CN107550909B - Application of 4-hydroxyethyl piperazine ethanesulfonic acid in xanthine oxidase inhibitory peptide - Google Patents
Application of 4-hydroxyethyl piperazine ethanesulfonic acid in xanthine oxidase inhibitory peptide Download PDFInfo
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- CN107550909B CN107550909B CN201710623309.XA CN201710623309A CN107550909B CN 107550909 B CN107550909 B CN 107550909B CN 201710623309 A CN201710623309 A CN 201710623309A CN 107550909 B CN107550909 B CN 107550909B
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Abstract
The invention discloses application of 4-hydroxyethyl piperazine ethanesulfonic acid in enhancing the activity of xanthine oxidase inhibitory peptide, wherein the xanthine oxidase inhibitory peptide comprises peptide obtained by fermenting food-borne protein or performing protease enzymolysis, and preferably tuna peptide, saury peptide or walnut meal protein peptide. The research of the patent finds that the 4-hydroxyethyl piperazine ethanesulfonic acid has the effect of remarkably enhancing the activity of the polypeptide xanthine oxidase inhibitor, and compared with the existing buffer system (Tris-HCl and phosphate buffer solution (PB)) commonly used for screening the xanthine oxidase inhibitory activity, the xanthine inhibitory peptide has the optimal xanthine oxidase inhibitory effect in the buffer system containing the 4-hydroxyethyl piperazine ethanesulfonic acid.
Description
Technical Field
The invention relates to a new application of 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES), in particular to an application of 4-hydroxyethyl piperazine ethanesulfonic acid in enhancing the activity of xanthine oxidase inhibitory peptide.
Background
Uric acid (Uric acid) is a product of purine substance metabolism in a human body, the content of Uric acid in normal human blood is 200-410 mu mol/L, and once Uric acid is formed excessively and/or excretion is reduced, the Uric acid level is increased, so that urate is deposited in soft tissues, and hyperuricemia is induced.
In recent years, with the development of society, people's life and eating patterns change, and people suffering from hyperuricemia are more and more due to the tense life rhythm and poor eating habits, and the situation is the ' fourth highest ' seriously harming human health after hypertension, hyperglycemia and hyperlipidemia. Epidemiological studies have shown that hyperuricemia is an important factor in the initiation of chronic diseases such as gout.
At present, the treatment of hyperuricemia is mainly realized by inhibiting the generation of uric acid and promoting the excretion of uric acid through two ways, and the representative medicines are respectively as follows: allopurinol and benzbromarone. However, the existing medicines can not radically cure hyperuricemia generally, and have serious toxic and side effects after long-term administration.
In the last two decades, with the research on the digestion and absorption mechanism of polypeptides, it is found that the polypeptides do not completely enter the body in the form of amino acids, some polypeptides enter the body in the form of dipeptides or even longer peptide chains through the cell bypass transportation, endocytosis and exocytosis, and the like, and have important biological activity functions in the body. Since bioactive peptides are natural biological molecules constituting the body, almost no toxic or side effect exists, and more bioactive peptides are developed and utilized, especially mainly antioxidant peptides and ACE (angiotensin converting enzyme) inhibitory peptides.
At present, research on peptide capable of reducing uric acid mainly includes screening peptide chains having xanthine oxidase inhibitory activity. Research has shown that some polypeptides, such as peptide chains containing serine at two ends, have good xanthine oxidase inhibitory activity and can significantly reduce uric acid levels of hyperuricemia rats.
Since biological macromolecules usually have a complex spatial structure and different conformations in different environments, the biological activity of a polypeptide is closely related to its spatial conformation. In the process of measuring the activity of the polypeptide, a proper buffer reagent is often required to be selected, and the buffer reagent selected at present is mainly directly used in the screening process of other xanthine oxidase inhibitors.
The structural formula of the 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) is as follows:
4-hydroxyethyl piperazine ethanesulfonic acid is a common component of buffers used in cell cultures of animal and human origin. Studies have demonstrated that HEPES has minimal cytotoxicity in all known buffers. Piperazine-based zwitterionic molecules have been approved by the FDA as food additives.
Disclosure of Invention
The invention aims to provide application of 4-hydroxyethyl piperazine ethanesulfonic acid in enhancing the activity of xanthine oxidase inhibitory peptide.
The purpose of the invention is realized by the following technical scheme:
the application of 4-hydroxyethyl piperazine ethanesulfonic acid in enhancing the activity of xanthine oxidase inhibitory peptide.
The research of the patent finds that the 4-hydroxyethyl piperazine ethanesulfonic acid has the effect of remarkably enhancing the activity of the polypeptide xanthine oxidase inhibitor, and compared with the existing buffer system (Tris-HCl and phosphate buffer solution (PB)) commonly used for screening the xanthine oxidase inhibitory activity, the xanthine inhibitory peptide has the optimal xanthine oxidase inhibitory effect in the buffer system containing the 4-hydroxyethyl piperazine ethanesulfonic acid.
Preferably, HEPES is added as an additive to the polypeptide having xanthine oxidase inhibitory function.
The polypeptide with the xanthine oxidase inhibition function comprises peptides obtained by fermenting or carrying out protease enzymolysis on food-borne protein.
The polypeptide with the xanthine oxidase inhibition function is preferably tuna peptide, saury peptide or walnut meal protein peptide;
the tuna peptide, the saury peptide and the walnut meal protein peptide are prepared by the method in the prior art;
the preparation of the polypeptide with the xanthine oxidase inhibition function comprises a filling agent corresponding to a solid medicine and a diluent corresponding to a liquid medicine.
Compared with the prior art, the invention has the following advantages and effects:
the 4-hydroxyethyl piperazine ethanesulfonic acid has the effect of remarkably enhancing the activity of polypeptide xanthine oxidase inhibitors, and compared with the existing buffer system (Tris-HCl and phosphate buffer solution (PB)) commonly used for screening the xanthine oxidase inhibition activity, the conformation of xanthine inhibitory peptides (including protein peptides such as tuna peptide, saury peptide and walnut meal protein peptide) in the buffer system containing the 4-hydroxyethyl piperazine ethanesulfonic acid has the effect of improving the xanthine oxidase inhibition rate.
Drawings
FIG. 1 is a graph showing the effect of three buffer systems on the production of uric acid by xanthine oxidase.
FIG. 2 shows the effect of tuna peptide on xanthine oxidase inhibition in three buffer systems.
FIG. 3 is a graph showing the effect of saury peptide on xanthine oxidase inhibition in three buffer systems.
FIG. 4 shows the effect of Juglans regia protein peptide on xanthine oxidase inhibition rate in three buffer systems.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
Example 1
The enhancement effect of 4-hydroxyethyl piperazine ethanesulfonic acid on tuna peptide xanthine oxidase inhibitor comprises the following specific experimental steps:
(1) preparing 150mM HEPES, Tris-HCl and PB buffer solution with the pH value of 7.4 at the temperature of 37 ℃, and preserving at the temperature of 4 ℃ for later use;
(2) preparing a required sample (tuna peptide) by using the 150mM buffer solution, wherein the sample concentration is 40 mg/ml;
(3) adding 50 μ L of tuna peptide or 50 μ L of buffer solution (blank) and 150 μ L of xanthine into 96-well enzyme label plate, performing 3 replicates for each sample, keeping the temperature at 37 deg.C for 5min, adding 50 μ L of xanthine oxidase, reading light absorption value once for 30s, and counting for 50 times for 25 min. After reading, terminating the reaction by 80 mu L of HCl, taking the reaction solution, diluting the reaction solution by 10 times by using a corresponding buffer solution, passing through a 0.25 mu m aqueous membrane, and measuring the concentration of uric acid;
(4) measuring the content of uric acid in the reacted sample by high performance liquid chromatography, and quantifying by using a uric acid standard substance;
(5) the xanthine oxidase inhibition rate is calculated by the following method:
I＝(CB-CS)/CB×100％
in the formula: i represents xanthine oxidase inhibition rate, CBThe peak area of uric acid peak of liquid phase chromatogram after blank group (without adding polypeptide) reaction, CSShowing the peak area of uric acid peak of the liquid phase map after the reaction of the sample group (added with polypeptide).
The preparation process of the tuna peptide in this example is as follows: the tuna meat is crushed according to the feed liquid mass ratio of 1: 1, adding deionized water, mixing, adjusting the pH value to 7.0, adding compound protease and papain respectively according to 0.6-1.2% of the mass of the tuna, carrying out enzymolysis for 4-7 hours at 50-60 ℃, heating to 95 ℃ after the enzymolysis is finished, keeping the temperature for 15min, inactivating enzyme, cooling, centrifuging, taking supernatant, concentrating, and carrying out spray drying to obtain the tuna peptide.
As shown in fig. 1 and 2, in HEPES buffer, Tris buffer and PB buffer, there was almost no difference in the content of uric acid produced by xanthine oxidase catalyzing xanthine (fig. 1), while HEPES was able to significantly increase the xanthine oxidase inhibition rate of tuna polypeptide (fig. 2), which indicates that HEPES acts to enhance the xanthine oxidase inhibitory activity of polypeptide.
Example 2
The enhancement effect of 4-hydroxyethyl piperazine ethanesulfonic acid on the saury peptide xanthine oxidase inhibitor comprises the following specific experimental steps:
(1) preparing 150mM HEPES, Tris-HCl and PB buffer solution with the pH value of 7.4 at the temperature of 37 ℃, and preserving at the temperature of 4 ℃ for later use;
(2) preparing a required sample (saury peptide) by using the 150mM buffer solution, wherein the sample concentration is 40 mg/ml;
(3) 50 mul of saury peptide or 50 mul of buffer (blank) and 150 mul of xanthine are sequentially added into a 96-well enzyme label plate, 3 samples are paralleled, 50 mul of xanthine oxidase is added after heat preservation at 37 ℃ for 5min, light absorption value is read once for 30s, and the total time is 25 min. After reading, terminating the reaction by 80 mu L of HCl, taking the reaction solution, diluting the reaction solution by 10 times by using a corresponding buffer solution, passing through a 0.25 mu m aqueous membrane, and measuring the concentration of uric acid;
(4) measuring the content of uric acid in the reacted sample by high performance liquid chromatography, and quantifying by using a uric acid standard substance;
(5) the xanthine oxidase inhibition rate is calculated by the following method:
I＝(CB-CS)/CB×100％
in the formula: i represents xanthine oxidase inhibition rate, CBThe peak area of uric acid peak of liquid phase chromatogram after blank group (without adding polypeptide) reaction, CSShowing the peak area of uric acid peak of the liquid phase map after the reaction of the sample group (added with polypeptide).
The process flow for preparing the saury peptide in the embodiment is as follows: mincing the saury fish meat according to the mass ratio of feed liquid of 1: 2, adding deionized water, mixing, adjusting the pH value to 7.0, adding neutral protease and trypsin according to 0.3-0.8% of the weight of the saury respectively, carrying out enzymolysis for 5-8 hours at 50-60 ℃, heating to 95 ℃ after the enzymolysis is finished, keeping the temperature for 15min, inactivating enzyme, cooling, centrifuging, taking supernate, concentrating, and carrying out spray drying to obtain the saury peptide.
As shown in FIGS. 1 and 3, HEPES has no significant effect on the process of producing uric acid by xanthine oxidase (FIG. 1), while HEPES can significantly increase the xanthine oxidase inhibition rate of saury peptide (FIG. 3), which indicates that HEPES can play a role in enhancing the xanthine oxidase inhibitory activity of polypeptide.
Example 3
The enhancement effect of the 4-hydroxyethyl piperazine ethanesulfonic acid on the walnut pulp protein peptide xanthine oxidase inhibitor comprises the following specific experimental steps:
(1) preparing 150mM HEPES, Tris-HCl and PB buffer solution with the pH value of 7.4 at the temperature of 37 ℃, and preserving at the temperature of 4 ℃ for later use;
(2) preparing a required sample (walnut pulp protein peptide) by using the 150mM buffer solution, wherein the sample concentration is 40 mg/ml;
(3) adding 50 μ L of walnut protein peptide or 50 μ L of buffer solution (blank) and 150 μ L of xanthine into 96-well enzyme-labeled plate, performing 3 parallel reactions on each sample, keeping the temperature at 37 deg.C for 5min, adding 50 μ L of xanthine oxidase, reading light absorption value once every 30s, and counting for 50 times for 25 min. After reading, stopping the reaction by LHCl of 80 mu, taking reaction liquid, diluting the reaction liquid by 10 times by using corresponding buffer solution, passing through a 0.25 mu m aqueous membrane, and measuring the concentration of uric acid;
(4) measuring the content of uric acid in the reacted sample by high performance liquid chromatography, and quantifying by using a uric acid standard substance;
(5) the xanthine oxidase inhibition rate is calculated by the following method:
I＝(CB-CS)/CB×100％
in the formula: i represents xanthine oxidase inhibition rate, CBThe peak area of uric acid peak of liquid phase chromatogram after blank group (without adding polypeptide) reaction, CSRepresenting the sample set (plus much)Peptide) peak area of uric acid peak of liquid phase chromatogram after reaction.
The preparation process flow of the walnut pulp protein peptide in the embodiment is as follows: and (3) mixing the walnut protein meal after oil extraction according to the mass ratio of feed liquid of 1: and 9, adding deionized water, mixing, heating to 95 ℃, keeping the temperature for 30min, cooling to 50-60 ℃, adding alkaline protease and cellulase respectively according to 0.6-1.2% of the mass of the walnut meal, carrying out enzymolysis for 8-10 hours at 55 ℃, heating to 95 ℃ after the enzymolysis is finished, keeping the temperature for 15min, inactivating the enzyme, cooling, centrifuging, taking supernatant, concentrating, and carrying out spray drying to obtain the walnut meal protein peptide.
As shown in FIGS. 1 and 4, HEPES has little effect on the content of uric acid produced by xanthine oxidase through xanthine oxidase (FIG. 1), while HEPES can significantly increase the xanthine oxidase inhibition rate of walnut protein peptide polypeptide (FIG. 4), which indicates that HEPES can play a role in enhancing the xanthine oxidase inhibitory activity of polypeptide.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (3)
- The application of 4-hydroxyethyl piperazine ethanesulfonic acid in preparing the medicine for enhancing the activity of xanthine oxidase inhibitory peptide is characterized in that: the xanthine oxidase inhibitory peptide is tuna peptide, saury peptide or walnut meal protein peptide.
- 2. Use according to claim 1, characterized in that: 4-hydroxyethyl piperazine ethanesulfonic acid is added as an additive to a polypeptide having a xanthine oxidase inhibitory function.
- 3. Use according to claim 1, characterized in that: the preparation of the xanthine oxidase inhibitory peptide comprises a filling agent corresponding to a solid medicament and a diluent corresponding to a liquid medicament.
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