CN115644455B - Application of bovine spleen peptide powder in improving intestinal function - Google Patents
Application of bovine spleen peptide powder in improving intestinal function Download PDFInfo
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Abstract
The application provides an application of bovine spleen peptide powder in improving intestinal functions. The application also provides application of the bovine spleen peptide powder in preparing health-care food for improving the intestinal function of a subject, and application of the bovine spleen peptide powder in preparing a medicine for improving the intestinal function of the subject.
Description
Technical Field
The present application relates generally to the field of biomedicine and health food, and in particular, to the use in the preparation of a health food for improving the intestinal function of a subject, or in the preparation of a medicament for improving the intestinal function of a subject.
Background
The bovine spleen peptide is small molecular bovine spleen peptide powder which is obtained by taking pure and natural bovine spleen as a raw material and performing the processing technologies of crushing, sterilization, biological enzymolysis, enzyme deactivation, purification, concentration, centrifugal spraying, drying and the like. The bovine spleen peptide powder has very high activity, is easy to be absorbed by human bodies, can play a role in relieving fatigue and enhancing immunity, has the efficacy of dispelling the effects of alcohol and protecting liver, and can help to reduce blood fat and blood pressure.
In view of the excellent activity of bovine spleen peptide powder, it is very significant to develop new biomedical and health food functions thereof in improving intestinal functions.
Disclosure of Invention
In a first aspect, the present application provides the use of bovine spleen peptide powder in the manufacture of a health food for improving intestinal function in a subject, or in the manufacture of a medicament for improving intestinal function in a subject.
In some embodiments of the first aspect, the bovine splenopeptide powder is used at a concentration of no more than 250 μ g/mL.
In some embodiments of the first aspect, the bovine splenopeptide powder, or the bovine splenopeptide powder together with edible adjuvants is prepared as a health food.
In some embodiments of the first aspect, the bovine splenopeptide powder is formulated in a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.
In some embodiments of the first aspect, the medicament is administered orally, subcutaneously, intramuscularly or intraperitoneally.
In some embodiments of the first aspect, the subject is a vertebrate.
In some embodiments of the first aspect, the subject is a fish, a mammal, a round-mouth, an amphibian, a reptile or a bird.
In some embodiments of the first aspect, the subject is a fish or a mammal.
In some embodiments of the first aspect, the improving intestinal function in the subject comprises lubricating the intestines, and/or preventing, ameliorating, or treating constipation.
In a second aspect, the present application provides a method of improving gut function in a subject, comprising administering to the subject an effective amount of bovine splenopeptide powder.
In a third aspect, the present application provides a method of preventing, ameliorating, or treating constipation in a subject comprising administering to the subject an effective amount of bovine splenopeptide powder.
Drawings
FIG. 1 shows a flow chart for the preparation of bovine spleen peptide powder;
FIG. 2 shows a standard curve for polypeptide detection of bovine spleen peptide powder;
FIG. 3 shows the result of SDS-PAGE electrophoresis of bovine spleen peptide powder;
FIG. 4 shows the HPLC detection results of amino acid standards;
FIG. 5 shows the HPLC assay of the amino acid content of bovine spleen peptide powder;
FIG. 6 shows a typical plot of the number of goblet cells in the intestinal tract of zebrafish after treatment with bovine spleen peptide powder, with the dashed line being the intestinal tract of the analysis region and the particles being goblet cells;
figure 7 shows a plot of the number of gut goblet cells after treatment with bovine spleen peptide powder, where p represents p <0.001 compared to the model control group;
FIG. 8 shows a typical plot of fluorescence intensity of zebrafish gastrointestinal tract after treatment with bovine spleen peptide powder, wherein the dashed box in the exemplary plot is the zebrafish gastrointestinal tract analysis site;
figure 9 shows a plot of fluorescence intensity of the gastrointestinal tract of zebrafish after treatment with bovine splenopeptide powder, where p represents <0.001 compared to the model control group.
Detailed Description
The inventors of the present application found through experiments that bovine spleen peptide powder has an effect of improving intestinal function (e.g., lubricating the intestinal tract, or preventing, ameliorating or treating constipation). Therefore, the bovine spleen peptide powder can be used for preparing health-care food for improving the intestinal function of a subject, or the bovine spleen peptide powder can be used for preparing a medicine for improving the intestinal function of the subject, so that the new application of the bovine spleen peptide powder is developed.
The practice of the present application employs, unless otherwise indicated, conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques.
Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.
The term "health food" as used herein refers to a food having a health function or for the purpose of supplementing nutrients such as vitamins, minerals, etc. The health food is suitable for specific people, has the function of regulating organism, does not aim at treating diseases, and does not produce any acute, subacute or chronic harm to human body.
In a first aspect, the present application provides the use of bovine spleen peptide powder in the preparation of a health food for improving intestinal function in a subject.
In some embodiments of the first aspect, the bovine splenopeptide powder, or the bovine splenopeptide powder together with edible adjuvants is prepared as a health food.
In some embodiments of the first aspect, the dietary supplement comprises a sweetener, an acidulant, and/or a preservative.
In some embodiments of the first aspect, the sweetener is selected from one or more of the following: sorbitol, fructose, glucose, lactose, mannitol, maltitol, and xylitol.
In some embodiments of the first aspect, the acidulant is selected from one or more of the following: citric acid, lemon concentrate, tartaric acid, malic acid, lactic acid and acetic acid.
In some embodiments of the first aspect, the preservative is selected from one or more of: benzoic acid, sodium benzoate, potassium sorbate and sodium lactate.
In some embodiments of the first aspect, the health food is a tablet, powder, granule, tea, hard capsule, soft capsule, oral liquid, pill, wine, paste, beverage, pastry, liquid milk, biscuit, candy, raw material extract or compounded nutrient.
In a second aspect, the present application provides the use of bovine spleen peptide powder in the preparation of a medicament for improving intestinal function in a subject.
In some embodiments of the second aspect, the bovine splenopeptide powder is formulated in a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.
In some embodiments of the second aspect, the pharmaceutically acceptable carrier refers to a carrier that does not interfere with the biological activity of the active ingredient, including those conventionally used in the pharmaceutical arts. The pharmaceutically acceptable carrier of the present application may be a solid or a liquid, including pharmaceutically acceptable excipients, buffers, emulsifiers, stabilizers, preservatives, diluents, encapsulating agents, fillers, and the like. For example, pharmaceutically acceptable buffers further include phosphates, acetates, citrates, borates, carbonates, and the like. In some embodiments, the medicament of the present application for improving intestinal function in a subject may be prepared by any method known in the pharmaceutical art. All methods include the step of bringing into association the active ingredients of the present application with one or more pharmaceutically acceptable carriers. Generally, compositions are prepared by combining the active ingredient with a liquid carrier, a solid carrier, or both, and then shaping the resulting product as desired.
In some embodiments of the second aspect, the bovine splenopeptide powder is formulated in an oral liquid, capsule, powder, tablet, granule, pill, syrup, suppository, or injection with a pharmaceutically acceptable carrier.
In some embodiments of the second aspect, the medicament is administered orally, subcutaneously, intramuscularly or intraperitoneally.
In some embodiments of the second aspect, the medicament is administered orally.
In some embodiments of any of the above aspects, the bovine splenopeptide powder is used at a concentration of no more than 250 μ g/mL, e.g., the bovine spleen peptide powder is used at a concentration of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 60.5, 61, 61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 241, 240, 244, 242, 244, 248, 245, 243, 249, 250, 246, or any two or more μ g/mL value.
In some embodiments of any of the foregoing aspects, the bovine splenopeptide powder is used at a concentration of 62.5-250 μ g/mL, e.g., 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 85, 90, 95, 100, 105, 110, 115, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250 μ g/mL, or a value or a range between any two or more of the foregoing values.
In some embodiments of any of the above aspects, the bovine splenopeptide powder is used at a concentration of 62.5, 125, or 250 μ g/mL.
In some embodiments of any of the aspects above, the subject is a vertebrate.
In some embodiments of any of the above aspects, the subject is a fish, a mammal, a round-mouth, an amphibian, a reptile or a bird.
In some embodiments of any of the above aspects, the subject is a fish (e.g., zebrafish) or a mammal (e.g., human).
In some embodiments of any of the above aspects, the bovine splenic peptide powder is capable of increasing the number of goblet cells in the gut, and the goblet cells are capable of synthesizing and secreting mucin, thereby serving to lubricate the gut.
In some embodiments of any of the above aspects, the improving intestinal function in the subject comprises lubricating the intestine.
In some embodiments of any of the aspects above, the improving intestinal function in the subject comprises preventing, ameliorating, or treating constipation.
In a second aspect, the present application provides a method of improving gut function in a subject, comprising administering to the subject an effective amount of bovine splenopeptide powder.
In a third aspect, the present application provides a method of preventing, ameliorating or treating constipation in a subject comprising administering to the subject an effective amount of bovine splenopeptide powder.
In some embodiments of any of the above aspects, the preparation of the bovine splenopeptide powder comprises the steps of: selection of bovine spleen, disruption of the homogenate, freeze-thawing, solid-liquid separation, filtration (e.g., coarse filtration and/or ultrafiltration), sterilization and spray drying of the supernatant.
In some embodiments of any of the aspects above, the amount of polypeptide in the bovine spleen peptide powder is 195.8mg/g.
In some embodiments of any of the above aspects, the polypeptide molecules in the bovine spleen peptide powder have a molecular weight of less than 17KD.
In some embodiments of any of the above aspects, the bovine splenic peptide powder has an amino acid content of 444.5mg/g.
In the present description and claims, the words "comprise", "comprises" and "comprising" mean "including but not limited to", and are not intended to exclude other moieties, additives, components or steps.
It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application may be applied to any other aspect, embodiment or example described herein unless incompatible therewith.
The above disclosure generally describes the present application and the following examples are further illustrative of the present application and are not to be construed as limiting the present application. The invention discloses application of bovine spleen peptide powder in preparing health-care food for improving intestinal functions of a subject or medicines for improving the intestinal functions of the subject. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. Those skilled in the art will recognize that the invention may be implemented and practiced with modification, or with appropriate alteration and combination, of the methods and applications described herein without departing from the spirit, scope, and content of the present invention.
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to the embodiments.
Examples
Example 1 preparation and characterization of bovine spleen peptide powder
1. Preparation of bovine spleen peptide powder
The preparation method of the bovine spleen peptide powder comprises the following steps (the flow chart is shown in figure 1):
1) Crushing and homogenizing: cleaning fresh cattle spleen, removing fascia trachea, shearing, crushing by a homogenizer, adding purified water with the weight 5 times of that of the fresh spleen homogenate, and uniformly stirring to obtain spleen pulp;
2) Freezing and thawing: freezing the spleen slurry in a-20 deg.C refrigerator for more than 48 hr, thawing in water bath (upper limit thawing temperature is 40 deg.C), and repeatedly freezing and thawing for 3 times;
3) Separation: separating the supernatant with a sedimentation centrifuge or a tubular centrifuge;
4) Coarse filtration: filtering the supernatant obtained in the step 3) by a filter membrane with the aperture of 50 mu m to obtain a crude filtrate;
5) And (3) ultrafiltration: ultrafiltering the crude filtrate obtained in the step 4) by using an ultrafiltration membrane of 30KDa to obtain ultrafiltrate, namely the bovine spleen extracting solution;
6) Sterilizing, filtering with 0.2 μm sterilizing filter core, sterilizing, and packaging;
7) And (5) carrying out spray drying on the subpackaged solution obtained in the step 6) to obtain the bovine spleen peptide powder.
2. Characterization of bovine spleen peptide powder
2.1 And (3) polypeptide content detection: BCA method
An experimental instrument: ultraviolet spectrophotometer (model: UV1800; manufacturer: TOSOH).
Detection reagent: BCA rapid test kit (cat # A53225; manufacturer: thermo).
Sample preparation: weighing 0.05g of bovine spleen peptide powder sample, dissolving in 10mL of purified water, and uniformly mixing for later use.
The detection method comprises the following steps:
1) Bovine Serum Albumin (BSA) standard (provided by kit) was diluted in a gradient to 0.1, 0.2, 0.3, 0.4, and 0.5mg/mL;
2) Preparing a BCA working solution, and uniformly mixing 100mL of the working solution A and 2mL of the working solution B in the kit;
3) Respectively adding the diluted standard substance and the sample to be detected into an enzyme label plate (20 mu L/hole), adding 200 mu L of BCA working solution, and incubating for 30min at 37 ℃;
4) And reading the absorbance of each hole at 562nm, drawing a standard curve, and calculating the concentration of the sample to be detected.
The results of the experiment are shown in table 2: the polypeptide content in the bovine spleen peptide powder is 195.8mg/g.
Table 1. Standard curve test results.
| Standard substance concentration (mug/mL) | 2000 | 1000 | 500 | 250 | 125 | 62.5 | 31.25 | 0 |
| Absorbance (OD value) | 0.708 | 0.445 | 0.296 | 0.215 | 0.168 | 0.144 | 0.136 | 0.127 |
TABLE 2 polypeptide content test results of bovine spleen peptide powder samples
2.2 And (3) detecting the molecular weight of the polypeptide: SDS-PAGE method
An experimental instrument: mini vertical electrophoresis apparatus (model: BG-Power600; manufactured by Beijing Baijing Biotechnology Co., ltd.), and decolorizing shaking table (model: WD-9405D; manufactured by Beijing six Biotechnology Co., ltd.).
Detection reagent: 12% of bis-Tris precast electrophoresis gel (model: MP0342BOX; manufacturer: thermo Scientific), electrophoresis buffer (20X) (model: NP0002; manufacturer: thermo Scientific), protein staining solution (model: 46-5034; manufacturer: thermo Scientific), sample reducing agent (10X) (model: NP0009; manufacturer: thermo Scientific), sample buffer (model: NP0007; manufacturer: thermo Scientific), molecular weight standard for electrophoresis protein (Marker) (model: 26619; manufacturer: thermo Scientific).
Sample preparation: weighing 0.1g of bovine spleen peptide powder, dissolving in 10mL of PBS, and mixing uniformly for later use.
The detection method comprises the following steps:
1) Taking 50 mu L of a sample to be detected, adding 25 mu L of sample buffer solution, 10 mu L of sample reducing agent and 15 mu L of purified water, wherein the total volume is 100 mu L;
2) Heating the mixed solution obtained in the step 1) in boiling water for 3 minutes, taking out, and placing in a refrigerator at 4 ℃ for later use;
3) Balancing the electrophoretic precast gel to room temperature, opening the electrophoretic precast gel, washing, placing in a prepared electrophoretic buffer solution (1 x), adding a sample (10 mu L of a sample to be detected, 5 mu L of a Marker), opening an electrophoresis apparatus switch, and carrying out electrophoresis at a voltage of 120V until a bromophenol blue indicator is positioned in the middle of the gel; taking out the rubber plate, adding a dyeing solution, and dyeing for 60 minutes on a shaking table; the quality control product is Bovine Serum Albumin (BSA), and the molecular weight is 66.4KD;
4) After the dyeing was completed, decolorization was performed with purified water and the range of molecular weight was determined.
The experimental results are shown in fig. 3: the molecular weight of the polypeptide in the bovine spleen peptide powder is mainly concentrated below 17KD.
2.3 And (3) detecting the content of amino acid: HPLC method
An experimental instrument: high performance liquid chromatograph (model: SPD-20A; manufacturer: shimadzu, japan).
Detection reagent: acetonitrile (cat # 75-05-8; lot # 203096; manufacturer: sammer Feishell technology (China) Co., ltd.), methanol (cat # 67-56-1; lot # 211775; manufacturer: sammer Feishell technology (China) Co., ltd.), amino acid method kit (cat # AJS-01; manufacturer: shimadzu, japan).
Sample preparation: a bovine spleen peptide powder sample of 0.03g was weighed, added with 5mL of 0.1mol/L hydrochloric acid and mixed well, and filtered through a 0.45 μm filter membrane. Taking 400 mu L of filtrate, 100mL of 0.1mol/L hydrochloric acid and 50 mu L of internal standard substance, and mixing uniformly for later use.
The detection method comprises the following steps:
mobile phase: weighing 9.0g of disodium hydrogen phosphate dodecahydrate and 9.5g of sodium tetraborate decahydrate, adding 2000mL of water, adjusting the pH value to 8.2 by using hydrochloric acid, uniformly mixing, filtering by using a 0.45-micrometer filter membrane, and performing ultrasonic treatment to obtain a mobile phase A; taking 450mL of methanol, 450mL of acetonitrile and 100mL of purified water, mixing uniformly, filtering by using a 0.45-micron filter membrane and carrying out ultrasonic treatment to obtain a mobile phase B.
Wavelength: UV detectors 338nm and 262nm;
column temperature: 50 ℃;
a chromatographic column: c18;
flow rate: 1.0mL/min;
sample introduction amount: 1 mu L of the solution;
sample treatment: derivatisation
1) Precisely measuring 1 mu L of a mixed solution of 17 amino acid standard products, injecting the mixed solution into a liquid chromatograph, and recording a chromatogram, wherein an experimental result is shown in figure 4;
2) Precisely measuring the sample to obtain 1 μ L, injecting into liquid chromatographic column, and recording chromatogram.
The results of the experiment are shown in fig. 5 and table 3: the content of amino acid in the bovine spleen peptide powder is 444.5mg/g.
TABLE 3 amino acid content test results for bovine spleen peptide powder samples
Example 2 modulation of Calicides in the gut by bovine splenopeptide powder
1. Detection material
1.1 Sample preparation information
Bovine spleen peptide powder is prepared into 20.0mg/mL mother liquor by using standard dilution water, and is prepared for use immediately by ultrasonic treatment.
Positive control: prednisone, white powder, lot number C10016501, shanghai mclin biochemistry science and technology ltd, stored at 4 ℃. Prepared into a mother solution of 15.0mg/mL with DMSO and stored at-20 ℃.
1.2 Laboratory animal
The zebra fishes are all bred in fish breeding water at 28 ℃ (water quality: 200mg of instant sea salt is added in every 1L of reverse osmosis water, the conductivity is 450-550 muS/cm, the pH is 6.5-8.5, the hardness is 50-100mg/L CaCO 3), the zebra fishes are provided by breeding of a fish breeding center of Hangzhou Huantian Loop Biotechnology Limited company, and the use license number of experimental animals is as follows: SYXK (Zhe) 2022-0004. The feeding management meets the requirements of the international AAALAC certification (certification number: 001458).
Wild type AB strain zebrafish, in a natural mated breeding mode. Zebra fish aged 3 days (3 dpf) after fertilization are used for measuring the maximum detection concentration (MTC) of bovine spleen peptide powder and evaluating the efficacy of improving intestinal functions.
1.3 Instruments, consumables and reagents
Dissecting microscopy (SZX 7, OLYMPUS, japan); a CCD camera (VertA 1, shanghai, geosenson vision science and technology limited, china); microtomes (KD 2258, jinhua kedi medical devices, ltd., china); precision electronic balances (CP 214, OHAUS, USA); 6 well plates (Nest Biotech, china).
Trinitrobenzenesulfonic acid (TNBS, batch number SLCK4178, sigma, USA); alcian blue (lot No. BCBV8028, sigma, switzerland); dimethyl sulfoxide (DMSO, lot No. BCCD8942, sigma, switzerland); glacial acetic acid (batch No. C10323745, shanghai mclin biochemical technologies ltd, china); methylcellulose (batch No. G2106167, shanghai alading biochem technology ltd., china); 4% of a tissue cell fixing solution (batch No. 20210828, beijing Soilebao Tech Co., ltd., china); xylene (batch No. 20201113, chemical reagents of national drug group, inc., china); PBS buffer (batch No. 70115000, biosharp, china); mayer hematoxylin staining solution (batch No. 20220120, shanghai ihh biotechnology limited, china); eosin staining solution (batch No. 20220120, shanghai ihh biotechnology limited, china); neutral gums (batch No. 330a021, solarbio, china); huabao high-efficiency section paraffin (batch No. 20210828, shanghai hua yong paraffin limited, china); anhydrous ethanol (batch No. 20210107, chemical reagents of national drug group, ltd., china).
2. Detection method
2.1 MTC assays
3dpf wild type AB strain zebra fish was randomly selected in a 6-well plate, and 30 zebra fish were treated per well in the experimental group. Except for a normal control group (fed according to a conventional mode), all the other experimental groups are given water-soluble TNBS to establish a zebra fish gastrointestinal mucosa injury model. After 2 days of treatment at 28 ℃, TNBS was removed, and a water-soluble bovine splenopeptide powder was administered at the concentration shown in table 4 below, while a normal control group and a model control group were set at a capacity of 3mL per well. After further treatment for 2 days at 28 ℃, the MTC of the bovine spleen peptide powder on the zebra fish of the gastrointestinal mucosa injury model is determined.
2.2 Evaluation of intestinal Calicite regulatory efficacy
3dpf of wild type AB zebra fish was randomly selected in a 6-well plate, and 30 zebra fish were treated per well in the experimental group. Except for a normal control group (which is raised according to a conventional mode), water-soluble TNBS is given to other experimental groups to establish a zebra fish gastrointestinal mucosa injury model. After 2 days of treatment at 28 ℃, TNBS was removed, and water-soluble bovine splenic peptide powder was administered at a concentration as shown in table 5 below, with the concentration of the positive control prednisone being 15.0 μ g/mL, and with the normal control group and the model control group (i.e., a zebrafish gastrointestinal mucosa injury model established by TNBS) being set at the same time, the volume per well being 3mL. After treatment is carried out for 2 days at 28 ℃, the zebra fish is stained by alcian blue, 10 zebra fish are randomly selected from each experimental group and placed under an anatomical microscope for photographing, data are collected by NIS-Elements D3.20 advanced image processing software, the number of intestinal goblet cells of the zebra fish is analyzed, and the adjusting effect of the intestinal goblet cells of the bovine spleen peptide powder is evaluated according to the statistical analysis result of the index. Statistical treatment results are expressed as "mean ± standard deviation". Statistical analysis was performed using SPSS26.0 software and p <0.05 indicated that the differences were statistically significant.
3. The result of the detection
3.1 MTC
The maximum detectable concentration of bovine splenopeptide powder for the modulation of goblet cell efficacy (MTC) was 250 μ g/mL, as detailed in Table 4.
TABLE 4 Experimental results of Calicin efficacy concentration determination by bovine spleen peptide powder (n = 30)
3.2 Regulating effect of bovine spleen peptide powder on intestinal goblet cells
The results of table 5 and fig. 6-7 show that: the bovine spleen peptide powder has a regulating effect on goblet cells and can increase the number of intestinal goblet cells.
TABLE 5 evaluation of the efficacy of bovine splenopeptide powder in regulating intestinal goblet cells (n = 10)
Note: as compared to model control group,. Indicates p <0.001.
Example 3 evaluation of efficacy of bovine spleen peptide powder for preventing constipation
1. Detection material
1.1 Sample preparation information
Bovine spleen peptide powder is prepared into 20.0mg/mL mother liquor by using standard dilution water, and is prepared for use immediately by ultrasonic treatment.
Positive control: spirulina alfalfa meal, white powder, lot No. 145611, perfection (china) ltd, stored in the shade. The mother liquor is prepared into 2.00mg/mL by using standard dilution water and is prepared for use.
1.2 Laboratory animal
The zebra fishes are all bred in fish breeding water at 28 ℃ (water quality: 200mg of instant sea salt is added in each 1L of reverse osmosis water, conductivity is 450-550 muS/cm; pH is 6.5-8.5; hardness is 50-100mg/L CaCO 3), the water is provided by breeding of a fish breeding center of Hangzhou Huate Loop Biotechnology Ltd, and the use license numbers of experimental animals are as follows: SYXK (Zhe) 2022-0004. The feeding management meets the requirements of the international AAALAC certification (certification number: 001458).
Wild type AB strain zebrafish, in a natural mated mating breeding mode. Zebrafish aged 5 days (5 dpf) after fertilization were used for bovine spleen peptide powder maximum assay concentration (MTC) assay and evaluation of constipation prevention efficacy.
1.3 Instruments, consumables and reagents
Dissecting microscopes (SZX 7, OLYMPUS, japan); a CCD camera (VertA 1, shanghai, geosenson vision science and technology limited, china); an electric focusing continuous variable magnification fluorescence microscope (AZ 100, nikon, japan); precision electronic balances (CP 214, OHAUS, USA); 6 well plates (Nest Biotech, china).
Methylcellulose (batch No. C2004046, shanghai alading biochem technology ltd., china); aluminum sulfate (batch No. D1909026, shanghai alading biochemical science & technology, ltd., china); nile red (batch number SLBP9326V, sigma, india).
2. Detection method
2.1 MTC assay
5dpf wild type AB strain zebra fish was randomly selected in a 6-well plate, and 30 zebra fish were treated per well in the experimental group. Water-soluble bovine splenopeptide powder was administered at the concentrations shown in Table 6 below, while a normal control group (fed in a conventional manner) and a model control group (constipation model established by aluminum sulfate) were set at a volume of 3mL per well. After 2 days of treatment at 28 ℃, bovine splenin peptide powder was removed and the gastrointestinal tract was stained with water-soluble nile red given to each experimental group. After dyeing is finished, the other experimental groups except the normal control group are given water-soluble aluminum sulfate to establish a constipation model. After 6h of aluminum sulfate treatment, the MTC of the bovine spleen peptide powder on model zebra fish is determined.
2.2 Evaluation of Constipation prevention efficacy
And randomly selecting 5dpf wild AB strain zebra fish, randomly distributing the zebra fish into 6-hole plates, and treating 30 zebra fish in each hole of the experimental group. Water-soluble bovine spleen peptide powder was administered at a concentration as shown in table 7 below, the positive control spirulina alfalfa powder concentration was 667 μ g/mL, a normal control group (fed in a conventional manner) and a model control group (constipation model was established by aluminum sulfate) were set at the same time, and the volume per well was 3mL. After 24h of treatment at 28 ℃, bovine splenin peptide powder was removed and the gastrointestinal tract was stained with water-soluble nile red given to each experimental group. After dyeing, the other experimental groups except the normal control group are all given water-soluble aluminum sulfate to establish a constipation model. After the aluminum sulfate is treated for 6 hours, 10 zebra fish in each group are randomly selected to be photographed under a fluorescence microscope and stored, NIS-Elements D3.20 advanced image processing software is used for analyzing and collecting data, the gastrointestinal fluorescence intensity of the zebra fish is analyzed, and the constipation prevention effect of the bovine spleen peptide powder is evaluated according to the index statistical analysis result. Statistical treatment results are expressed as "mean ± standard deviation". Statistical analysis was performed with SPSS26.0 software and p <0.05 indicated that the differences were statistically significant.
3. The result of the detection
3.1 MTC
The Maximum Test Concentration (MTC) of the bovine spleen peptide powder for preventing constipation is 250 mug/mL, and is detailed in Table 6.
TABLE 6 results of experiments with concentration of bovine spleen peptide powder for constipation prevention efficacy (n = 30)
3.2. Evaluation of Constipation prevention efficacy
The results of table 7 and fig. 8-9 show that: the bovine spleen peptide powder has the effect of preventing constipation.
TABLE 7 Experimental results of evaluation of constipation preventing efficacy of bovine spleen peptide powder (n = 10)
Note: p <0.001 compared to model control.
All publications and patent documents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the disclosure. Unless the context indicates otherwise, any feature, step or embodiment of an embodiment of the present disclosure may be used in combination with any other feature, step or embodiment.
Claims (7)
1. Use of bovine spleen peptide powder for the preparation of a health food for improving constipation or lubricating the intestinal tract of a subject, or for the preparation of a medicament for preventing, improving or treating constipation or lubricating the intestinal tract of a subject;
wherein the using concentration of the bovine spleen peptide powder is 62.5-250 mug/mL; and
the subject is a zebrafish or a human.
2. The use of claim 1, wherein the bovine splenopeptide powder, or the bovine splenopeptide powder with edible excipients is prepared into a health food.
3. The use according to claim 2, wherein the health food is a tablet, powder, granule, tea, hard capsule, soft capsule, oral liquid, pill, medicated wine, paste, beverage, pastry, liquid milk, biscuit, candy, raw material extract or compounded nutrient.
4. The use of claim 1, wherein the bovine splenopeptide powder is formulated in a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier.
5. The use of claim 4, wherein the pharmaceutically acceptable dosage form is an oral liquid, capsule, powder, tablet, granule, pill, syrup, suppository, or injection.
6. The use of claim 1, wherein the medicament is administered orally, subcutaneously, intramuscularly or intraperitoneally.
7. The use of claim 1, wherein the medicament is administered orally.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211570539.1A CN115644455B (en) | 2022-12-08 | 2022-12-08 | Application of bovine spleen peptide powder in improving intestinal function |
| PCT/CN2023/136683 WO2024120423A1 (en) | 2022-12-08 | 2023-12-06 | Use of bovine spleen peptide powder in improving sleep, preventing or treating depression, or improving intestinal function |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2006126574A1 (en) * | 2005-05-24 | 2008-12-25 | 国立大学法人 熊本大学 | ES cell differentiation induction method |
| CN103892166A (en) * | 2014-04-11 | 2014-07-02 | 江西德泰医药生物技术有限公司 | Method for preparing health food by polypeptide factor and Chinese herb extracts |
| CN103989041A (en) * | 2014-04-11 | 2014-08-20 | 南昌大学 | Multi-biological activity function food and preparation process thereof |
| CN113584108A (en) * | 2021-07-19 | 2021-11-02 | 湖南益旺生物科技有限公司 | Preparation method of spleen aminopeptide |
| CN113647556A (en) * | 2021-08-19 | 2021-11-16 | 江西傲龙优可健康产业有限公司 | Rice flour capable of improving immunity of infants and preparation method thereof |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPWO2006126574A1 (en) * | 2005-05-24 | 2008-12-25 | 国立大学法人 熊本大学 | ES cell differentiation induction method |
| CN103892166A (en) * | 2014-04-11 | 2014-07-02 | 江西德泰医药生物技术有限公司 | Method for preparing health food by polypeptide factor and Chinese herb extracts |
| CN103989041A (en) * | 2014-04-11 | 2014-08-20 | 南昌大学 | Multi-biological activity function food and preparation process thereof |
| CN113584108A (en) * | 2021-07-19 | 2021-11-02 | 湖南益旺生物科技有限公司 | Preparation method of spleen aminopeptide |
| CN113647556A (en) * | 2021-08-19 | 2021-11-16 | 江西傲龙优可健康产业有限公司 | Rice flour capable of improving immunity of infants and preparation method thereof |
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