protein powder dispersion from yeast-made yeast protein powder.

[0012] (2) Oscillate the yeast protein powder dispersion at the enzymolysis reaction temperature.

[0013] (3) Add papain to the yeast protein powder dispersion for enzymolysis, and obtain an enzymolyzed solution after the enzymolysis ends.

[0014] (4) Heat the enzymolyzed solution to the protease inactivation temperature and keep it warm, then cool the enzymolyzed solution and adjust the pH of the enzymolyzed solution.

[0015] (5) Centrifuge the enzymolyzed solution and take the supernatant, and successively perform ultrafiltration and freeze-drying on the supernatant to obtain a yeast peptide with a molecular weight greater than or equal to 1 kDa and less than 3 kDa.

[0016] (6) Disperse yeast peptide and hydroxytyrosol in phosphate buffer to form a composite system, and stir to obtain a yeast peptide-hydroxytyrosol complex dispersion;

[0017] (7) Purify and freeze-dry the yeast peptide-hydroxytyrosol complex dispersion to obtain a yeast peptide-hydroxytyrosol complex, and the yeast peptide-hydroxytyrosol complex is the above-mentioned complex with stroke treatment characteristics.

[0018] For the preparation method of the above-mentioned complex with stroke treatment characteristics, in step (1), prepare a yeast protein powder dispersion using phosphate buffer, and the pH of the phosphate buffer is equal to the pH during enzymatic hydrolysis; the content of yeast protein in the yeast protein powder is greater than or equal to 80 wt%, and in the yeast protein powder dispersion, the concentration of yeast protein powder is 48-50 g / L.

[0019] For the preparation method of the above-mentioned complex with stroke treatment characteristics, in step (2), the oscillation time is 10-15 min; in step (4), the protease inactivation temperature is 95-100 °C, and the holding time is 10-15 min; after the enzymatic hydrolysate is naturally cooled, adjust the pH of the enzymatic hydrolysate to 7.0.

[0020] For the preparation method of the above-mentioned complex with stroke treatment characteristics, in step (5), when centrifuging the enzymatic hydrolysate, the centrifugation temperature is 4-8 °C, the rotation speed during centrifugation is 10000-12000 rpm, and the centrifugation time is 10-20 min;

[0021] In step (6), the molar ratio of yeast peptide to hydroxytyrosol in the composite system is 1:1, and the concentration of hydroxytyrosol in the composite system is 1-6 mg / mL; after preparing the composite system, add lecithin and / or a phospholipid membrane made of lecithin to the composite system; the amount of the phospholipid membrane in the composite system is calculated according to the amount of substance of lecithin, and the molar concentration of lecithin in the composite system is 0.1-0.5 mmol / L.

[0022] For the preparation method of the above-mentioned complex with stroke treatment characteristics, in step (6), during the stirring process, perform cyclic heating on the composite system; the method of cyclic heating is to heat the composite system to 48-52 °C at a heating rate of 3-7 °C / min, hold for 20-30 min, and then naturally cool to room temperature until the stirring ends; the stirring time is 20-26 h.

[0023] For the preparation method of the above-mentioned complex with stroke treatment characteristics, in step (1), a yeast protein powder dispersion is prepared using a phosphate buffer solution with a pH of 7.0; the yeast protein content in the yeast protein powder is greater than or equal to 80 wt%, and in the yeast protein powder dispersion, the concentration of the yeast protein powder is 50 g / L;

[0024] In step (2), the oscillation time is 10 min;

[0025] In step (3), the enzymatic hydrolysis time is 4 h, the enzymatic hydrolysis temperature is 50 °C, the pH during enzymatic hydrolysis is 7, and the enzyme amount / mass ratio of papain to yeast protein in the enzymatic hydrolysis system is 9000 U / g;

[0026] In step (4), the protease inactivation temperature is 100 °C and the heat preservation time is 10 min; after the enzymatic hydrolysis solution is naturally cooled, the pH of the enzymatic hydrolysis solution is adjusted to 7.0;

[0027] In step (5), when centrifuging the enzymatic hydrolysis solution, the centrifugation temperature is 4 °C, the rotation speed during centrifugation is 10000 rpm, and the centrifugation time is 15 min;

[0028] In step (6), the molar ratio of yeast peptide to hydroxytyrosol in the composite system is 1:1, and the concentration of hydroxytyrosol in the composite system is 5 mg / mL; after preparing the composite system, lecithin and / or a phospholipid membrane made of lecithin is added to the composite system; the amount of the phospholipid membrane in the composite system is calculated according to the amount of lecithin, and the molar concentration of lecithin in the composite system is 0.1 mmol / L; during the stirring process, the composite system is heated in a cycle; the method of cyclic heating is to heat the composite system to 50 °C at a heating rate of 5 °C / min, keep it warm for 30 min, and then naturally cool it to room temperature until the stirring ends; the stirring time is 24 h. When yeast peptide and hydroxytyrosol are combined in a molar ratio of 1:1, the contact and combination between yeast peptide and hydroxytyrosol are relatively efficient. Cyclic heating can promote the full contact and combination of yeast peptide and hydroxytyrosol, and improve the formation efficiency of the complex. The control of temperature helps to improve the stability of the complex, avoid the degradation or inactivation of components caused by high temperature, and thus enhance the curative effect of the complex.

[0029] For the application of the above-mentioned complex with stroke treatment characteristics, the complex is used to manufacture a drug for treating ischemic stroke.

[0030] The technical solution of the present invention has achieved the following beneficial technical effects:

[0031] 1. The present invention provides a complex with stroke treatment characteristics. This complex is prepared by compounding yeast peptide and hydroxytyrosol, and exhibits obvious treatment characteristics for ischemic stroke. The results of animal experiments show that the cerebral infarction area of mice injected with this yeast peptide-hydroxytyrosol complex is significantly decreased, and the neurological score is also significantly reduced; this yeast peptide-hydroxytyrosol complex can reduce the content of reactive oxygen species in the mouse brain tissue, enhance the activity of superoxide dismutase, enhance the content of the antioxidant glutathione, reverse the oxidative damage during ischemia-reperfusion, and has no obvious toxic and side effects. This complex is expected to become a new drug for the treatment of stroke, providing a safer and more effective treatment means for clinical practice.

[0032] 2. The complex of the present invention is prepared by enzymatically hydrolyzing yeast protein with papain. The process is simple, the raw materials are easy to obtain, and the production cost is low. Compared with some polypeptide drugs that need to be extracted from special organisms, the complex of the present invention is more economical and feasible. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The results of TCC staining of the brains of sham-operated mice and MCAO mice injected with PBS buffer, yeast peptide, and yeast peptide-hydroxytyrosol complex in Example 1 of the present invention;

[0034] Figure 2 The statistical results of the cerebral infarction area of sham-operated mice and MCAO mice injected with PBS buffer, yeast peptide, and yeast peptide-hydroxytyrosol complex in Example 1 of the present invention;

[0035] Figure 3 The neurological score results of sham-operated mice and MCAO mice injected with PBS buffer, yeast peptide, and yeast peptide-hydroxytyrosol complex in Example 1 of the present invention;

[0036] Figure 4 The measurement results of the ROS content in the brain tissues of sham-operated mice and tMCAO model mice injected with PBS buffer, yeast peptide, and yeast peptide-hydroxytyrosol complex in Example 2 of the present invention;

[0037] Figure 5 The measurement results of the SOD activity in the brain tissues of sham-operated mice and tMCAO model mice injected with PBS buffer, yeast peptide, and yeast peptide-hydroxytyrosol complex in Example 2 of the present invention;

[0038] Figure 6 The measurement results of the GSH content in the brain tissues of sham-operated mice and tMCAO model mice injected with PBS buffer, yeast peptide, and yeast peptide-hydroxytyrosol complex in Example 2 of the present invention;

[0039] Figure 7Results of determination of MDA content in the brain tissues of mice in the sham operation group and tMCAO model mice injected with PBS buffer, yeast peptide, and yeast peptide-hydroxytyrosol complex in Example 2 of the present invention. Detailed implementation mode

[0040] Example 1

[0041] First, the complex was prepared. The specific preparation method is as follows:

[0042] (1) Angel AnPro80 yeast protein powder was selected. This yeast protein powder was made from yeast. The nominal yeast protein content of this yeast protein powder was 80 wt%. 50 g of yeast protein powder was weighed and formulated into a yeast protein powder dispersion. The specific method was to add 50 g of yeast protein powder to 1 L of phosphate buffer (PBS buffer) with a pH of 7.0, and shake well to obtain a yeast protein powder dispersion with a yeast protein powder concentration of 50 g / L.

[0043] (2) The yeast protein powder dispersion was placed in a water bath thermostatic oscillator at 50 °C and shaken for 10 min. After this step, the temperature of the yeast protein powder dispersion would stabilize at 50 °C, ensuring that the subsequent enzymatic hydrolysis reaction started under suitable temperature conditions from the beginning, thereby improving the reaction efficiency and effect.

[0044] (3) Papain was added to the yeast protein powder dispersion to enzymatically hydrolyze yeast protein. 9000 U of papain was added per gram of yeast protein. Calculated according to the yeast protein content of 80 wt% in the yeast protein powder in this example, the concentration of yeast protein in the yeast protein powder dispersion was 40 g / L, that is, 360000 U of papain was added to each liter of yeast protein powder dispersion. The enzymatic hydrolysis time was 4 h, and the enzymatic hydrolysis temperature was 50 °C. After the enzymatic hydrolysis was completed, an enzymatic hydrolysate was obtained.

[0045] (4) The enzymatic hydrolysate was heated to 100 °C (denaturation temperature) and kept warm for 10 min to inactivate the protease. After it naturally cooled to room temperature, the pH was adjusted to 7.0 with 1 mol / L NaOH or 1 mol / L HCl.

[0046] (5) The pH-adjusted enzymatic hydrolysate was centrifuged using a low-temperature high-speed centrifuge. The centrifugation temperature was 4 °C, the rotation speed was 10000 rpm, and the centrifugation time was 15 min. After centrifugation, the supernatant was retained, and an enzymatic hydrolysis product with a molecular weight greater than or equal to 1 kDa and less than 3 kDa was ultrafiltered from the supernatant using an ultrafiltration centrifugal tube, and freeze-dried to obtain yeast peptide.

[0047] (6) Calculate the dosages of yeast peptide and hydroxytyrosol, and prepare a composite system by adding yeast peptide and hydroxytyrosol to PBS buffer according to a molar ratio of yeast peptide to hydroxytyrosol of 1:1 (calculate the amount of substance of yeast peptide based on the average molecular mass of yeast peptide being 1133.29 Da). The concentration of hydroxytyrosol in the composite system is 5 mg / mL, and the concentration of yeast peptide in the composite system is 36.8 mg / mL. In some other embodiments, the concentration of hydroxytyrosol in the composite system can also be other values within the range of 1 - 6 mg / mL.

[0048] To enable better binding of yeast peptide and hydroxytyrosol, add lecithin or a phospholipid membrane made from lecithin to the composite according to a concentration of 0.1 mmol / L (it can also be prepared according to any value between 0.1 - 0.5 mmol / L for the concentration of lecithin or phospholipid membrane, and the amount of the phospholipid membrane is calculated based on the amount of substance of lecithin used to prepare the phospholipid membrane). The preparation method of the phospholipid membrane is: dissolve lecithin in chloroform and then evaporate the chloroform. The relatively hydrophilic head of lecithin can respectively bind to the alcohol hydroxyl group in hydroxytyrosol and the hydrophilic group in yeast peptide, and the tails of lecithin can bind to each other. In addition, some hydrophobic groups in yeast peptide can bind to the tails of lecithin (while without adding phospholipids, these hydrophobic groups cannot bind to hydroxytyrosol). Therefore, adding lecithin or the phospholipid membrane can play a role in promoting the mutual binding of hydroxytyrosol and yeast peptide and forming a complex.

[0049] After adding lecithin, heat the composite system in a cycle while stirring. Cyclic heating means that first heat the composite system to 50 °C at a heating rate of 5 °C / min, keep it warm for 30 min, and then naturally cool it to room temperature. Repeat this cycle and continue stirring for 24 h. After the stirring is completed, a yeast peptide-hydroxytyrosol complex dispersion is obtained.

[0050] In some other embodiments, the heating rate for cyclic heating is within the range of 3 - 7 °C / min, the temperature after heating is within the range of 48 - 52 °C, and the stirring time is within the range of 20 - 26 h.

[0051] (7) Purify the yeast peptide-hydroxytyrosol complex dispersion. Transfer the above yeast peptide-hydroxytyrosol complex dispersion to a centrifuge tube equipped with a 1 kDa cut-off molecular weight ultrafiltration membrane and centrifuge. The conditions for the centrifugation operation are 4 °C, a rotation speed of 10000 rpm, and a centrifugation time of 15 minutes. After centrifugation is completed, carefully discard the filtrate passing through the ultrafiltration membrane and retain the filtrate above the ultrafiltration membrane. To further remove unbound yeast peptide and hydroxytyrosol, add an appropriate amount of ultrapure water above the ultrafiltration membrane, mix well, and then centrifuge again. Repeat this washing and centrifugation step twice, each time centrifuging at 4 °C and 10000 rpm for 15 minutes.

[0052] To test whether the prepared yeast peptide-hydroxytyrosol complex has a therapeutic effect on ischemic cerebral infarction, an animal experiment was conducted. First, a mouse model of ischemic stroke (MCAO mouse model) was constructed through surgery. The specific steps are as follows:

[0053] 1. Anesthetize C57BL / 6J mice by intraperitoneal injection of sodium pentobarbital (50 mg / kg) to ensure that the mice are completely unconscious.

[0054] 2. Make an incision about 1 cm long in the midline of the mouse neck. Use forceps and scissors to gently separate the skin and muscle to expose the right common carotid artery, internal carotid artery, and external carotid artery.

[0055] 3. Gently separate and clamp the common carotid artery, internal carotid artery, and external carotid artery with hemostatic forceps to prevent blood flow. Make a small incision at the end of the external carotid artery (near the branching point) (this incision is the stump of the external carotid artery), insert a 6-0 suture with a length of 30 mm into the internal carotid artery through the stump of the external carotid artery, and slowly push it forward to block the middle cerebral artery.

[0056] During the operation, maintain the mouse body temperature between 37.0 and 37.5 °C using a heating pad.

[0057] In addition, set up a sham operation group. In the sham operation group, no suture is inserted into the arterial blood vessels, and the remaining steps are the same as those for constructing the mouse model of ischemic stroke above.

[0058] Randomly divide the MCAO mice into 3 groups, with 6 mice in each group. After 60 min of MCAO (middle cerebral artery occlusion), for the 3 groups of MCAO mice, inject PBS buffer, yeast peptide dispersion (prepared with PBS buffer, concentration 0.01 M), and yeast peptide-hydroxytyrosol complex dispersion (prepared with PBS buffer, concentration 0.01 M) into the tail vein respectively, and the injection volume is 200 μL for all. At the same time, inject an equal volume of PBS buffer into the mice in the sham operation group.

[0059] After 1.5 h of injection, remove the suture in the blood vessel to simulate clinical treatment intervention for thrombectomy and restore the blood flow of the middle cerebral artery.

[0060] Evaluate the neurological score of the mice, and then sacrifice the mice and take the brain tissue for TCC staining. The staining results are as Figure 1 shown. The white part in the figure represents the cerebral infarction area. Sham represents the sham operation group, PBS represents the MCAO mice injected with PBS buffer, yeast peptide represents the MCAO mice injected with yeast peptide dispersion, and "LP@complex" represents the MCAO mice injected with yeast peptide-hydroxytyrosol complex dispersion.

[0061] The area of the cerebral infarction region was measured, and the proportion of the infarction area was calculated. The results are as Figure 2 shown. In the figure, Sham represents the sham operation group, PBS represents the MCAO mice injected with PBS buffer, yeast peptide represents the MCAO mice injected with yeast peptide dispersion, and "LP@Complex" represents the MCAO mice injected with yeast peptide-hydroxytyrosol complex dispersion. From Figure 2 the figure, it can be seen that the infarction area of the mice in the PBS group (i.e., the control group) was about 40% (i.e., the area of the infarction region accounted for 40% of the cerebral surface area), the infarction area of the mice in the yeast peptide group was about 15%, and the infarction area of the mice in the yeast peptide-hydroxytyrosol complex group was about 5%. From the experimental results, injecting the yeast peptide-hydroxytyrosol complex dispersion had an obvious therapeutic effect on MCAO mice, and it could reduce the cerebral infarction area of MCAO mice by about 87.5% (even compared with using hydroxytyrosol alone, the cerebral infarction area of the mice also decreased by 58.3%).

[0062] As Figure 3 shown in the figure are the evaluation results of the neurological scores of each group of mice. In the figure, Sham represents the sham operation group, PBS represents the MCAO mice injected with PBS buffer, yeast peptide represents the MCAO mice injected with yeast peptide dispersion, and "LP@Complex" represents the MCAO mice injected with yeast peptide-hydroxytyrosol complex dispersion. The neurological score is to evaluate the neurological function of the mice, and the scoring standard is the five-point method of Bederson. According to this scoring standard, the score of the mice in the PBS group was 3.2, the score of the mice in the yeast peptide group was 1.5, and the score of the mice in the yeast peptide-hydroxytyrosol complex group was 1.2. From the scoring results, the neurological score of the MCAO mice injected with the yeast peptide-hydroxytyrosol complex dispersion was significantly lower.

[0063] The above results indicate that the yeast peptide-hydroxytyrosol complex prepared in this example is a complex with stroke treatment characteristics.

[0064] Example 2

[0065] When ischemic stroke occurs, the interruption of cerebral blood flow causes local cerebral tissue ischemia and hypoxia, and the restoration of cerebral blood flow will further cause cerebral ischemia-reperfusion injury (CIRI). As is well known, oxidative stress is an important factor that exacerbates CIRI. Therefore, inhibiting oxidative stress is particularly important for reducing neurological function damage in stroke.

[0066] Experimental animals: SPF-grade C57BL / 6 male mice, 8 weeks old, with a body weight of 20 - 25 g.

[0067] Experimental design:

[0068] 1. Establishment of the mouse tMCAO model: After anesthesia, the heads and limbs of the mice were fixed in the supine position. After disinfecting the neck with iodine tincture, a 1-cm midline incision was made. The right common carotid artery, external carotid artery, and internal carotid artery were sequentially dissected using forceps. The proximal ends of the common carotid artery and external carotid artery were ligated, and the distal end of the internal carotid artery was clamped with an artery clip. A suture was reserved at the distal end of the common carotid artery, and an inverted V-shaped notch was cut at the proximal end and the arterial bifurcation to allow the insertion of the suture plug. The suture plug was inserted into the internal carotid artery through the incision. After tying the reserved suture tightly without affecting the movement of the suture plug, the artery clip was opened. The suture plug was pushed to the bifurcation of the internal and external arteries and then fixed. The wound was sutured and iodine tincture was applied to prevent infection. After 2 hours of MCA occlusion, the suture plug was removed by 10 mm to achieve blood reperfusion for 72 hours. The modeling process of the sham operation group was the same as that of the tMCAO group, but the suture plug was not inserted.

[0069] 2. Grouping and administration: The mice were randomly divided into 4 groups, with 10 mice in each group. The corresponding reagents were injected through the tail vein, and the injection volume was 200 μL for all. Experimental group 1 was injected with the yeast peptide-hydroxytyrosol complex YP@HT prepared in Example 1 (prepared with PBS buffer, concentration 0.2 mg / mL), experimental group 2 was injected with the yeast peptide YP prepared in Example 1 (concentration 0.2 mg / mL), experimental group 3 was injected with hydroxytyrosol HT (concentration 0.2 mg / mL), and the sham operation group and the control group were respectively injected with an equal volume of PBS buffer.

[0070] Evaluation of oxidative brain injury:

[0071] Seven days after administration, the mice were anesthetized and sacrificed by cervical dislocation. Subsequently, the brain tissues were quickly removed and the olfactory bulbs were removed in physiological saline and the residual blood stains were washed away. 0.5 g of brain tissue was taken, the surface moisture was blotted dry with filter paper, and physiological saline was added according to a ratio of 1:9. The homogenate was centrifuged to obtain the supernatant. The contents of ROS (reactive oxygen species), SOD (superoxide dismutase) activity, GSH (glutathione), and MDA (malondialdehyde) in the brain tissues were measured according to the instructions of the kits (all produced by Beyotime Biotechnology).

[0072] Result analysis:

[0073] As Figure 4 shows the ROS detection results (the vertical axis represents the multiple of the ROS content compared to the sham operation group), Figure 5 shows the SOD activity detection results, Figure 6 shows the GSH content detection results, Figure 7 shows the MDA content detection results. Figures 5 to 7 In

[0074] The occurrence and development of CIRI are closely related to oxidative stress. The burst of reactive oxygen species (ROS) generates oxidative stress. SOD is an endogenous antioxidant enzyme that plays a role in scavenging free radical activity; MDA is a lipid peroxidation product caused by oxidative stress (the content of MDA is positively correlated with the degree of oxidative damage to cells); GSH can maintain the normal immune activity of the body and has antioxidant activity. The above indicators can be used as biomarkers for the degree of oxidative damage to nerve tissue.

[0075] It can be seen from Figures 4 to 7 that compared with the Sham group, the PBS group had low levels of SOD and GSH, and an increased level of MDA, indicating that oxidative damage occurred in the mouse brain tissue; the yeast peptide-hydroxytyrosol complex could reverse the above oxidative damage, and the effect was better than that of yeast peptide and hydroxytyrosol alone.

[0076] Comparative Example 1

[0077] In this comparative example, a yeast peptide was prepared. The difference between this yeast peptide and the yeast peptide prepared in Example 1 was that during the enzymatic hydrolysis process, alkaline protease was used to replace papain, and the pH during enzymatic hydrolysis was adjusted to 9, and the remaining conditions and methods were the same as those in Example 1.

[0078] Comparative Example 2

[0079] In this comparative example, a yeast peptide was prepared. The difference between this yeast peptide and the yeast peptide prepared in Example 1 was that during the enzymatic hydrolysis process, flavor protease was used to replace papain, and the remaining conditions and methods were the same as those in Example 1.

[0080] Comparative Example 3

[0081] In this comparative example, a yeast peptide was prepared. The difference between this yeast peptide and the yeast peptide prepared in Example 1 was that when papain was used to enzymatically hydrolyze yeast protein, the enzymatic hydrolysis temperature was 60 °C and the enzymatic hydrolysis time was 8 h.

[0082] Comparative Example 4

[0083] In this comparative example, a yeast peptide was prepared. The difference between this yeast peptide and the yeast peptide prepared in Example 1 was that the enzyme amount / mass ratio of papain to yeast protein in the enzymatic hydrolysis system was 12000 U / g.

[0084] When preparing yeast peptides by enzymatic hydrolysis of yeast proteins, the type of enzyme, the amount of enzyme, and the conditions during enzymatic hydrolysis can all affect the fragment size of the prepared yeast peptides, and further affect the amino acid composition of the yeast peptides with a molecular weight greater than or equal to 1 kDa and less than 3 kDa collected. Yeast peptides with different amino acid compositions also have different biological properties. In order to compare the effects of the yeast peptide-hydroxytyrosol complexes prepared in Example 1, Comparative Example 1, and Comparative Example 2 on reducing oxidative stress damage to nerve tissues in CI RI, using the same method as in Example 2, the nerve repair effects of the yeast peptides in Comparative Examples 1, 2, 3, and 4 combined with hydroxytyrosol were tested in a mouse tMCAO model.

[0085] Table 1 shows the measurement results of reducing oxidative stress damage to the brain tissues of mice in the tMCAO model after injection of each yeast peptide-hydroxytyrosol complex.

[0086] Table 1

[0087]

[0088] From the results in the table, it can be seen that the ROS content in the mice injected with the yeast peptide-hydroxytyrosol complex prepared in Example 1 (mice in the Example 1 group) is lower, the SOD activity is higher, the GSH content is higher, and the MDA content is lower, indicating that the degree of oxidative stress damage to the nerve tissues of the mice in the Example 1 group is lower. The effect of the yeast peptide-hydroxytyrosol complex prepared in Example 1 on reducing oxidative stress damage to the nerve tissues of mice in the tMCAO model is better than that of the yeast peptide-hydroxytyrosol complexes prepared in Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4.

[0089] Obviously, the above examples are only illustrations clearly made and not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the claims of this patent application.

Claims

1. A complex with stroke treatment characteristics, characterized in that, The complex contains yeast peptide and hydroxytyrosol; the yeast peptide is a yeast peptide with a molecular weight of not less than 1 kDa and less than 3 kDa produced by papain enzymolysis of yeast protein; when papain enzymolyzes yeast protein, the pH is 6.8 - 7.2, the enzymolysis time is 3.8 - 4.2 h, the enzymolysis temperature is 48 - 52 °C, and the enzyme amount / mass ratio of papain to yeast protein in the enzymolysis system is 8800 - 9200 U / g; The complex further contains lecithin and / or a phospholipid membrane prepared from lecithin; the preparation method of the phospholipid membrane is to dissolve lecithin in chloroform and then evaporate the chloroform to dryness; When lecithin is added to the complex: the molar ratio of yeast peptide, hydroxytyrosol and lecithin is (6.5 - 38.9):(6.5 - 38.9):(0.1 - 0.5); when the phospholipid membrane is added to the complex, the amount of the phospholipid membrane is calculated according to the amount of lecithin used for preparing the phospholipid membrane: the molar ratio of yeast peptide, hydroxytyrosol and the phospholipid membrane is (6.5 - 38.9):(6.5 - 38.9):(0.1 - 0.5); When preparing the complex, the complex system is prepared by adding yeast peptide and hydroxytyrosol to PBS buffer according to the molar ratio of yeast peptide and hydroxytyrosol of 1:1; lecithin or a phospholipid membrane prepared from lecithin is added to the complex system, and then the complex system is stirred while being heated in a cycle.

2. The preparation method of the complex with stroke treatment characteristics as described in claim 1, characterized in that, It includes the following steps: (1) Prepare a yeast protein powder dispersion from yeast - made yeast protein powder; (2) Oscillate the yeast protein powder dispersion at the enzymolysis reaction temperature; (3) Add papain to the yeast protein powder dispersion for enzymolysis, and an enzymolysis solution is obtained after the enzymolysis ends; (4) Heat the enzymolysis solution to the protease inactivation temperature and keep it warm, then cool the enzymolysis solution and adjust the pH of the enzymolysis solution; (5) Centrifuge the enzymolysis solution and take the supernatant, and successively perform ultrafiltration and freeze - drying on the supernatant to obtain a yeast peptide with a molecular weight of not less than 1 kDa and less than 3 kDa; (6) Disperse yeast peptide and hydroxytyrosol in phosphate buffer to form a complex system, stir to obtain a yeast peptide - hydroxytyrosol complex dispersion; during the stirring process, the complex system is heated in a cycle; the method of heating in a cycle is to heat the complex system to 48 - 52 °C at a heating rate of 3 - 7 °C / min, keep it warm for 20 - 30 min, and then naturally cool it to room temperature until the stirring ends; the stirring time is 20 - 26 h; (7) Purify and freeze - dry the yeast peptide - hydroxytyrosol complex dispersion to obtain a yeast peptide - hydroxytyrosol complex, and the yeast peptide - hydroxytyrosol complex is the complex with the characteristics of treating stroke.

3. The preparation method of the complex with stroke treatment characteristics according to claim 2, characterized in that, In step (1), a yeast protein powder dispersion is prepared using phosphate buffer, and the pH of the phosphate buffer is equal to the pH during enzymolysis; the content of yeast protein in the yeast protein powder is not less than 80 wt%, and in the yeast protein powder dispersion, the concentration of yeast protein powder is 48 - 50 g / L.

4. The preparation method of the complex with stroke treatment characteristics according to claim 2, characterized in that, In step (2), the oscillation time is 10 - 15 min; in step (4), the protease inactivation temperature is 95 - 100 °C and the heat preservation time is 10 - 15 min; after the enzymatic hydrolysate is naturally cooled, the pH of the enzymatic hydrolysate is adjusted to 7.

0.

5. The preparation method of the complex with stroke treatment properties according to claim 2, characterized in that, In step (5), when centrifuging the enzymatic hydrolysate, the centrifugation temperature is 4 - 8 °C, the rotation speed during centrifugation is 10000 - 12000 rpm, and the centrifugation time is 10 - 20 min; In step (6), the molar ratio of yeast peptide to hydroxytyrosol in the composite system is 1:1, and the concentration of hydroxytyrosol in the composite system is 1 - 6 mg / mL; after preparing the composite system, lecithin and / or a phospholipid membrane made of lecithin is added to the composite system; the amount of the phospholipid membrane in the composite system is calculated according to the amount of substance of lecithin, and the molar concentration of lecithin in the composite system is 0.1 - 0.5 mmol / L.

6. The preparation method of the complex with stroke treatment characteristics according to claim 2, characterized in that, In step (1), a yeast protein powder dispersion is prepared using a phosphate buffer solution with a pH of 7.0; the yeast protein content in the yeast protein powder is greater than or equal to 80 wt%, and in the yeast protein powder dispersion, the concentration of the yeast protein powder is 50 g / L; In step (2), the oscillation time is 10 min; In step (3), the enzymatic hydrolysis time is 4 h, the enzymatic hydrolysis temperature is 50 °C, the pH during enzymatic hydrolysis is 7, and the enzyme amount / mass ratio of papain to yeast protein in the enzymatic hydrolysis system is 9000 U / g; In step (4), the protease inactivation temperature is 100 °C and the heat preservation time is 10 min; after the enzymatic hydrolysate is naturally cooled, the pH of the enzymatic hydrolysate is adjusted to 7.0; In step (5), when centrifuging the enzymatic hydrolysate, the centrifugation temperature is 4 °C, the rotation speed during centrifugation is 10000 rpm, and the centrifugation time is 15 min; In step (6), the molar ratio of yeast peptide to hydroxytyrosol in the composite system is 1:1, and the concentration of hydroxytyrosol in the composite system is 5 mg / mL; after preparing the composite system, lecithin and / or a phospholipid membrane made of lecithin is added to the composite system; the amount of the phospholipid membrane in the composite system is calculated according to the amount of substance of lecithin, and the molar concentration of lecithin in the composite system is 0.1 mmol / L; during the stirring process, the composite system is subjected to cyclic heating; the method of cyclic heating is to heat the composite system to 50 °C at a heating rate of 5 °C / min, keep it warm for 30 min, and then naturally cool it to room temperature until the stirring ends; the stirring time is 24 h.

7. Use of the complex having stroke treatment characteristics as described in claim 1, characterized in that, The complex is used for manufacturing a drug for treating ischemic stroke.

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