CN106692123B - Application of gamma-aminobutyric acid in preparation of heart protection pharmaceutical preparation - Google Patents
Application of gamma-aminobutyric acid in preparation of heart protection pharmaceutical preparation Download PDFInfo
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- CN106692123B CN106692123B CN201611203181.3A CN201611203181A CN106692123B CN 106692123 B CN106692123 B CN 106692123B CN 201611203181 A CN201611203181 A CN 201611203181A CN 106692123 B CN106692123 B CN 106692123B
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Images
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/005—Amino acids other than alpha- or beta amino acids, e.g. gamma amino acids
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of biomedicine, and particularly relates to application of gamma-aminobutyric acid (GABA) in preparing a heart protection medicinal preparation. Fermenting low-value fish or processing waste thereof serving as a raw material by using a microorganism with glutamate decarboxylase activity, and separating and purifying the obtained fermentation product to obtain GABA. The experimental results show that: (1) GABA has obvious nutrition and protection effects on cardiac cells; (2) the oxidation resistance of GABA can inhibit the apoptosis of heart cells and prevent the aging of myocardial cells; (3) GABA can increase the content of high-density lipoprotein in heart blood; (4) GABA can increase the nerve growth factor content of heart tissue.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to application of gamma-aminobutyric acid in preparation of a heart protection pharmaceutical preparation.
Background
The marine edible fish has rich nutrition, delicious taste, and many health functions, and has important edible and economic values. With the development of modern separation technology and biotechnology, the utilization of fish resources by people is no longer limited to the mariculture industry, and the research content relates to a plurality of fields of ecology, cultivation, medicine, toxicology, heredity, molecular biology and the like. The method carries out high-valued development and utilization on low-value fish resources, provides ideal marine food and marine medicine for human beings, creates macroscopic economic benefit, and is beneficial to promoting the rapid development of marine industry in China.
The heart is one of the most important organs in the vertebrate body. The heart functions to promote blood flow, provide sufficient blood flow to organs and tissues, supply oxygen and various nutrients (such as water, inorganic salts, glucose, proteins, various water-soluble vitamins, etc.), and remove metabolic end products (such as carbon dioxide, urea, uric acid, etc.), so that cells maintain normal metabolism and functions. Various endocrine hormones and other body fluid factors in the body are also transported to target cells through blood circulation to realize body fluid regulation and maintain the internal environment of the body relatively constant. In addition, the implementation of blood defense functions, and the regulation of body temperature that is relatively constant, also relies on the continuous circulation of blood within the blood vessels, which is achieved by the action of the heart "pump". The key of protecting heart is to strengthen the antioxidation performance of heart cells, promote blood circulation and reduce the cholesterol content in blood.
Gamma-aminobutyric acid (GABA) is an important inhibitory neurotransmitter in the nervous system of mammals, crustaceans, insects and certain parasitic worms. A few plants contain such non-protein amino acids. GABA is involved in various metabolic activities and has the effects of resisting oxidation, lowering blood pressure, improving sleep, resisting anxiety, improving lipid metabolism, reducing weight, preventing arteriosclerosis and the like, so that the GABA is paid more and more attention by scientific workers.
Disclosure of Invention
The invention aims to provide application of gamma-aminobutyric acid in preparation of a heart protection pharmaceutical preparation.
The scheme for achieving the purpose of the invention is as follows:
use of gamma-aminobutyric acid (GABA) for the preparation of a cardioprotective pharmaceutical preparation.
Preferably; application of gamma-aminobutyric acid (GABA) in preparing medicine for protecting heart damage caused by fluorine.
Preferably; the preparation method of GABA comprises the following steps: fermenting low-value fish or processing waste thereof serving as a raw material by using microorganisms with glutamate decarboxylase activity, and separating and purifying the obtained fermentation product to obtain the feed.
The method comprises the following steps:
1) grinding raw material low-value fish or processing waste thereof into homogenate, adding distilled water with 5-10 times volume (w/v) of homogenate mass into the homogenate to prepare homogenate, adding 0-5 wt% glucose (v/w) of the volume of the homogenate into the homogenate, and adjusting the pH value of the solution to 4.5-6.0 to obtain a microorganism culture solution. Sterilizing the culture solution, cooling, inoculating a microorganism with glutamate decarboxylase activity, and culturing at 30-40 ℃ for 24-72 hours to obtain a bacterial suspension (solution A) rich in the microorganism with the glutamate decarboxylase activity;
2) adjusting the pH value of the solution A to 5.5-6.5, adding 0.5-2 wt% of sodium glutamate (v/w) of the volume of the solution A to prepare a culture solution (solution B) rich in sodium glutamate, and culturing the solution B at 35-40 ℃ for 72-120 hours to obtain a fermentation liquid rich in GABA;
3) and (3) removing thallus and degreasing of GABA-rich fermentation liquor: centrifuging the fermentation liquid obtained in the step 2) for 5-20 minutes at the speed of 2000-;
4) and (3) decoloring thallus-free and grease-free fermentation liquor: decolorizing with active carbon or SD300 resin to obtain colorless transparent fermentation liquid;
5) and (3) separating and purifying GABA in colorless and transparent fermentation liquor: fully performing exchange adsorption on 732 resin (hydrogen type) exchange columns pretreated on the colorless and transparent fermentation liquor obtained in the step 4) at the flow rate of 2-5mL/min, washing the column to pH6.0 by deionized water after adsorption, washing the column by 100-200mL of 0.1-0.2mol/L alkaline solution, and then eluting the column by 1-2mol/L alkaline solution at the flow rate of 2-5 mL/min; and (3) developing with ninhydrin acetone solution during the elution process, collecting effluent with ninhydrin reaction showing blue color and pH of about 6.0, detecting GABA content in each tube of effluent by high performance liquid chromatography, combining the effluent with high GABA content, and concentrating under reduced pressure to obtain separated and purified GABA.
Preferably; the step 4) of adopting the activated carbon to decolor the thallus-free and grease-free fermentation liquor is to add 0.5 to 5 percent (w/v) of the activated carbon into the thallus-free and grease-free fermentation liquor, carry out water bath for 10 to 30 minutes at a temperature of between 60 and 90 ℃, and carry out vacuum filtration when the liquor is hot to obtain colorless and transparent fermentation liquor;
the SD300 resin decolorization is to decolorize thallus-free and grease-free fermentation liquor by adding 5-10% SD300 resin into the thallus-free and grease-free fermentation liquor, decolorizing at 20-30 ℃ for 3 hours by using a shaking table at 100rpm, and filtering to obtain colorless transparent fermentation liquor.
Preferably; and 5) concentrating under reduced pressure to 1/20-1/10 volume to obtain separated and purified GABA concentrated solution, and detecting the purity of GABA in the concentrated solution by using high performance liquid chromatography.
Preferably; and 5) the alkaline solution is a sodium hydroxide solution or ammonia water.
The heart damage model is fed with GABA (gamma-aminobutyric acid) which is separated and purified and has the concentration of 0.1-2.0g/L and is fed to a heart damage model mouse which is made of sodium fluoride for 5-20 days, and the GABA has obvious heart protection effect through the change of the morphological structure of heart cells, the malondialdehyde level of heart tissues and the change conditions of high-density lipoprotein and neurotrophic factor in heart blood.
Detecting the heart protection capability of the isolated and purified GABA:
1) a heart-damaged mouse model was established with sodium fluoride.
2) Feeding the separated and purified GABA with the concentration of 0.1-2.0g/L for 5-20 days.
3) The change conditions of the cell morphological structure of the heart cells, the malondialdehyde of the heart tissue, the high-density lipoprotein and the level of the neurotrophic factor show that the GABA has obvious heart protection effect.
The invention has the advantages that:
1) the twelfth order food grade GABA production process of Ministry of health in 2009: the L-sodium glutamate is used as a substrate and is produced by the steps of safe strain fermentation, heating sterilization, cooling, active carbon treatment, filtration, spray drying and the like. The present production line generally produces food grade GABA in batches with 20% purity, and GABA obtained through separation and purification of the present invention is prepared through biotransformation of L-sodium glutamate with safe strain, and has purity over 60% and purity higher than that of food grade GABA sold in the market.
2) The present invention provides a new use of GABA, the protection effect of GABA to heart can be realized from several aspects: (1) GABA has obvious nutrition and protection effects on cardiac cells; (2) the oxidation resistance of GABA can inhibit the apoptosis of heart cells and prevent the aging of myocardial cells; (3) GABA can increase the content of high density lipoprotein in heart blood. The high-density lipoprotein is plasma lipoprotein for resisting atherosclerosis, and is a protective factor for coronary heart disease. Commonly known as "blood vessel scavenger", it plays a key role in maintaining the normal function of the heart. (4) GABA can increase the nerve growth factor content of heart tissue. The movements of the vascular smooth muscle and the cardiac muscle in the heart are regulated by the autonomic nervous system. The nerve growth factor has important regulation and control functions on the development, differentiation, growth and regeneration of neurons distributed in the heart. By nourishing nerve cells in the heart, the blood flow in the heart is sufficient, and the myocardial contraction function can be normally exerted.
3) The GABA obtained by the invention is separated and purified from low-value fish or processing waste fermentation liquor thereof, and has the obvious characteristics of no toxicity and easy absorption.
Drawings
FIG. 1 is a microscopic structure diagram of cardiac cells under a 40-fold objective lens of a microscope, wherein (a) cardiac microstructures of a normal group, (b) cardiac microstructures of a negative control group, (c) cardiac microstructures of a positive control group, and (d) cardiac microstructures of a GABA group are isolated and purified;
FIG. 2 shows the detection of the purity of GABA separated and purified by high performance liquid chromatography.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
1. Grinding fresh red bonito into homogenate by a grinder, adding distilled water with the mass of 10 volume (w/v) of the homogenate of the red bonito to prepare homogenate, adding glucose (v/w) with the volume of 1 wt% of the homogenate into the homogenate, adjusting the pH value of the solution to 5.5, and preparing a microorganism culture medium. The medium was sterilized at 121 ℃ for 15 minutes. After cooling, inoculating streptococcus lactis according to the inoculation amount (v/v) of 1 percent, and fermenting and culturing for 1 day at the temperature of 30 ℃ to obtain bacterial suspension rich in streptococcus lactis.
2. Adjusting the pH value of the bacterial suspension rich in the streptococcus lactis to 6.0, adding 1 wt% of sodium glutamate (v/w) of the volume of the bacterial suspension into the bacterial suspension, placing the bacterial suspension in a thermostat at 37 ℃, and performing static culture for 72 hours to obtain the fermented liquor of the rhodotorula gracilis with the GABA content of 4.8 g/L.
The streptococcus lactis is purchased from China general microbiological culture collection center, Beijing, and is numbered bio-52538.
3. And (3) separating and purifying GABA in the fermented liquor of the rhodomyrtus tomentosa rich in GABA.
1) And (3) removing thalli and degreasing of the fermented liquor of the rhodotorula gracilis rich in GABA: and (3) centrifuging the fermentation liquor obtained in the step (2) at the speed of 4000r/min for 10 minutes, taking the upper layer liquid, and carrying out autoclaving to obtain the sterile fermentation liquor. And removing grease from the sterile fermentation liquor by a Soxhlet extraction method to obtain thallus-free and grease-free fermentation liquor.
2) And (3) decoloring thallus-free and grease-free fermentation liquor: and (3) carrying out decolorization treatment by using SD300 resin, specifically adding 5% SD300 resin (w/v) into the obtained thallus-free and grease-free fermentation liquor, decolorizing for 3 hours at 25 ℃ by using a shaking table at 100rpm, and carrying out suction filtration to obtain colorless transparent fermentation liquor.
3) And (3) separating and purifying GABA in colorless and transparent fermentation liquor: the colorless transparent liquid was put on a 732 resin (hydrogen type) pretreated exchange column and sufficiently subjected to exchange adsorption at a flow rate of 3 mL/min. After adsorption, the mixture is washed by deionized water until the pH value is 6.0. The column was washed with 150mL of 0.1mol/L NaOH and then eluted with 1mol/L NaOH at a flow rate of 3 mL/min. The color development test was carried out with ninhydrin acetone solution continuously during the elution, and the change in pH was detected with pH paper. When the ninhydrin reaction is blue, and the pH value is about 6.0, retaining the effluent liquid until the ninhydrin blue reaction disappears, detecting the GABA content in the collection tube by high performance liquid chromatography, combining the effluent liquid in the collection tube with higher GABA content (the pH value of GABA aqueous solution is about 6, and the effluent liquid reacts with ninhydrin to be blue, so that the pH value of the effluent liquid can be judged to be 6, the effluent liquid reacts with ninhydrin to be blue, the amino acid contained in the effluent liquid is GABA), and concentrating under reduced pressure to 1/20 volumes to obtain the isolated and purified GABA concentrated solution.
Example 2
1. Adding 5 times (w/v) volume of distilled water into viscera (waste product) of the red-spotted maigre to prepare solution, adding 2.0 wt% of glucose (v/w) of the solution volume into the solution, adjusting pH value to 5.0, and preparing into microorganism culture solution. The culture broth was sterilized at 121 ℃ for 20 minutes. Cooling, inoculating enterococcus avium according to 1% inoculum size (v/v), fermenting and culturing at 37 deg.C for 36 hr to obtain suspension rich in enterococcus avium.
2. Adding 2 wt% of sodium glutamate (v/w) of the volume of the bacterial suspension into the bacterial suspension rich in the enterococcus avium, and adjusting the pH value to 5.5 to prepare a sodium glutamate-rich culture solution. The culture solution is placed in a thermostat with the temperature of 37 ℃ for static culture for 96 hours, and the fermented liquid of the internal organs of the red-brown mackerel with the GABA content of 5.7g/L is obtained.
The enterococcus avium is purchased from China general microbiological culture Collection center, Beijing, and the serial number is CGMCC 9184.
3. And (3) separating and purifying GABA in the fermented liquor of the viscera of the rhodomyrtus tomentosa rich in GABA.
1) And (3) removing thallus and degreasing from the fermented liquor of the internal organs of the rhodotorula gracilis rich in GABA: centrifuging at 3000r/min for 15 min, and sterilizing the upper layer liquid at high temperature and high pressure to obtain the sterile fermentation liquid. And removing grease from the thallus-free fermentation liquor by a Soxhlet extraction method to obtain the thallus-free and grease-free fermentation liquor.
2) And (3) decoloring thallus-free and grease-free fermentation liquor: and (3) carrying out decolorization treatment on SD300 resin, specifically adding 10% SD300 resin (w/v) into the thallus-free and grease-free fermentation liquor obtained above, decolorizing for 4 hours at 25 ℃ by using a shaking table at 100rpm, and carrying out suction filtration to obtain colorless transparent fermentation liquor.
3) And (3) separating and purifying GABA in colorless and transparent fermentation liquor: the colorless transparent liquid was passed through a 732 resin (hydrogen type) column which had been pretreated, and the flow rate was 3mL/min, and the column was sufficiently subjected to exchange adsorption. After adsorption, the mixture is washed by deionized water until the pH value is 6.0. The column was washed with 150mL of 0.1mol/L ammonia water, and then eluted with 1mol/L ammonia water at a flow rate of 3 mL/min. The color development test was carried out with ninhydrin acetone solution continuously during the elution, and the change in pH was detected with pH paper. When the ninhydrin reaction is blue, and the pH value is about 6.0, retaining the effluent, detecting the GABA content in the collecting tube by high performance liquid chromatography, combining the effluents in the collecting tube with higher GABA content (the pH value of the GABA aqueous solution is about 6, and the pH value of the GABA aqueous solution reacts with the ninhydrin to be blue, so that the effluent can be judged to have the pH value of 6, and reacts with the ninhydrin to be blue, and the effluent contains GABA, and concentrating under reduced pressure to 1/10 volume to obtain the separated and purified GABA concentrated solution.
Example 3
1. Decocting Engraulis japonicus Temminck et Schlegel in water, concentrating the waste liquid, adding 5 times (v/v) of distilled water to obtain diluent, adding 1 wt% of glucose (v/w) of the diluent, adjusting pH to 6.0, and making into microorganism culture solution. The culture broth was sterilized at 121 ℃ for 20 minutes. After cooling, inoculating lactobacillus brevis according to the inoculation amount (v/v) of 1 percent, and fermenting and culturing for 24 hours at the temperature of 37 ℃ to obtain bacterial suspension rich in lactobacillus brevis.
2. Adding 1.5 wt% of sodium glutamate (v/w) of the volume of the bacterial suspension into the bacterial suspension rich in the lactobacillus brevis, and adjusting the pH value to be 6.5 to prepare a sodium glutamate-rich culture solution. The culture solution is placed in a thermostat with the temperature of 37 ℃ for static culture for 100 hours, and anchovy poaching waste liquid fermentation liquor with the GABA content of 5.81g/L is obtained.
The lactobacillus brevis is purchased from China general microbiological culture collection center, Beijing, and has the number of CGMCC 1.2028.
3. And (3) separating and purifying GABA in the anchovy water-boiling waste liquid fermentation liquor rich in GABA.
1) Removing thallus and degreasing from the anchovy poaching waste liquid fermentation liquor rich in GABA: centrifuging at 5000r/min for 10 min, and sterilizing the upper layer liquid at high temperature and high pressure to obtain sterile fermentation liquid. And removing grease from the thallus-free fermentation liquor by a Soxhlet extraction method to obtain the thallus-free and grease-free fermentation liquor.
2) Activated carbon decolorization of thallus-free and grease-free fermentation liquor: adding 1.5% active carbon (w/v) into the thallus-free and grease-free fermentation liquor, decoloring for 30 minutes at 80 ℃, and filtering to obtain colorless transparent fermentation liquor.
3) And (3) separating and purifying GABA in colorless and transparent fermentation liquor: the colorless transparent fermentation liquid is put on a 732 resin (hydrogen type) pretreated exchange column, and the flow rate is 3mL/min, so that the colorless transparent fermentation liquid is fully subjected to exchange adsorption. After adsorption, the mixture is washed by deionized water until the pH value is 6.0. The column was washed with 150mL of 0.1mol/L ammonia water, and then eluted with 1mol/L ammonia water at a flow rate of 3 mL/min. The color development test was carried out with ninhydrin acetone solution continuously during the elution, and the change in pH was detected with pH paper. When the ninhydrin reaction is blue, and pH is about 6.0, retaining the effluent, detecting GABA content in the collection tube by high performance liquid chromatography, and combining the effluents in the collection tube with higher GABA content (the pH of GABA aqueous solution is about 6, and reacts with ninhydrin to be blue, so that the pH of the effluent can be judged to be 6, and the effluent reacts with ninhydrin to be blue, and contains GABA). The GABA concentrated solution is separated and purified by concentrating under reduced pressure to 1/10 volumes.
The GABA concentrated solutions separated and purified in the above examples 1, 2 and 3 were combined, and the purity of GABA was measured by HPLC, and the results are shown in FIG. 2, in which the GABA content was 63%, and the other detectable amino acids were: ala alanine 2%, Gly glycine 8%, Arg arginine 5%. Alanine, glycine and arginine are all common amino acids in human bodies, have low content, are all in a safe intake range, and cannot generate adverse physiological influence on human bodies or tested animals.
Example 4
The combined GABA concentrated solution obtained by separation and purification is used for preparing the heart protection pharmaceutical preparation, and the test and the effect of the heart protection effect are as follows:
cardioprotection test of isolated and purified GABA:
experimental animals: 18-22 g/male mouse of 120 Kunming species.
Animal model: 100 mice were fed 0.1g/L NaF for 30 days, creating heart-damaged model mice.
Preparing the medicine: the GABA concentrated solutions separated and purified in the above examples 1, 2 and 3 were combined, and then purified water was added to the combined solution to prepare a solution having GABA contents of 0.1g/L, 0.5g/L and 1.0 g/L.
Grouping experiments: 20 normal groups (healthy mice were fed purified water daily); 20 negative control groups (heart-impaired mice were fed purified water daily); 20 positive control groups (heart-damaged mice were fed daily with American GNC Jiananxi coenzyme Q10, dosage according to the instructions); 20 low dose isolated and purified GABA groups (heart-damaged mice are fed with GABA content of 0.1g/L solution every day); the GABA group is separated and purified by 20 medium doses (the heart-damaged mice are fed with GABA content of 0.5/L solution every day); the GABA group was isolated and purified at 20 high doses (heart-damaged mice were fed with 1.0g/L GABA content solution per day).
The experimental method comprises the following steps: feeding pure water and American GNC Jiananxi coenzyme Q10 to heart-damaged mice, separating and purifying GABA solution with different concentrations for 14 days, and taking heart tissues to detect the morphological structure of heart cells, the change of malondialdehyde level of the heart tissues and the change of high-density lipoprotein and neurotrophic factor content in heart blood. Detecting the change of the malondialdehyde content in the heart tissue by using a malondialdehyde detection kit (Nanjing Biotechnology Co., Ltd., reference specification of the detection method); detecting the change of the content of the high-density lipoprotein by using a high-density lipoprotein detection kit (Nanjing Biotechnology Co., Ltd., reference specification of detection method); detecting the content of the cardiac nerve growth factor by using a neurotrophic factor Elisa kit (Houston corporation, USA, a reference instruction of a detection method); the cardiac cell structure is observed by conventional slice HE staining and microscopic examination.
Detection indexes are as follows: the change of the morphological structure of heart cells and the change of the contents of malondialdehyde, high-density lipoprotein and neurotrophic factor in heart tissues.
(1) The results of the morphological structure change of the heart cells are shown in fig. 1: the normal group of myocardial cells are closely arranged, the cell nucleus is positioned in the center of the cell, the cell membrane boundary is clear, the number of blood cells is large, and the blood flow is full; the density of the myocardial cells of the negative control group is reduced, the arrangement is loose, the cell membrane boundary is not clear, and the density of the blood cells is low; the heart damage phenomena of the positive control group and the group fed with 1g/L of separated and purified GABA are obviously improved, the density of cardiac muscle cells and blood cells is higher than that of the negative control group, the cell membrane boundary is clear, and the cell nucleus is positioned in the center of the cell. Therefore, the high concentration GABA (1g/L) has obvious improvement effect on the heart injury induced by NaF, leads the heart cell tissue structure to tend to be normalized, and has no obvious difference with a positive control group.
(2) The results of the change in the neurotrophic factor content in the heart homogenate showed: the content of neurotrophic factors of mice fed with 1g/L of separated and purified GABA group is increased, is close to the value of a normal group, is obviously higher than that of a negative control group, has no obvious difference with the positive control group, and the result is shown in table 1; the content of the neurotrophic factor of mice fed with 0.1g/L and 0.5g/L of GABA separated and purified groups has no significant difference with that of a negative control group; proved that GABA separated and purified from fermentation liquor of low-value fish or processing waste thereof has obvious promotion effect on the synthesis of neurotrophic factors under high concentration (1g/L), can ensure the normal play of the regulation and control function of the autonomic nervous system in the heart, and ensures the blood flow of the heart and the rhythmicity of myocardial beating.
TABLE 1 neurotrophic factor content in cardiac homogenate
1. normal group mice were fed purified water daily; 2. the negative control group is the heart-damaged model mouse which is fed with purified water every day; 3. positive control group is heart damaged model mouse fed with American GNC JIAN' ANXI coenzyme Q10 (dosage is referred to the instruction); 4. the GABA group is that GABA obtained by separating and purifying is fed to heart damaged model mice by 1g/L every day.
(3) Malondialdehyde is one of the most important products of membrane lipid peroxidation, and the generation of the malondialdehyde can also aggravate membrane damage, so that the content of the malondialdehyde is a common index in physiological researches on aging physiology and resistance of tissues and organs, and the degree of membrane lipid peroxidation can be known through the malondialdehyde so as to indirectly measure the damage degree of a membrane system and the stress resistance of the tissues and organs. The content of malondialdehyde in heart tissues of mice taking 1g/L of the separately purified GABA group is obviously lower than that of a negative control group, and no significant difference exists between the heart tissues of the mice taking the separately purified GABA group and the positive control group, and the results are shown in Table 2; there was no significant difference between the malondialdehyde content in the heart tissue of mice in the groups administered with 0.1g/L and 0.5g/L of separately purified GABA and the negative control group. Proves that the separated and purified GABA has a protective effect on heart tissues damaged by fluoride ions under a high concentration (1g/L), can reduce the damage degree of a heart cell membrane system and improve the stress resistance of the heart tissues.
TABLE 2 influence of GABA on malondialdehyde content in cardiac homogenate
Normal group mice were fed purified water daily; the negative control group is the heart-damaged model mouse which is fed with purified water every day; positive control group is heart damaged model mouse fed with American GNC JIAN' ANXI coenzyme Q10 (dosage is referred to the instruction); the GABA group is that GABA obtained by separating and purifying is fed to heart damaged model mice by 1g/L every day.
(4) The high-density lipoprotein is plasma lipoprotein for resisting atherosclerosis, is a protective factor for coronary heart disease, and is commonly called as blood vessel scavenger. The content of high-density lipoprotein in heart tissues of mice fed with 1g/L of isolated and purified GABA groups is increased, and the result is shown in Table 3; the content of high-density lipoprotein in heart tissues of mice fed with 0.1g/L and 0.5g/L of the separated and purified GABA groups has no significant difference with that of a negative control group.
TABLE 3 high Density lipoprotein content in cardiac tissue
Normal group mice were fed purified water daily; the negative control group is the heart-damaged model mouse which is fed with purified water every day; positive control group is heart damaged model mouse fed with American GNC JIAN' ANXI coenzyme Q10 (dosage is referred to the instruction); the GABA group is that GABA obtained by separating and purifying is fed to heart damaged model mice by 1g/L every day.
The test results show that GABA biologically converted from low-value fish or processing waste thereof has obvious heart protection effect.
Claims (6)
1. The application of gamma-aminobutyric acid (GABA) in preparing the medicine for protecting heart damage caused by fluorine ion induction comprises the following steps:
normalizing the tissue structure of heart cells, promoting the synthesis of neurotrophic factors, ensuring the normal performance of the regulation and control function of the autonomic nervous system in the heart, and ensuring the blood flow of the heart and the rhythmicity of the beating of cardiac muscle;
reducing the malondialdehyde content in heart tissue;
increasing the content of high density lipoprotein in heart tissue.
2. Use according to claim 1, characterized in that: the preparation method of GABA comprises the following steps: fermenting low-value fish or processing waste thereof serving as a raw material by using microorganisms with glutamate decarboxylase activity, and separating and purifying the obtained fermentation product to obtain the feed.
3. The use according to claim 2, wherein the GABA is prepared by a process comprising:
1) grinding raw material low-value fish or processing waste thereof into homogenate, adding distilled water with 5-10 times volume of homogenate into the homogenate to prepare homogenate, adding 0-5 wt% of glucose in the volume of the homogenate into the homogenate, adjusting the pH value of the solution to 4.5-6.0 to obtain a microorganism culture solution, sterilizing the culture solution, cooling, inoculating a microorganism with glutamate decarboxylase activity, and culturing at 30-40 ℃ for 24-72 hours to obtain a bacterial suspension rich in glutamate decarboxylase activity microorganism;
2) adjusting the pH value of the bacterial suspension rich in the glutamic acid decarboxylase active microorganisms obtained in the step 1) to be 5.5-6.5, adding 0.5-2 wt% of sodium glutamate in the volume of the bacterial suspension to prepare a culture solution rich in sodium glutamate, and culturing at the temperature of 35-40 ℃ for 72-120 hours to obtain fermentation liquor rich in GABA;
3) and (3) removing thallus and degreasing of GABA-rich fermentation liquor: centrifuging the fermentation liquid obtained in the step 2) for 5-20 minutes at the speed of 2000-;
4) and (3) decoloring thallus-free and grease-free fermentation liquor: decolorizing with active carbon or SD300 resin to obtain colorless transparent fermentation liquid;
5) and (3) separating and purifying GABA in colorless and transparent fermentation liquor: fully performing exchange adsorption on the 732 resin hydrogen type exchange column pretreated on the colorless transparent fermentation liquid obtained in the step 4) at the flow rate of 2-5mL/min, washing the column to pH6.0 by deionized water after adsorption, washing the column by using 100-200mL of 0.1-0.2mol/L alkaline solution, and then eluting the column by using 1-2mol/L alkaline solution at the flow rate of 2-5 mL/min; and (3) developing with ninhydrin acetone solution during the elution process, collecting effluent with ninhydrin reaction showing blue color and pH of about 6.0, detecting GABA content in each tube of effluent by high performance liquid chromatography, combining the effluent with high GABA content, and concentrating under reduced pressure to obtain separated and purified GABA.
4. Use according to claim 3, characterized in that:
the step 4) of adopting the activated carbon to decolor the thallus-free and grease-free fermentation liquor is to add 0.5 to 5 percent (w/v) of the activated carbon into the thallus-free and grease-free fermentation liquor, carry out water bath for 10 to 30 minutes at a temperature of between 60 and 90 ℃, and carry out vacuum filtration when the liquor is hot to obtain colorless and transparent fermentation liquor;
the SD300 resin decolorization is to decolorize thallus-free and grease-free fermentation liquor by adding 5-10% SD300 resin into the thallus-free and grease-free fermentation liquor, decolorizing at 20-30 ℃ for 3 hours by using a shaking table at 100rpm, and filtering to obtain colorless transparent fermentation liquor.
5. Use according to claim 3, characterized in that: and 5) concentrating under reduced pressure to 1/20-1/10 volume to obtain separated and purified GABA concentrated solution, and detecting the GABA content in the concentrated solution by using high performance liquid chromatography.
6. Use according to claim 3, characterized in that: and 5) the alkaline solution is a sodium hydroxide solution or ammonia water.
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