Lyophilized cardiomyopeptide composition as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly discloses a cardiomyopeptide freeze-dried composition as well as a preparation method and application thereof. The cardiomyopeptide freeze-dried composition comprises the following components in parts by mass: 12-15 parts of cardiomyopeptide, 8-11 parts of radix ophiopogonis, 6-8 parts of schisandra chinensis, 3-5 parts of hawthorn, 1-2 parts of spirulina powder and 1-2 parts of blueberry powder. The invention discloses a cardiomyopeptide freeze-dried composition and a preparation method and application thereof. The cardiomyopeptide freeze-dried composition is high in activity and good in stability, can relieve myocardial damage and can be applied to preparation of drugs for nourishing myocardium and protecting blood vessels.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a freeze-dried composition of myocardial peptides and a preparation method and application thereof. BACKGROUND

[0002] Myocardial health is the core of maintaining the normal function of the human cardiovascular system. At present, products for nourishing myocardium and protecting blood vessels mainly include two categories: one is chemical synthetic drugs, such as some energy metabolism regulators, which can take effect quickly but may have side effects and are mostly therapeutic drugs, not suitable for long-term use as nutritional health products; the other is natural product extracts, such as traditional Chinese medicine compounds or some single plant active ingredients. Traditional Chinese medicine compounds have strong systemic regulation, but have limitations in the explicitness of the action mechanism, the standardization of the components and the speed of taking effect. Single active ingredients often have a single action target, and it is difficult to achieve the effect of multi-pathway and synergistic protection.

[0003] Myocardial peptides are a group of low-molecular-weight active peptides extracted from animal hearts, which have multiple physiological functions such as improving myocardial metabolism, enhancing myocardial contractility and antioxidant activity, and are considered to be one of the ideal components for nourishing myocardium. At present, the preparation of myocardial peptides mostly adopts direct enzymatic hydrolysis. However, this method has some inherent defects. First, the enzymatic hydrolysis efficiency is limited, and the peptide yield is not high; second, the enzymatic hydrolysis process is not strong in specificity, and some bitter peptides or ineffective fragments may be produced, affecting the taste and activity of the product, and the molecular weight distribution of myocardial peptides prepared by conventional processes is wide, and the purity and concentration of the target active peptides need to be improved. SUMMARY

[0004] The application aims to provide a freeze-dried composition of myocardial peptides and a preparation method and application thereof, which has high activity, good stability, can relieve myocardial injury and can be applied to the preparation of drugs for nourishing myocardium and protecting blood vessels.

[0005] To solve the above technical problems, the technical scheme adopted by the application is as follows: A freeze-dried composition of myocardial peptides, comprising the following components in mass parts: 12-15 parts of myocardial peptides, 8-11 parts of radix ophiopogonis, 6-8 parts of schisandra chinensis, 3-5 parts of hawthorn, 1-2 parts of spirulina powder and 1-2 parts of blueberry powder.

[0006] The application further provides a preparation method of the freeze-dried composition of myocardial peptides, comprising the following steps: S1, mixing and grinding radix ophiopogonis and schisandra chinensis, sieving, adding hawthorn, adding 8-10 times the amount of deionized water, decocting 2-3 times, each time for 1-2 hours, and combining the extract; S2, mixing blueberry powder and spirulina powder, adding deionized water, stirring uniformly, and ultrasonic treatment to obtain a mixture; S3, mixing the extract obtained in S1 with the mixture obtained in S2, low-temperature vacuum concentration to obtain a reaction product; S4, mixing the reaction product obtained in S3 with the myocardial peptide, stirring uniformly, freeze-drying, crushing, sieving to obtain a freeze-dried myocardial peptide composition.

[0007] Preferably, in S2, the stirring speed is 300-450 rpm, and the stirring time is 20-30 min.

[0008] Preferably, in S2, the ultrasonic treatment power is 300-400 W, the temperature is 30-40 , and the time is 10-15 min.

[0009] Preferably, in S3, the low-temperature vacuum concentration temperature is 45-50 , the vacuum degree is -0.06 to -0.08 MPa, and the time is 1-2 h.

[0010] Preferably, in S4, the stirring speed is 300-500 rpm, and the stirring time is 35-45 min.

[0011] Preferably, in S3, the specific process parameters of freeze-drying are as follows: first frozen to -45 ~-30 , keep for 2-4 h, open the vacuum system, reduce the pressure in the tank to 10-30 Pa, increase the temperature to -20 ~-10 , and maintain for 10-20 h.

[0012] Preferably, in S4, the preparation method of the myocardial peptide comprises the following steps: T1, taking a healthy pig myocardium with connective tissue removed, washing with physiological saline until no blood color, crushing, mixing with deionized water, adjusting the pH to 6.5-7.5, grinding to obtain a homogenate; T2, inoculating the complex strain into the homogenate obtained in T1, and fermenting to obtain a fermentation liquor; T3, ultrasonic treatment of the fermentation liquor obtained in T2, adjusting the pH to neutral, adding neutral protease, enzyme hydrolysis, enzyme hydrolysis, heating to inactivate the enzyme, centrifugation, filtration, discarding the precipitate to obtain an enzyme hydrolysis liquor; T4, adding L-arginine with a weight of 1.5%-2% of the weight of the homogenate to the enzyme hydrolysis liquor obtained in T3, ultrasonic stirring, passing through 10 kDa and 3 kDa ultrafiltration membranes in sequence to obtain a myocardial peptide.

[0013] In T1, the pig myocardium is mixed with deionized water at a weight ratio of (0.4-0.6):(1.2-1.5).

[0014] Preferably, in T2, the compound microbial strain is composed of lactic acid bacteria and Aspergillus in a live count ratio of (1:3) to (2:4), and the fermentation temperature is 30-35°C. The fermentation time is 24 hours.

[0015] Preferably, in T3, the specific process parameters for the ultrasonic treatment are: ultrasonic power of 550~600W and extraction temperature of 40~50°C. The extraction time is 15-20 minutes, and the enzymatic hydrolysis temperature is 45-50°C. The enzymatic hydrolysis time is 2-6 hours.

[0016] Preferably, in T3, the temperature for inactivating the enzyme by heating is 85-90°C. The time is 10-15 minutes.

[0017] Preferably, in T4, the ultrasonic stirring power is 300~500W, the time is 20~30min, and the temperature is 30~35℃. The stirring speed is 300~450 rpm.

[0018] The present invention also provides the use of the lyophilized cardiac peptide composition as described above in the preparation of a medicament for nourishing the myocardium and protecting blood vessels.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects: This invention discloses a lyophilized composition of cardiac peptides, its preparation method, and its applications. By combining cardiac peptides with Ophiopogon japonicus, Schisandra chinensis, hawthorn, spirulina powder, and blueberry powder, the components exhibit a clear synergistic effect, resulting in a product with both high peptide activity and excellent stability. Animal experiments have confirmed that the lyophilized composition of cardiac peptides has a clear protective effect against myocardial injury. Compared with the model control group, the example group significantly reduced the levels of myocardial injury markers cTnI, inflammatory factors, and oxidative stress, with effects comparable to the positive control drug, fully validating its application value in nourishing the myocardium and protecting blood vessels.

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0021] Fig. 1 This is a statistical chart showing the changes in moisture content in different groups according to the present invention; Fig. 2 This is a line graph showing the change of polypeptide content over time in different groups of this invention; Fig. 3 This is a graph showing the trend of DPPH clearance rate over time in different groups according to the present invention. Detailed Implementation

[0022] The technical solutions of the present application are further described below by means of the drawings and examples.

[0023] Unless otherwise defined, the technical terms or scientific terms used in the present application shall have the usual meanings understood by those with ordinary skills in the art to which the present application belongs.

[0024] Sources of test materials: Lactic acid bacteria were purchased from Weifang Yihao Biotechnology Co., Ltd.; and Aspergillus was purchased from Yishui Jinrun Biotechnology Co., Ltd.

[0025] In the present application, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0026] Example 1 Prepare the following parts by mass: 13.5 parts of myocardial peptide, 9.5 parts of Ophiopogon japonicus, 7 parts of Schisandra chinensis, 4 parts of hawthorn, 1.5 parts of spirulina powder, and 1.5 parts of blueberry powder.

[0027] The preparation method of myocardial peptide includes the following steps: T1. Take 1 kg of healthy pig myocardium with connective tissue removed, rinse with physiological saline until no blood color, then grind, mix with 3 kg of deionized water, adjust the pH to 7.0, and grind into a homogenate; T2. Inoculate the homogenate obtained in T1 with complex strains (the ratio of lactic acid bacteria to Aspergillus viable bacteria is 2:3), and ferment at 32 for 24 h to obtain a fermentation broth; T3. Treat the fermentation broth obtained in T2 at an ultrasonic power of 575 W for 45 min, adjust the pH to 7.0, add neutral protease, and enzymatically hydrolyze at 47 for 4 h, heat at 88 for 12 min to inactivate the enzyme, centrifuge and filter, discard the precipitate, and obtain an enzymatic hydrolysate; T4. Add L-arginine at 1.8% of the weight of the homogenate to the enzymatic hydrolysate obtained in T3, treat at an ultrasonic power of 400 W for 32 min at a stirring speed of 375 rpm, pass through 10 kDa and 3 kDa ultrafiltration membranes in sequence, collect the filtrate, and obtain a myocardial peptide solution.

[0028] The preparation method of the myocardial peptide freeze-dried composition includes the following steps: S1. Mix and grind Ophiopogon japonicus and Schisandra chinensis through an 80-mesh sieve, add hawthorn, add 9 times the amount of deionized water, decoct twice, each time for 1.5 hours, and combine the two extraction solutions; S2. Mix blueberry powder and spirulina powder, add 20 mL of deionized water, stir at a speed of 375 rpm for 25 min, then treat at an ultrasonic power of 350 W for 35 Process for 12 min, get the mixture; S3, S1 extract and S2 mixture is combined, in 47 , under the vacuum-0.07 MPa low temperature vacuum concentration 1.5 h, get the reactant; S4, S3 get the reactant and myocardial peptide mixture, stirring speed 400 rpm 40 min, first frozen to-38 , keep 3 h, open the vacuum system, the pressure in the warehouse to 20 Pa, heating to-15 , and maintain 15 h, crushing, 100 mesh sieve, get myocardial peptide freeze-dried composition.

[0029] Example 2 According to the following mass parts of raw materials: myocardial peptide 12 parts, 11 parts of ophiopogon, 6 parts of schisandra, 5 parts of hawthorn, 1 part of spirulina powder, 2 parts of blueberry powder.

[0030] Myocardial peptide preparation method includes the following steps: T1, take the healthy pig myocardium 0.4 kg of connective tissue, washed with physiological saline until no blood color, then twisted, mixed with 1.2 kg of deionized water, adjusted to pH 7.0, and ground into homogenate; T2, inoculate the complex strain (lactic acid bacteria: Aspergillus viable bacteria ratio 1:2) into the homogenate obtained in T1, ferment at 30 for 24 h, get the fermentation broth; T3, the fermentation broth obtained in T2 is treated under ultrasonic power 550 W, 40 for 20 min, adjust the pH to 7.0, add neutral protease, and enzymolysis at 45 for 6 h, heat at 85 for 15 min to inactivate the enzyme, centrifugal filtration, discard the precipitate, and get the enzyme solution; T4, add L-arginine 1.5% of the weight of the homogenate to the enzyme solution obtained in T3, treat under ultrasonic power 300 W, 30 rpm stirring speed 300 rpm for 30 min, pass through 10 kDa and 3 kDa ultrafiltration membrane in turn, collect the filtrate, and get the myocardial peptide solution.

[0031] Myocardial peptide freeze-dried composition preparation method includes the following steps: S1, mix ophiopogon and schisandra, grind through 80 mesh sieve, add hawthorn, add 8 times the amount of deionized water, decoct 3 times, 1 hour each time, combine the two extractives; S2, mix blueberry powder and spirulina powder, add 20 mL deionized water, stir at 300 rpm for 30 min, then treat under ultrasonic power 300 W, 30 The mixture was processed for 15 minutes to obtain a mixture; S3. Combine the extract from S1 with the mixture from S2, and heat at 45°C. The reactants were concentrated under low-temperature vacuum at a vacuum degree of -0.07 MPa for 1.5 h to obtain the reactants. S4. Mix the reactants obtained in S3 with cardiac peptides, stir at 300 rpm for 45 min, and then freeze to -30°C. Maintain this for 4 hours, then activate the vacuum system to reduce the pressure inside the chamber to 30 Pa and raise the temperature to -10°C. The mixture was kept for 20 hours, then pulverized and passed through a 100-mesh sieve to obtain a myocardial peptide lyophilized composition.

[0032] Example 3 Prepare the ingredients according to the following proportions by weight: 15 parts myocardial peptide, 8 parts Ophiopogon japonicus, 8 parts Schisandra chinensis, 3 parts hawthorn, 2 parts spirulina powder, and 1 part blueberry powder.

[0033] The preparation method of cardiac peptides includes the following steps: T1. Take 0.6 kg of healthy pig myocardium with connective tissue removed, rinse with physiological saline until there is no blood, mince it, mix it with 1.5 kg of deionized water, adjust the pH to 7.0, and grind it into a homogenate. T2. Inoculate the homogenate obtained in T1 with a compound bacterial strain (lactic acid bacteria: Aspergillus viable count ratio of 3:4), and incubate at 35°C. Ferment for 24 hours to obtain fermentation broth; T3. The fermentation broth obtained in T2 was subjected to ultrasonic treatment at a power of 600W and a temperature of 50°C. After 15 minutes of treatment, adjust the pH to 7.0, add neutral protease, and incubate at 45°C. Enzymatic hydrolysis for 6 hours at 90°C Heat for 10 minutes to inactivate the enzyme, centrifuge and filter, discard the precipitate to obtain the enzyme hydrolysate; T4. Add 2.0% (by weight) L-arginine of the homogenate to the enzymatic hydrolysate obtained in T3, and sonicate at 500W and 35°C. The solution was processed at a stirring speed of 450 rpm for 20 min, and then passed through 10 kDa and 3 kDa ultrafiltration membranes in sequence. The filtrate was collected to obtain a myocardial peptide solution.

[0034] The method for preparing the myocardial peptide lyophilized composition includes the following steps: S1. Grind Ophiopogon japonicus and Schisandra chinensis together and pass through an 80-mesh sieve. Add hawthorn and 10 times the amount of deionized water. Decoction twice, 2 hours each time. Combine the two extracts. S2. Mix blueberry powder and spirulina powder, add 20mL of deionized water, stir at 450rpm for 20min, then sonicate at 400W for 40 minutes. The mixture was processed for 10 minutes to obtain a mixture; S3. Combine the extract from S1 with the mixture from S2, and heat at 50°C. The reactants were concentrated at low temperature under vacuum of -0.08 MPa for 1 hour to obtain the reactants. S4. Mix the reactants obtained in S3 with cardiac peptides, stir at 500 rpm for 35 min, and then freeze to -45°C. Maintain this temperature for 2 hours, then activate the vacuum system to reduce the pressure inside the chamber to 10 Pa and raise the temperature to -20°C. The mixture was kept for 10 hours, then pulverized and passed through a 100-mesh sieve to obtain a myocardial peptide lyophilized composition.

[0035] Comparative Example 1 The preparation method is the same as in Example 1, except that when preparing myocardial peptides, the porcine myocardial homogenate obtained in step T1 is directly taken and subjected to ultrasonic treatment and neutral protease hydrolysis in step T3, omitting the fermentation step T2.

[0036] Comparative Example 2 The preparation method is the same as in Example 1, except that when preparing cardiac peptides, the fermentation broth obtained in T2 is subjected to ultrasonic treatment in step T3, and the neutral protease hydrolysis is omitted.

[0037] Comparative Example 3 The preparation method is the same as in Example 1, except that Schisandra chinensis and hawthorn are not added to the raw materials.

[0038] Comparative Example 4 The preparation method is the same as in Example 1, except that no spirulina powder or blueberry powder is added to the raw materials.

[0039] In the preparation of the composition, step S2 is omitted, and the herbal extract obtained in step S1 is directly concentrated under low temperature vacuum in step S3, and then mixed with myocardial peptides to prepare a myocardial peptide freeze-dried composition.

[0040] The efficacy of the myocardial peptide lyophilized compositions prepared in Examples 1-3 and Comparative Examples 1-4 was verified.

[0041] 1. The above-mentioned myocardial peptide lyophilization composition was subjected to 60°C. The samples were placed under the specified conditions for 10 days, and samples were taken on days 0, 5, and 10 for testing to examine the stability of the samples. The results are as follows: Figs. 1-3 .

[0042] Depend on Fig. 1 It can be seen that the moisture content of all groups increases with time. The moisture content growth curves of Examples 1 to 3 are relatively flat, while the moisture content of the comparative groups increases faster and the final moisture content is significantly higher than that of the example groups, indicating that the products of the comparative examples are more prone to moisture absorption and have poor stability.

[0043] Depend onFig. 2 It can be seen that the polypeptide content of all groups decreases with time, and the polypeptide content of Examples 1-3 decreases more gently, and still maintains a high content retention rate after 10 days, showing excellent stability. The polypeptide content of Comparative Examples 1-4 decreases greatly, and the content decreases to a low level after 10 days, indicating that it has poor stability.

[0044] By Fig. 3 It can be seen that the initial antioxidant activity of the myocardial peptide freeze-dried composition provided by Examples 1-3 and the activity retention rate after 10-day accelerated test are significantly better than those of all comparative examples, indicating that the myocardial peptide freeze-dried composition prepared by Examples 1-3 has high antioxidant activity and stability.

[0045] 2. Animal experiment: Select 10-12-week-old healthy male C57BL / 6 mice, weighing 25-30g.

[0046] The myocardial disease model was established by intraperitoneal injection of adriamycin at a dose of 5mg / kg per week for 4 weeks, with a total cumulative dose of 20mg / kg. After the modeling was completed, the mice with stable myocardial injury were selected for subsequent experiments by measuring the degree of body weight loss.

[0047] Grouping: (use 10mL physiological saline to prepare a uniform suspension of the required concentration of each freeze-dried composition), 10 mice in each group.

[0048] Blank control group: gavage with equal volume of physiological saline.

[0049] Model control group: adriamycin modeling + gavage with equal volume of physiological saline.

[0050] Positive drug control group: adriamycin modeling + gavage with 10mg / kg trimetazidine per day.

[0051] Example 1 group: adriamycin modeling + gavage with 200mg / kg of the freeze-dried composition prepared in Example 1 per day.

[0052] Comparative Example 1 group: adriamycin modeling + gavage with 200mg / kg of the freeze-dried composition prepared in Comparative Example 1 per day.

[0053] Comparative Example 2 group: adriamycin modeling + gavage with 200mg / kg of the freeze-dried composition prepared in Comparative Example 2 per day.

[0054] Comparative Example 3 group: adriamycin modeling + gavage with 200mg / kg of the freeze-dried composition prepared in Comparative Example 3 per day.

[0055] Comparative Example 4 group: adriamycin modeling + gavage with 200mg / kg of the freeze-dried composition prepared in Comparative Example 4 per day.

[0056] Dosing cycle: once a day, continuously for 21 days after the end of modeling.

[0057] After the end of the test, the contents of myocardial troponin I (cTnI), inflammatory factors (IL-6, TNF- ), and oxidative stress markers (MDA, SOD) of the mice in each group were detected, and the results are shown in Table 1.

[0058] Table 1: Results of blood index detection of mice

[0059] As can be seen from Table 1, compared with the model control group, the myocardial injury marker cTnI, inflammatory factors, and oxidative stress product MDA of the model mice orally administered with the product of Example 1 of the application were significantly reduced, while the activity of the antioxidant enzyme SOD was significantly improved, and the effect was comparable to that of the positive drug.

[0060] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the application rather than limit them, and although the application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the application can still be modified or replaced by equivalents, and these modifications or equivalent replacements should not make the modified technical solutions deviate from the spirit and scope of the technical solutions of the application.

Claims

1. A lyophilized composition of cardiac peptides, characterized in that, It contains the following components by weight: 12-15 parts myocardial peptide, 8-11 parts Ophiopogon japonicus, 6-8 parts Schisandra chinensis, 3-5 parts hawthorn, 1-2 parts spirulina powder, and 1-2 parts blueberry powder.

2. The method for preparing the myocardial peptide lyophilized composition according to claim 1, characterized in that, Includes the following steps: S1. Grind Ophiopogon japonicus and Schisandra chinensis together, sift, add hawthorn, add 8-10 times the amount of deionized water, decoct 2-3 times, 1-2 hours each time, and combine the extracts. S2. Mix blueberry powder and spirulina powder, add deionized water, stir well, and sonicate to obtain a mixture; S3. Mix the extract obtained in S1 with the mixture obtained in S2, and concentrate under low temperature and vacuum to obtain the reactants; S4. Mix the reactants obtained in S3 with cardiac peptides, stir evenly, freeze dry, pulverize, and sieve to obtain a cardiac peptide freeze-dried composition.

3. The preparation method according to claim 2, characterized in that, In S2, the ultrasonic processing power is 300~400W and the temperature is 30~40℃. The time is 10-15 minutes.

4. The preparation method according to claim 2, characterized in that, In S3, the low-temperature vacuum concentration temperature is 45~50℃. The vacuum degree is -0.06 to -0.08 MPa, and the time is 1 to 2 hours.

5. The preparation method according to claim 2, characterized in that, In S3, the specific process parameters for freeze-drying are: first freeze to -45°C. ~-30 Maintain this state for 2-4 hours, then activate the vacuum system to reduce the pressure inside the chamber to 10-30 Pa and raise the temperature to -20°C. ~-10 And maintain for 10-20 hours.

6. The preparation method according to claim 2, characterized in that, In S4, the method for preparing the cardiac peptide includes the following steps: T1. Take healthy pig heart muscle with connective tissue removed, rinse with physiological saline until no blood is visible, mince, mix with deionized water, adjust pH to 6.5~7.5, grind to obtain homogenate; T2. Inoculate the compound bacterial strain into the homogenate obtained in T1 and ferment to obtain the fermentation broth; T3. The fermentation broth obtained in T2 is subjected to ultrasonic treatment, the pH is adjusted to neutral, neutral protease is added, and enzymatic hydrolysis is performed. After the enzymatic hydrolysis is completed, the enzyme is inactivated by heating, centrifugation is performed, filtration is carried out, and the precipitate is discarded to obtain the enzymatic hydrolysate. T4. Add 1.5% to 2% L-arginine (by weight of homogenate) to the enzymatic hydrolysate obtained in T3, sonicate and stir, and then pass through 10kDa and 3kDa ultrafiltration membranes sequentially to obtain cardiac peptides.

7. The preparation method according to claim 6, characterized in that, In T2, the compound microbial strain consists of lactic acid bacteria and Aspergillus at a live count ratio of (1:3) to (2:4), and the fermentation temperature is 30-35°C. The fermentation time is 24 hours.

8. The preparation method according to claim 6, characterized in that, In T3, the specific process parameters for the ultrasonic treatment are: ultrasonic power of 550~600W, and extraction temperature of 40~50℃. The extraction time is 15-20 minutes, and the enzymatic hydrolysis temperature is 45-50°C. The enzymatic hydrolysis time is 2-6 hours.

9. The preparation method according to claim 6, characterized in that, In T4, the ultrasonic stirring power is 300~500W, the time is 20~30min, and the temperature is 30~35℃. The stirring speed is 300~450 rpm.

10. The use of the myocardial peptide lyophilized composition of claim 1 in the preparation of a medicament for nourishing the myocardium and protecting blood vessels.

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