CN107929711B - Application of blood-derived enzymolysis peptide in preparation of medicine for inhibiting animal intestinal inflammation - Google Patents
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Abstract
The invention provides an application of blood-derived enzymolysis peptide in preparation of a medicine for inhibiting animal intestinal inflammation, and particularly relates to a biological active peptide with an animal intestinal inflammation inhibiting effect, which is obtained by taking pig blood as a raw material and purifying the pig blood through processes of grinding, enzymolysis, suction filtration, centrifugation, ultrafiltration and the like. The dried active peptide is thin layer, powder and powder with lighter color. The bioactive peptide has effect in inhibiting intestinal inflammation, and can be used as feed, feed additive, and medicinal adjuvant. The blood source enzymolysis peptide for inhibiting the intestinal inflammation has no toxic or side effect, can not be left in animal bodies, and has obvious economic and social contributions. The invention can promote the rapid development of bioactive peptide biological products in the pharmaceutical industry and the feed industry.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of blood-derived enzymolysis peptide in preparation of a medicine for inhibiting animal intestinal inflammation.
Background
A great amount of meat food including livestock and poultry and aquatic food is produced in China every year, and the main factor causing the unsafe animal food in China is the serious overproof problem of veterinary drug residue. In the process of breeding livestock and poultry, lawless persons do not consider national regulations to add clenbuterol into the livestock and poultry feed to cause harm to human bodies; in the process of breeding livestock and poultry, antibiotics such as chloramphenicol, oxytetracycline and the like are used for preventing or treating diseases, although the antibiotics have effective antiseptic and antibacterial effects, the drug remains in the animal body, and after the drug is taken into the human body, the drug cannot be metabolized in the body at all, and the drug causes cancer and serious harm to the human body. However, modern breeding tends to be high-density intensive breeding, so that breeding bodies are more prone to diseases, and the problem of drug residues is further increased. However, no literature report has been found so far when zymolytic peptides, i.e., zymolytic peptides derived from blood sources, are used as drugs for inhibiting intestinal inflammation in animals. Therefore, the development of the bioactive peptide for inhibiting the intestinal inflammation of the animals and the application thereof have very important significance and value.
The invention obtains the blood source small peptide or protein mixture by a proper enzymolysis and purification method, verifies whether the existence activity of the extracted zymolyte is higher than that of the simulated and synthesized target peptide, and confirms the specific activity of the simulated and synthesized target peptide by the inhibition effect on the intestinal inflammation of the mouse animal. Therefore, the development of the blood source enzymolysis peptide for inhibiting the intestinal inflammation of the animals has important economic and social benefits.
Disclosure of Invention
The invention relates to an application of blood-derived enzymolysis peptide in preparation of a medicament for inhibiting animal intestinal inflammation. Pig blood is used as a raw material, and the optimal enzymolysis time and enzyme dosage are determined. A DSS acute intestinal inflammation model of a C57BL/6 mouse is established, and the inhibition effect of the model on intestinal inflammation is measured by indexes such as weight change, excrement state, intestinal mucosa injury condition, inflammatory factors and the like of the mouse. The blood source proteolytic peptide has no toxic or side effect, plays a certain role in inhibiting animal intestinal inflammation, and has wide application prospect. The blood source enzymolysis peptide is applied to feed additives and pharmaceutical auxiliary materials.
In order to realize the purpose, the invention adopts the following technical scheme:
a method for using blood-derived proteolytic peptide is characterized by taking pig blood as a raw material, adding phosphate buffer solution into the pig blood, then adding food-grade protease for enzymolysis, carrying out centrifugal filtration and ultrafiltration to obtain filtrate, and carrying out freeze drying to obtain the blood-derived proteolytic peptide.
A preparation method of blood source enzymolysis peptide for inhibiting animal intestinal inflammation comprises the following specific steps:
1) adding pig blood to Phosphate Buffered Saline (PBS) at a pH of 7.2-7.4, wherein the weight of pig blood is 9% of the volume of the buffer;
2) adding trypsin and pepsin into the pig blood mixed solution according to the proportion of 1:1, wherein the total adding mass of the trypsin and the pepsin is 3 percent of the mass of the pig blood;
3) the enzymolysis conditions are as follows: the rotation speed is 150r/min at 37 ℃, and the enzymolysis time is 6 hours;
4) centrifuging the enzymolysis liquid, collecting supernatant, vacuum filtering the supernatant, filtering the obtained filtrate with 0.22um water system membrane, filtering the twice filtered enzymolysis supernatant with ultrafiltration centrifuge tube (aperture 3000 Da) to obtain filtrate with aperture below 3000Da, and freeze drying in vacuum freeze dryer to obtain blood source enzymolysis peptide.
The invention has the following remarkable advantages: the invention relates to an application of blood source enzymolysis peptide for inhibiting animal intestinal inflammation, which has the following remarkable characteristics:
1) in the aspects of medicine and feed, the screening of the anti-inflammatory blood-derived zymolytic peptide lays a foundation for the research and development of new medicine synthesis, and especially, more and more people in the modern society use antibiotics more and more commonly, and the drug resistance of some pathogenic microorganisms to the antibiotics is higher and higher; excessive antibiotic use can be harmful to animals and humans;
the active peptide has the function of remarkably inhibiting intestinal inflammation, has no toxicity or side effect on animals, can not be enriched in the bodies of the animals, can also participate in metabolism, and can not cause harm after being taken into human bodies.
Drawings
FIG. 1 is a comparison of the body weight changes of PBS, blood-derived enzymatic products, blood-derived powder and norfloxacin mice.
FIG. 2 is a comparison of the hematochezia of PBS, blood-derived enzymolysis products, blood-derived powder and norfloxacin mice.
FIG. 3 intestinal morphology comparison of PBS, blood-derived enzymatic hydrolysate, blood-derived powder and norfloxacin.
FIG. 4 shows a comparison of the intestinal length of PBS, blood-derived enzymatic products, blood-derived powder and norfloxacin.
Figure 5 PBS, blood derived zymolyte, blood derived powder and norfloxacin intestinal tissue score comparisons.
FIG. 6 is a comparison of pathological forms of PBS, blood-derived enzymolysis products, blood-derived powder and norfloxacin intestinal mucosa.
FIG. 7 is a comparison of the detection of inflammatory factors by PBS, blood-derived enzymatic products, blood-derived powder and norfloxacin Q-PCR.
Detailed Description
The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention.
Example 1
Weighing 5.0g of pig blood, 0.15mg of trypsin, 0.15mg of pepsin and 40mL of prepared phosphoric acid buffer solution into a conical flask, and carrying out enzymolysis in a shaking table, wherein the temperature of the shaking table is set to be 37 ℃, the stirring speed is 150r/min, and the enzymolysis time is 6 hours. Centrifuging the enzymolysis solution to obtain filtrate, setting the rotation speed of the centrifuge at 5000 rpm/min, and centrifuging at normal temperature for 10 min. Removing precipitate, taking supernatant, and repeatedly centrifuging twice; vacuum filtering the obtained filtrate, filtering the obtained filtrate with 0.22um water system membrane, centrifuging and filtering the enzymolysis supernatant after twice filtration with an ultrafiltration centrifugal tube (aperture is 3000 Da) at 2500 rpm/min to obtain filtrate below 3000Da, and freeze-drying in a vacuum freeze dryer to obtain blood source enzymolysis peptide powder. The acute intestinal inflammation model of C57BL/6 mice was established using DSS: the prepared DSS solution is filled in drinking bottles for free drinking of 4 groups of mice by using the DSS modeling with the concentration of 2.5%, and the state, the weight and the hematochezia condition of each mouse are observed every day to ensure that the modeling is successfully established. After 5 days, the DSS solution was removed and the drinking water with tap water was replaced. Dividing mice into 4 groups which are respectively a negative control group, a blood source powder group, a blood source enzymolysis peptide group and a positive control group, wherein the dosages of the negative control group, the blood source powder group, the enzymolysis product group and the positive control group are respectively 0 and 500C/mg/kg for the first day of drinking DSS (direct sequence spread spectrum) from the beginning of the experiment-1、500 C/mg˙kg-1、8 C/mg˙kg-1Each group was dosed at the same time with different doses of each group, each mouse was weighed daily after the DSS solution was removed and replaced with normal drinking water, and the average change in body weight of each group of mice was calculated. The results are shown in fig. 1, the weight reduction range of the initial blood-derived enzymolysis product group is lower than that of the positive group, and the mice of the blood-derived enzymolysis peptide group are recovered to the initial weight most quickly in the recovery period, so that the blood-derived enzymolysis peptide has the effects of inhibiting and treating intestinal inflammation.
Example 2
Weighing 5.0g of pig blood, 0.15mg of trypsin, 0.15mg of pepsin and 40mL of prepared phosphoric acid buffer solution into a conical flask, and carrying out enzymolysis in a shaking table, wherein the temperature of the shaking table is set to be 37 ℃, the stirring speed is 150r/min, and the enzymolysis time is 6 hours. Centrifuging the enzymolysis solution to obtain filtrate, setting the rotation speed of the centrifuge at 5000 rpm/min, and centrifuging at normal temperature for 10 min. Removing precipitate, taking supernatant, and repeatedly centrifuging twice; vacuum filtering the obtained filtrate, filtering the obtained filtrate with 0.22um water system membrane, centrifuging and filtering the enzymolysis supernatant after twice filtration with an ultrafiltration centrifugal tube (aperture is 3000 Da) at 2500 rpm/min to obtain filtrate below 3000Da, and freeze-drying in a vacuum freeze dryer to obtain blood source enzymolysis peptide powder. The acute intestinal inflammation model of C57BL/6 mice was established using DSS: the prepared DSS solution is filled in drinking bottles for free drinking of 4 groups of mice by using the DSS modeling with the concentration of 2.5%, and the state, the weight and the hematochezia condition of each mouse are observed every day to ensure that the modeling is successfully established. After 5 days, the DSS solution was removed and the drinking water with tap water was replaced. Dividing the mice into 4 groups which are respectively a negative control group, a blood source powder group, an enzymolysis product group and a positive control group, and respectively adding 0, 500C/mg and kg of dosage into the first day of the experiment, namely the first day of drinking DSS (direct sequence sodium sulfide), the negative control group, the blood source powder group, the enzymolysis product group and the positive control group-1、500 C/mg˙kg-1、8 C/mg˙kg-1Each group was dosed at the same time with different doses of the formulated solutions, and the mice were semi-quantitatively examined for fecal occult blood after withdrawal of the DSS solution and replacement with normal drinking water. The specific operation method comprises the following steps:
1) opening a sealing cover printed with a Development Window with the back side of the test card facing upwards;
2) taking fresh excrement of each mouse to be tested, and quickly and uniformly smearing the excrement on a test area;
3) adding a first drop of color developing agent A to the fecal sample, and adding a drop of color developing agent B after the reagent is completely permeated;
4) after the color-developing agent B is added, the interpretation is finished after 2 min.
5) And (3) judging a test result:
(a) after addition of colour developer B, a bluish purple colour is produced immediately, reported as (4 +);
(b) after addition of developer B, bluish violet color is produced within 10s, reported as (3 +);
(c) after addition of developer B, bluish violet color is produced within 1min, reported as (2 +);
(d) after the color developing agent B is added, purple red is generated within 1-2 min and is reported as (1 +);
(e) after addition of developer B, there was no bluish purple or purplish red color reaction for the interpretation time, reported as (-).
According to the method, fecal occult blood detection, comparison and observation are carried out on each mouse at the same time every day. The results are shown in FIG. 2, in which four groups of mice had varying degrees of hematochezia, demonstrating that weight loss in mice was caused by intestinal inflammation. The blood-derived enzymolysis peptide and the norfloxacin have hematochezia conditions to a certain degree, but the condition is obviously lighter than that of blood-derived powder and PBS solution, the enzymolysis product group is light purple, the relative color of the norfloxacin group is slightly darker, and the treatment effect of the blood-derived enzymolysis product on the intestinal inflammation is better than that of the norfloxacin.
Example 3
Weighing 5.0g of pig blood, 0.15mg of trypsin, 0.15mg of pepsin and 40mL of prepared phosphoric acid buffer solution into a conical flask, and carrying out enzymolysis in a shaking table, wherein the temperature of the shaking table is set to be 37 ℃, the stirring speed is 150r/min, and the enzymolysis time is 6 hours. Centrifuging the enzymolysis solution to obtain filtrate, setting the rotation speed of the centrifuge at 5000 rpm/min, and centrifuging at normal temperature for 10 min. Removing precipitate, taking supernatant, and repeatedly centrifuging twice; vacuum filtering the obtained filtrate, filtering the obtained filtrate with 0.22um water system membrane, centrifuging and filtering the enzymolysis supernatant after twice filtration with an ultrafiltration centrifugal tube (aperture is 3000 Da) at 2500 rpm/min to obtain filtrate below 3000Da, and freeze-drying in a vacuum freeze dryer to obtain blood source enzymolysis peptide powder. The acute intestinal inflammation model of C57BL/6 mice was established using DSS: using a 2.5% DSS model, the prepared DSS solution was filled in drinking bottles and was freely drunk by 4 groups of mice, each mouse being subjected to daily measurement of state, body weight,The hematochezia condition was observed to ensure that the modeling was successfully established. After 5 days, the DSS solution was removed and the drinking water with tap water was replaced. Dividing the mice into 4 groups which are respectively a negative control group, a blood source powder group, an enzymolysis product group and a positive control group, and respectively adding 0, 500C/mg and kg of dosage into the first day of the experiment, namely the first day of drinking DSS (direct sequence sodium sulfide), the negative control group, the blood source powder group, the enzymolysis product group and the positive control group-1、500 C/mg˙kg-1、8 C/mg˙kg-1Each group was fed with the prepared solutions of different dosages at the same time, 3 of them were randomly taken from each group after the DSS solution was removed and changed to normal drinking water, and after ether anesthesia, the neck was cut off and sacrificed, and the intestinal tract was dissected and taken for observation.
The results are shown in fig. 3, the lengths of the four groups of intestinal tracts are greatly different, the intestinal tracts of the mice in the PBS group have very obvious atrophy phenomenon, the length is short and thin, no excrement is accumulated in the intestines, the normal eating cannot be realized, and only coagulated blood can be seen to adhere to the inner wall of the intestinal tract; the atrophy condition of the blood source powder group is slightly better than that of the PBS group, the difference of other conditions is not large, and excrement residue does not exist; the norfloxacin group and the blood-derived enzymolysis peptide group both find residual excrement in intestinal tracts, the excrement is mixed with a small amount of red blood, but the coagulation phenomenon is avoided, and the condition is far better than that of the other two groups. The intestinal length of the blood-derived zymolytic peptide group is slightly longer than that of the norfloxacin group, and no obvious atrophy phenomenon occurs.
Fig. 4 shows the average length of the intestinal tract of each group of mice, and the intestinal tract length of the blood-derived enzymolysis group is obviously longer than that of the other 3 groups, so that the blood-derived enzymolysis peptide product can well inhibit intestinal inflammation, prevent or relieve intestinal tract atrophy of the mice, stabilize the condition of the mice and has better treatment effect than norfloxacin.
Example 4
Weighing 5.0g of pig blood, 0.15mg of trypsin, 0.15mg of pepsin and 40mL of prepared phosphoric acid buffer solution into a conical flask, and carrying out enzymolysis in a shaking table, wherein the temperature of the shaking table is set to be 37 ℃, the stirring speed is 150r/min, and the enzymolysis time is 6 hours. Centrifuging the enzymolysis solution to obtain filtrate, setting the rotation speed of the centrifuge at 5000 rpm/min, and centrifuging at normal temperature for 10 min. Removing precipitate, collecting supernatant, and repeatedly centrifugingSecondly; vacuum filtering the obtained filtrate, filtering the obtained filtrate with 0.22um water system membrane, centrifuging and filtering the enzymolysis supernatant after twice filtration with an ultrafiltration centrifugal tube (aperture is 3000 Da) at 2500 rpm/min to obtain filtrate below 3000Da, and freeze-drying in a vacuum freeze dryer to obtain blood source enzymolysis peptide powder. The acute intestinal inflammation model of C57BL/6 mice was established using DSS: the prepared DSS solution is filled in drinking bottles for free drinking of 4 groups of mice by using the DSS modeling with the concentration of 2.5%, and the state, the weight and the hematochezia condition of each mouse are observed every day to ensure that the modeling is successfully established. After 5 days, the DSS solution was removed and the drinking water with tap water was replaced. Dividing the mice into 4 groups which are respectively a negative control group, a blood source powder group, an enzymolysis product group and a positive control group, and respectively adding 0, 500C/mg and kg of dosage into the first day of the experiment, namely the first day of drinking DSS (direct sequence sodium sulfide), the negative control group, the blood source powder group, the enzymolysis product group and the positive control group-1、500 C/mg˙kg-1、8 C/mg˙kg-1Feeding each group with prepared solution with different dosage at the same time, randomly taking 3 pieces from each group after removing DSS solution and changing to normal drinking water, killing neck after ether anesthesia, dissecting and taking out intestinal canal, and performing intestinal canal histopathology experiment.
Normal mice had normal colonic mucosa structure and should not appear atrophic. Degeneration, necrosis, inflammatory cell infiltration, ulcer, etc. As shown in fig. 5, the PBS group had typical pathological changes of ulcerative colitis, the intestinal mucosa layer was substantially completely disappeared, goblet cells were completely invisible, and the ulcerative phenomenon was very severe; the blood source powder group has a small amount of goblet cells remained at the tail end, but the arrangement is loose and disordered, and the mucous membrane layer at the tail end is destroyed, which indicates that the group of mice is damaged to a greater extent; the norfloxacin group also has a small amount of goblet cells, but the cells are arranged relatively closely and tidily, so that the intestinal tract of the mice in the group can keep a part of functions to prevent the disease from deteriorating; the intestinal mucosa of the mice in the blood source enzymolysis group has relatively best condition, the ulcer with too large area does not appear at the near tail end and the far tail end, only the local small area is infiltrated by inflammation, the goblet cells are reserved, and certain physiological function can be executed. Therefore, the treatment effect of the blood-derived enzymolysis group on the intestinal inflammation is better. Fig. 6 shows that the conversion to pathology scores based on residual goblet cells, higher scores, indicate that the greater the number of residual goblet cells, the less the intestinal mucosa is damaged. Thus, the blood source enzymolysis products have obvious protection effect on the intestinal mucosa.
Example 4
Weighing 5.0g of pig blood, 0.15mg of trypsin, 0.15mg of pepsin and 40mL of prepared phosphoric acid buffer solution into a conical flask, and carrying out enzymolysis in a shaking table, wherein the temperature of the shaking table is set to be 37 ℃, the stirring speed is 150rpm/min, and the enzymolysis time is 6 hours. Centrifuging the enzymolysis solution to obtain filtrate, setting the rotation speed of the centrifuge at 5000 rpm/min, and centrifuging at normal temperature for 10 min. Removing precipitate, taking supernatant, and repeatedly centrifuging twice; vacuum filtering the obtained filtrate, filtering the obtained filtrate with 0.22um water system membrane, centrifuging and filtering the enzymolysis supernatant after twice filtration with an ultrafiltration centrifugal tube (aperture is 3000 Da) at 2500 rpm/min to obtain filtrate below 3000Da, and freeze-drying in a vacuum freeze dryer to obtain blood source enzymolysis peptide powder. The acute intestinal inflammation model of C57BL/6 mice was established using DSS: the prepared DSS solution is filled in drinking bottles for free drinking of 4 groups of mice by using the DSS modeling with the concentration of 2.5%, and the state, the weight and the hematochezia condition of each mouse are observed every day to ensure that the modeling is successfully established. After 5 days, the DSS solution was removed and the drinking water with tap water was replaced. Dividing mice into 4 groups which are respectively a negative control group, a blood source powder group, a blood source enzyme degradation group and a positive control group, and respectively adding 0, 500C/mg kg of dosage into the first day of the experiment, namely the first day of drinking DSS, the negative control group, the blood source powder group, the blood source enzyme degradation group and the positive control group-1、500 C/mg˙kg-1、8 C/mg˙kg-1Quantitatively feeding each group with prepared solution with different dosage at the same time, randomly taking 3 pieces from each group after DSS solution is removed and normal drinking water is changed, breaking neck after ether anesthesia and killing, dissecting and taking intestinal tract, extracting intestinal mucosa RNA, and performing inflammation factor Q-PCR detection to quantitatively analyze common inflammation factors TNF-alpha, IL-1 beta, IL-6 and IL-10.
As shown in figure 7, in the quantitative detection of four inflammatory factors of TNF-alpha, IL-1 beta, IL-6 and IL-10, the content of the blood-derived enzymolysis group is the lowest and is far lower than that of the PBS group and the blood-derived powder group, and the content of the norfloxacin group is slightly higher than that of the blood-derived enzymolysis group but lower than that of the other two groups, which indicates that the inflammation inhibition effect of the blood-derived enzymolysis peptide is better than that of norfloxacin.
Claims (1)
1. An application of blood-derived enzymolysis peptide in preparing a medicine for inhibiting animal acute intestinal inflammation is disclosed, wherein the preparation method of the blood-derived enzymolysis peptide comprises the following steps:
1) adding pig blood into a phosphate buffer solution system;
2) adding trypsin and pepsin into a phosphate buffer solution system for double-enzyme enzymolysis;
3) centrifuging the enzymolysis liquid, taking supernatant, carrying out suction filtration, filtering a membrane again, and desalting;
4) taking the filtrate, freeze-drying, and concentrating to obtain a powder to obtain the bioactive peptide;
wherein, the pH of the phosphate buffer solution system is 7.2-7.4, and the adding mass of the pig blood is 12.5% of the volume of the buffer solution;
the mass ratio of the trypsin to the pepsin is 1:1, and the total added mass of the trypsin and the pepsin is 0.006 percent of the mass of the pig blood;
the enzymolysis conditions are as follows: the rotation speed is 150r/min at 37 ℃, and the enzymolysis time is 6 hours;
the vacuum filtration is to use vacuum filtration supernatant, and the obtained filtrate is filtered by a 0.22um water system filter membrane to remove macromolecules;
the filtered molecular weight cutoff is below 3000 Da;
the freeze drying is to freeze the enzymolysis liquid in a refrigerator at minus 86 ℃, and then freeze-dry the enzymolysis liquid in a vacuum freeze drier to prepare powder to be stored in the refrigerator at 4 ℃;
the blood-derived enzymolysis peptide has a remarkable inhibiting effect on animal acute intestinal inflammation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101250572A (en) * | 2007-10-29 | 2008-08-27 | 中国农业大学 | Method for extracting pig blood antibiotic peptide |
| CN107126551A (en) * | 2017-05-18 | 2017-09-05 | 福州大学 | Application of the microalgae proteolysis peptide in prevention and treatment enteritis medicine is prepared |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101250572A (en) * | 2007-10-29 | 2008-08-27 | 中国农业大学 | Method for extracting pig blood antibiotic peptide |
| CN107126551A (en) * | 2017-05-18 | 2017-09-05 | 福州大学 | Application of the microalgae proteolysis peptide in prevention and treatment enteritis medicine is prepared |
Non-Patent Citations (3)
| Title |
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| Bioactive dietary peptides and amino acids in inflammatory bowel disease;Zhang hua等;《Amino Acids》;20151031;第47卷(第10期);第2127-2147页 * |
| 猪血浆酶解肽的制备工艺及免疫调节功能的初步研究;吴涵等;《吉林农业大学学报》;20100630;第32卷(第3期);摘要，第350-351页的材料与方法，第354页的第2.6节和结论 * |
| 细胞因子在炎症性肠病的发病机制与治疗进展;李慕然等;《天津医药》;20061030;第34卷(第10期);第748-749页的第1-2节 * |
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