CN115974979B - Donkey-hide gelatin oligopeptide, donkey-hide gelatin oligopeptide composition and application thereof in preparation of health care products related to qi tonifying, blood nourishing or miscarriage prevention - Google Patents
Donkey-hide gelatin oligopeptide, donkey-hide gelatin oligopeptide composition and application thereof in preparation of health care products related to qi tonifying, blood nourishing or miscarriage prevention Download PDFInfo
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- CN115974979B CN115974979B CN202211696943.3A CN202211696943A CN115974979B CN 115974979 B CN115974979 B CN 115974979B CN 202211696943 A CN202211696943 A CN 202211696943A CN 115974979 B CN115974979 B CN 115974979B
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- FPAFDBFIGPHWGO-UHFFFAOYSA-N dioxosilane;oxomagnesium;hydrate Chemical compound O.[Mg]=O.[Mg]=O.[Mg]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O.O=[Si]=O FPAFDBFIGPHWGO-UHFFFAOYSA-N 0.000 description 1
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- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
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- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
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- JDNTWHVOXJZDSN-UHFFFAOYSA-N iodoacetic acid Chemical compound OC(=O)CI JDNTWHVOXJZDSN-UHFFFAOYSA-N 0.000 description 1
- 239000000797 iron chelating agent Substances 0.000 description 1
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 1
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- TVBSSDNEJWXWFP-UHFFFAOYSA-N nitric acid perchloric acid Chemical compound O[N+]([O-])=O.OCl(=O)(=O)=O TVBSSDNEJWXWFP-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/16—Inorganic salts, minerals or trace elements
- A23L33/165—Complexes or chelates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Peptides Or Proteins (AREA)
Abstract
The application discloses donkey-hide gelatin oligopeptide and a composition thereof, and application thereof in preparing health care products related to tonifying qi, nourishing blood or preventing miscarriage. The donkey-hide gelatin oligopeptide and the composition thereof comprise at least one of polypeptides with amino acid sequences shown as SEQ ID NO. 1-11. The donkey-hide gelatin peptide-iron chelate and the solid particle preparation prepared by the method not only can improve chemically induced anemia and radiation induced anemia, but also can enhance the immune function of mice at the same time, and provide a wide prospect for further improving and developing products with health care functions related to donkey-hide gelatin for application in the fields of supplementing qi, nourishing blood, preventing miscarriage and the like.
Description
Technical Field
The application relates to the technical field of donkey-hide gelatin, in particular to donkey-hide gelatin peptide and application thereof in preparing health-care products related to tonifying qi, nourishing blood or preventing miscarriage.
Background
Donkey-hide gelatin (Colla Corii Asini) is used as a traditional Chinese medicinal material, is known as a blood-replenishing "holy medicine", and is a solid gum prepared by removing hair from dried or fresh skin of donkey of equine animals, decocting, adding appropriate amount of yellow wine, soybean, crystal sugar and the like, and concentrating into thick paste. The main components of colla Corii Asini include proteins, amino acids, microelements, polysaccharides, chondroitin sulfate, hyaluronic acid, etc.
Donkey-hide gelatin has the effects of replenishing blood, nourishing yin, moistening dryness and stopping bleeding, and is clinically applied to blood deficiency and sallow complexion, dizziness and palpitation, muscle weakness, vexation and insomnia, deficiency wind internal movement, lung dryness and cough, cough with hemoptysis, hematemesis, hematuria, metrorrhagia and metrostaxis of stool and pregnancy. However, the full excavation of specific active ingredients with physiological effects and efficacy in donkey-hide gelatin still has very practical significance.
Disclosure of Invention
Therefore, the application aims to extract an active ingredient of donkey-hide gelatin different from the prior art so as to fully utilize the health-care resources of donkey-hide gelatin.
In a first aspect, the embodiment of the application discloses donkey-hide gelatin oligopeptide and a composition thereof, wherein the donkey-hide gelatin oligopeptide comprises at least one of polypeptides with amino acid sequences shown as SEQ ID NO. 1-11.
In a second aspect, the embodiment of the application discloses a donkey-hide gelatin peptide-iron chelate, which is formed by chelating at least one donkey-hide gelatin peptide shown in SEQ ID NO. 1-11 with an iron atom or an iron ion.
In a fourth aspect, the embodiment of the application discloses a donkey-hide gelatin peptide preparation, which comprises donkey-hide gelatin peptide-iron chelate compounds formed by chelating at least one donkey-hide gelatin peptide shown in SEQ ID NO. 1-11 and iron atoms or iron ions, and auxiliary materials acceptable in health care.
In embodiments of the present application, the healthcare acceptable excipients include fruit powder, diluents, binders, lubricants, sweeteners and flavoring agents.
In a fifth aspect, the embodiment of the application discloses a preparation method of the donkey-hide gelatin oligopeptide and the composition thereof in the first aspect, which comprises the following steps:
Obtaining melted donkey-hide gelatin;
obtaining donkey-hide gelatin enzymatic hydrolysate which is prepared from melted donkey-hide gelatin through first enzymatic hydrolysis, degreasing, second enzymatic hydrolysis and third enzymatic hydrolysis; wherein, lipase is used for the first enzymolysis, glycosyl peptidase is used for the second enzymolysis, and papain and trypsin are used for the third enzymolysis;
And (3) performing gel chromatography and reversed phase preparation chromatography purification on the donkey-hide gelatin enzymatic hydrolysate to obtain the donkey-hide gelatin oligopeptide and the composition thereof.
In the embodiment of the application, the specific steps of the first enzymolysis include:
Taking melted donkey-hide gelatin, adding lipase to make the concentration of the melted donkey-hide gelatin be 5-15U/mL, stirring at 40 ℃ for 90 min, inactivating enzyme in water bath at 100 ℃, centrifuging at 8000rpm for 30min, taking supernatant, leaching with 95% ethanol water solution for 48h, and concentrating to obtain extract.
In the embodiment of the application, the degreasing specific steps include:
Mixing the extract into petroleum ether, performing ultrasonic treatment for 10min under the conditions of 25 ℃ and ultrasonic power density of 35W/L, stirring thoroughly, standing for 10min, and removing petroleum ether to obtain petroleum ether treated solid; adding ethyl acetate again, stirring, mixing thoroughly, performing ultrasonic treatment for 10min under the conditions of 25deg.C and ultrasonic power density of 15W/L, and removing ethyl acetate to obtain defatted material.
In the embodiment of the application, the specific steps of the second enzymolysis include:
dissolving the degreased matter in water, adding glycosyl peptidase E-EF01, E-EF02 and E-EF03, stirring at 42 ℃ for 180 min, inactivating enzyme in water bath at 100 ℃, centrifuging at 8000rpm for 30min, collecting supernatant, leaching with 95% ethanol water solution for 48h, and concentrating to obtain extract.
In the embodiment of the application, the specific steps of the third enzymolysis include:
And adding the extract subjected to the third enzymolysis into a PBS buffer solution containing 800-1200U of papain and 100-300U of trypsin and having a pH=7.5, stirring at 40 ℃ for 180 min, performing enzyme deactivation in a water bath at 100 ℃ for 15min, centrifuging at 8000rpm for 30min, and taking the supernatant to obtain the final enzymolysis solution.
In a sixth aspect, the embodiment of the application discloses application of the donkey-hide gelatin oligopeptide and the composition thereof in the first aspect, the donkey-hide gelatin peptide-iron chelate in the second aspect or the donkey-hide gelatin preparation in the third aspect in preparation of health care products related to qi invigorating, blood nourishing or miscarriage prevention.
Compared with the prior art, the application has at least the following beneficial effects:
According to the embodiment of the application, through secondary development of donkey-hide gelatin, 11 donkey-hide gelatin peptides with molecular weight lower than 3000 are obtained by utilizing enzymolysis, gel chromatography and preparation chromatography technologies, and the donkey-hide gelatin peptide-iron chelate is prepared. The donkey-hide gelatin peptide-iron chelate not only has better stability and is suitable for high-humidity environment, but also proves that the solid granular preparation prepared from the donkey-hide gelatin peptide-iron chelate can not improve chemically induced anemia and radiation induced anemia, can also enhance the immune function of mice at the same time, and provides a wide prospect for further improving and developing products with health care functions related to donkey-hide gelatin, so as to be applied to the application fields of reinforcing vital energy, nourishing blood, preventing miscarriage and the like.
Drawings
FIG. 1 is a gel chromatography purification elution profile of examples 1-2 and comparative examples 1-3 of the present application.
FIG. 2 is a SDS-PAGE electrophoresis of F1-F11 fractions obtained in the gel chromatography purification process of the present application.
FIG. 3 is a preparative chromatogram of an F1 fraction obtained by the gel chromatography purification process of the present application.
FIG. 4 is a preparative chromatogram of an F2 fraction obtained by the gel chromatography purification process of the present application.
FIG. 5 is a preparative chromatogram of an F3 fraction produced by the gel chromatography purification process of the present application.
FIG. 6 is a preparative chromatogram of an F5 fraction obtained by the gel chromatography purification process of the present application.
FIG. 7 is a preparative chromatogram of an F6 fraction obtained by the gel chromatography purification process of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
Donkey-hide gelatin oligopeptide
1. Materials and methods
1. Related materials
Donkey-hide gelatin, product number: b13000154505, dongan donkey-hide gelatin Co., ltd.
Lipase, cat# L3001, sigma-Aldrich.
Glycosylpeptidase, brand Ludger, E-EF01: selectively releasing high mannose and partial mixed N-polysaccharide from polypeptide and protein, 1 Unit/60 μl; E-EF02: selectively releasing bilinear and high mannose N-polysaccharide (reduction rate 40X), 0.3 Unit/60. Mu.L from polypeptides and proteins; E-EF03: tri-linear and fucosylated double-linear N-polysaccharide (0.33 Unit/60. Mu.L) was selectively released from the polypeptides and proteins.
Papain, cat No. P4762, sigma-Aldrich; trypsin, cat No. Y0002311, sigma-Aldrich.
2. Enzymolysis
One specific example 1 was performed as follows:
(1) Sieving colla Corii Asini with 60 mesh sieve, adding 20 times of distilled water, and melting in water bath at 80deg.C for 30min to obtain melted colla Corii Asini 3L;
(2) First enzymolysis
Taking melted donkey-hide gelatin, adding lipase to lead the final concentration to be 10U/mL, stirring at 40 ℃ for 90min, inactivating enzyme in water bath at 100 ℃ for 15min, centrifuging at 8000rpm for 30min, taking supernatant, leaching with 95% ethanol water solution for 48h, concentrating for 3 times to obtain 253g extract;
(3) Degreasing
Mixing 253g of extract into 1.5L of petroleum ether, carrying out ultrasonic treatment for 10min under the conditions of 25 ℃ and ultrasonic power density of 35W/L, stirring and fully mixing, standing for 10min, and removing petroleum ether to obtain a solid substance treated by petroleum ether; after 1.5L of ethyl acetate was added again and stirred and thoroughly mixed, the mixture was subjected to ultrasonic treatment for 10 minutes under conditions of 25℃and an ultrasonic power density of 15W/L, and the ethyl acetate was removed to obtain 227g of a defatted material.
(4) Second enzymolysis
10G of degreased matter is taken, 30mL of water is added, after fully and uniformly mixing, glycosyl peptidase E-EF01 2U, E-EF02 1U and E-EF03 1U are added so that the concentrations are respectively, after stirring treatment is carried out at 42 ℃ for 180 min, enzyme deactivation is carried out after treatment in a water bath at 100 ℃ for 15min, centrifugation is carried out at 8000rpm for 30min, the supernatant is taken, leaching is carried out for 48h by 95% ethanol water solution, concentration is carried out, and total concentration is carried out for 3 times, thus obtaining 6.23g of extract;
(5) Third enzymolysis
The extract of the second enzymolysis is added into PBS buffer solution (20 mL) with pH=7.5 containing 1000U papain and 200U trypsin, and after stirring treatment at 40 ℃ for 180 min, the extract is treated at water bath 100 ℃ for 15min for enzyme deactivation, and the supernatant is taken after centrifugation at 8000rpm for 30min, so as to obtain the final enzymolysis solution.
One specific example 2 is implemented as follows:
Taking 10g of the degreased product obtained in the example 1, adding 30mL of water, fully and uniformly mixing, adding glycosyl peptidase E-EF01 4U, stirring at 42 ℃ for 180 min, inactivating enzyme by treating in a water bath at 100 ℃ for 15min, centrifuging at 8000rpm for 30min, taking the supernatant, leaching with 95% ethanol aqueous solution for 48h, concentrating for 3 times to obtain 6.23g of extract; the subsequent steps are the same as in example 1.
One specific comparative example 1 was carried out as follows:
10g of the defatted material obtained in example 1 was added to a PBS buffer (20 mL) containing 1000U of papain and 200U of trypsin and having pH=7.5, and after 180 min of stirring treatment at 40℃the defatted material was subjected to enzyme deactivation by centrifugation at 8000rpm for 30min in a water bath at 100℃to obtain a final enzyme hydrolysate.
One specific comparative example 2 was carried out as follows:
Taking 10g of the degreased product obtained in the example 1, adding 30mL of water, fully and uniformly mixing, adding glycosyl peptidase E-EF02 4U, stirring at 42 ℃ for 180 min, inactivating enzyme by treating in a water bath at 100 ℃ for 15min, centrifuging at 8000rpm for 30min, taking the supernatant, leaching with 95% ethanol aqueous solution for 48h, concentrating for 3 times to obtain 6.23g of extract; the subsequent steps are the same as in example 1.
One specific comparative example 3 was carried out as follows:
Taking 10g of the degreased product obtained in the example 1, adding 30mL of water, fully and uniformly mixing, adding glycosyl peptidase E-EF03 4U, stirring at 42 ℃ for 180 min, inactivating enzyme by treating in a water bath at 100 ℃ for 15min, centrifuging at 8000rpm for 30min, taking the supernatant, leaching with 95% ethanol aqueous solution for 48h, concentrating for 3 times to obtain 6.23g of extract; the subsequent steps are the same as in example 1.
3. Gel chromatography purification
Filtering the obtained enzymolysis solution with filter paper, collecting filtrate, ultrafiltering and concentrating with hollow fiber filter membrane with molecular weight cut-off of 3kD, specifically, for example, ultrafiltering and concentrating with hollow fiber ultrafilter cup (model number C0005552, pellicon small size tangential flow ultrafilter with nominal molecular weight 3000), collecting concentrate, and separating by gel chromatography.
Gel chromatographic separation conditions: taking 5mL of the ultrafiltration concentrated solution, loading the solution onto a gel chromatographic column (1.5 cm multiplied by 80 cm) of Sephadex G-50 (G50150, sigma-Aldrich), standing for 15min, eluting with PBS with pH=6 as a mobile phase, collecting chromatographic peaks at the light absorption position of A210 step by step, combining the collecting pipes, concentrating under reduced pressure, and freeze-drying to obtain lyophilized coarse powder of the donkey-hide gelatin peptide.
4. RP-HPLC separation and purification
Concentrating the above obtained lyophilized coarse powder by dialysis with dialysis bag with molecular weight cut off of 5000, filtering with 0.22 μm, purifying by HPLC, collecting sufficient eluent according to peaks, lyophilizing, dissolving in 0.15% formic acid water solution to obtain RP-HPLC test sample, loading on C18 chromatographic column, collecting chromatographic peak eluent, concentrating, and lyophilizing to obtain colla Corii Asini peptide lyophilized powder.
The conditions for the preparation chromatography were: the column was PuriFlash% Bio 100C 18N (5 μm, 30mm ID), agilent HPLC1200 series system (Agilent, waldebrand, germany), diode Array Detector (DAD).
The mobile phase is: phase a 0.1% trifluoroacetic acid, phase B acetonitrile; the gradient procedure is: 0.fwdarw.5 min, linear gradient 5.fwdarw.15% phase B; 5 to 15min, linear gradient 15 to 40% B; 15-25 min,40% B; 25-30 min, linear gradient 30-50% B; a pre-equilibration period of 20min was employed between each run. The flow rate was 0.6ml/min, the column temperature was 25 ℃, the injection volume was 10 μl, and the DAD wavelength was set to 214nm.
5. Donkey-hide gelatin peptide sequence identification
A suitable amount of sample was dissolved in 0.1% formic acid aqueous solution and analyzed by HPLC-MS.
Sample pretreatment:
(1) Taking lmg of donkey-hide gelatin peptide sample, placing into a centrifuge tube, adding 1mL of a 6M Guanidine (prepared in 100mMNH HC 03) solution with pH of 8.0 to obtain a 1mg/mL sample solution; adding 20 mu L of 1M DTT into the sample solution, and reacting for 1h at 37 ℃ to reduce disulfide bonds in the donkey-hide gelatin peptide;
(2) After the reaction is completed, the reaction solution is divided into two parts, 25 mu L of 1M iodoacetic acid and 1M sodium hydroxide aqueous solution are added respectively, and the mixture is placed for 30 minutes at room temperature in the absence of light;
(3) Again, centrifugation was performed at 12000rpm for 50min in a Centricon ultrafiltration tube with isolated proteins below 3 kDa. Then adding 200 mu L of 0.1M ammonium bicarbonate into the centrifuge tube respectively, and centrifuging again for 30min; repeating the operation a plurality of times to reduce Guanidine content in the sample;
(4) The donkey-hide gelatin peptide samples on the filter layers of the two centrifuge tubes are respectively added with 0.1M ammonium bicarbonate solution with the total amount of about 500 mu L for complete dissolution, the solution containing the donkey-hide gelatin peptide samples is transferred into a Trypsin test tube with 20 mu L, then 500 mu L of 0.1M ammonium bicarbonate solution is added, the total amount of liquid in the test tube is 1mL, the reaction is carried out for 16h in a 37 ℃ water bath, and 500 mu L of each reaction solution is respectively centrifuged for 50min by using a Centricon ultrafiltration tube at 15000 rpm.
(5) The filtrate was collected and concentrated by vacuum drying at 35 c, which aids in the decomposition and volatilization of the ammonium bicarbonate and reduces the salt concentration in the sample. The mixture was further dried and concentrated to a sample size of about 100. Mu.L.
(6) The desired sample concentration was quantified with 0.15% formic acid solution and care was taken to see if the sample solution was clear. If the sample solution is turbid, the sample solution is centrifuged for 10min at the rotation speed of 12000-14000rmp, and the supernatant is taken for sample injection analysis.
Chromatographic conditions:
HPLC detection mode: ultraviolet; scanning range is 50-2000 m/z; capillary outlet voltage: 166.0 V, skimmer in combination with the system voltage: 40.0V,Oct 1 DC 12.00 V,Oct 2 DC 2.70 V,Ampl separation width 4.0 m/z, fragmenter voltage: 1.00Vt; ion polarity: a positive ion; ion source type: ESI (electrospray ionization); drying temperature: 325 ℃, atomizer pressure: 15.00 psi, dryer flow rate: 5.00 L/min. The mass-to-charge ratio of the polypeptides and fragments of the polypeptides was obtained by collecting 10 fragment patterns after each full scan. The original file was analyzed using de navy algorithm in Mascot 2.3 software. The result filter parameter with the correlation parameter being :Enzyme = none, Variable modifi-canon：Oxidation(M),Peptides tolerance:20 ppm,MS/MS tolerance：0.1u,Mascot is FDR less than or equal to 0.01.
6. Agarose gel chromatography
The main chromatographic peak fractions of RP-HPLC described above were collected and subjected to SDS-PAGE electrophoresis.
2. Results
Gel chromatography purification elution curves of examples 1 to 2 and comparative examples 1 to 3 are shown in fig. 1. The fractions obtained in example 1 were F1 to F3, the fractions obtained in example 2 were F4 to F6, the fractions obtained in comparative example 1 were F7, the fractions obtained in comparative example 2 were F8 to F9, and the fractions obtained in comparative example 3 were F10 to F11.
F1-F11 is detected by SDS-PAGE electrophoresis, and as shown in FIG. 2, F1, F2, F3, F5 and F6 have peptide fragments with the size smaller than 5 Ku. Further, F1, F2, F3, F5 and F6 are subjected to RP-HPLC chromatography to prepare the products, and the preparation results are shown in figures 3-7. Purification of F1 by RP-HPLC gave four main fractions of 7.04min, 8.23min, 11.46min and 14.94 min. F2 was purified by RP-HPLC to give two main fractions of 15.14min and 15.87 min. F3 was purified by RP-HPLC to give three main fractions of 18.26min, 19.36min and 20.14 min. F5 was purified by RP-HPLC to give two main fractions of 15.11min and 15.69 min. Purification of F6 by RP-HPLC gave three main fractions of 18.31min, 19.27min and 20.07 min.
LS-MS detection was performed on each of these fractions, search analysis was performed by both the SEQUEST and Mascot search engines, and the primary structure of this fraction was determined by comparison with reference to the NCBI database, and the results are shown in Table 1.
TABLE 1
As can be seen from Table 1, F2 (15.14 min) and F2 (15.87 min) correspond to the primary structures of F5 (15.11 min) and F5 (15.69 min) being relatively close, while the three fractions of F3 are identical to the three fractions of F6. And through calculation, the molecular weight of the peptide shown in SEQ ID NO. 1-11 is 1000-3000.
Preparation of donkey-hide gelatin peptide-iron chelate
The 11 donkey-hide gelatin peptides obtained by extraction and enzymolysis of donkey-hide gelatin are further utilized to prepare a donkey-hide gelatin peptide-iron chelate, and therefore, the embodiment of the application discloses the donkey-hide gelatin peptide-iron chelate which is formed by chelating at least one donkey-hide gelatin peptide shown in SEQ ID NO. 1-11 with an iron atom or an iron ion.
The preparation implementation process of the specific donkey-hide gelatin peptide-iron chelate comprises the following steps:
200mg of donkey-hide gelatin peptide (F1 (7.04 min)) is taken, dissolved in 1L of an aqueous solution containing 0.1wt% of ascorbic acid, the proper pH=5 is regulated by 10wt% of NaOH or HCl, feCL 2·4H2 O is added to lead the final concentration to be 10 mg/L, the mixture is placed on a magnetic stirrer to be chelated for 20min at 25 ℃, the mixture is centrifuged for 5min at 4500 r/min, the precipitate is removed, the mixture is concentrated under reduced pressure (60-65 ℃ and minus 0.07-0.08 MPa), and the mixture is freeze-dried (-50 ℃ and minus 0.01 MPa) for 24h, thus obtaining the donkey-hide gelatin peptide-iron chelate.
The above-obtained 11 kinds of donkey-hide gelatin peptides were also used to prepare donkey-hide gelatin peptide-iron chelate complexes, respectively, by referring to the above-mentioned methods, and the chelation rate during the chelation process was evaluated, and the preliminary stability of the donkey-hide gelatin peptide-iron chelate complexes was evaluated.
The chelation rate evaluation method comprises the following steps:
and measuring the content of iron element in the donkey-hide gelatin peptide-iron chelate by adopting an atomic absorption photometry.
(1) Preparation of test article: 200mg of donkey-hide gelatin peptide-iron chelate is precisely weighed, placed in a graphite digestion instrument, 8mL of nitric acid-perchloric acid (4:1) liquid solution is added, and the mixture is gently shaken and mixed uniformly, and placed in the graphite digestion instrument for heating. The temperature programming method is adopted, the micro-boiling state is kept at the temperature of 220 ℃ for 20 min ℃, the temperature is increased after the solution is clear, the micro-boiling state is kept at the temperature of 280 ℃ for 30 min continuously, the sample solution emits thick smoke, after the white smoke is dispersed, the digestion solution is in a colorless transparent or slightly yellowish state, the digestion solution is placed at room temperature and transferred into a 50 mL measuring flask, the container is washed by 2% nitric acid solution, the washing solution is combined in the measuring flask, diluted to a scale, and the sample digestion solution is shaken uniformly.
(3) Preparing ferrous chloride solutions of 0.2, 0.4, 0.6, 0.8 and 1mg/L, and respectively preparing standard digestion solutions with different concentrations by the method; and detecting the light absorption values of the samples respectively by an atomic absorption spectrometer (ICE 3500 of thermoelectric instruments, america), drawing a standard curve according to the light absorption values, and calculating the content of the iron element in the samples according to the standard curve. The detection conditions are as follows: 238nm, air flow rate of 6.5L/min, spectral power of 0.2nm, lamp current of 8nm, acetylene flow rate of 2.0L/min,
(4) Calculation of chelation rate: chelation ratio = weight of iron in donkey-hide gelatin peptide-iron chelate/weight of donkey-hide gelatin peptide-iron chelate.
Stability evaluation method:
(1) Test article: the 11 donkey-hide gelatin peptide-iron chelates prepared by the method are respectively named as T1-T11, and are packaged by adopting an aluminum foil composite film to serve as a test sample for stability investigation.
(2) High temperature test: precisely weighing 20mg of the sample, spreading in a clean weighing bottle, standing at 60 ℃ for 10 days for sampling, measuring the total iron content, calculating the chelation rate, and observing the appearance.
(3) High humidity test: precisely weighing 20mg of the sample, spreading in clean weighing bottles, placing in a drier with relative humidity of 90% + -5%, placing in an incubator with set temperature of 25deg.C, sampling on day 10, measuring total iron content, calculating chelation rate, and observing appearance.
(4) Strong light irradiation test: precisely weighing 20mg of the sample, spreading in a clean weighing bottle, placing in an illumination box or other suitable illumination device with fluorescent lamp, standing for 10 days under the condition of 45001 x+ -500 lx illumination, measuring total iron content, calculating chelation rate, and observing appearance.
TABLE 2
As shown in Table 2, the initial chelation rate of the donkey-hide gelatin peptide-iron chelate prepared by the steps to iron is more than 7 per mill, and the chelation rate of T7-T11 is higher. However, in the high-temperature test, the iron chelating rate of the donkey-hide gelatin iron-iron chelate is reduced to different degrees, which represents that the stability of the donkey-hide gelatin iron-iron chelate at 60 ℃ is poor, and the damage to iron element chelating action caused by high temperature may exist. In the high-humidity test and the strong light irradiation test, the chelation rate of T1-T11 to iron is reduced, but the chelation rate is not obviously reduced in the relatively high-temperature test. In a high-humidity environment, the chelation rates of T1-T11 on iron are almost the same as the respective initial iron chelation rates, which indicates that the donkey-hide gelatin peptide-iron chelate provided by the embodiment of the application can resist the high-humidity environment and has better stability.
Animal experiment
1. Materials and methods
1. Experimental animal
Kunming mice, cat number hnslkjd002, stokes Jingda, normal diet.
2. Test article
For the purpose of carrying out related experiments, the embodiment of the application also provides a donkey-hide gelatin peptide preparation which comprises donkey-hide gelatin peptide-iron chelate compounds formed by chelating at least one donkey-hide gelatin peptide shown in SEQ ID NO. 1-11 and iron atoms or iron ions, and auxiliary materials acceptable in health care.
Wherein the auxiliary materials acceptable in health care comprise fruit powder, diluent, adhesive, lubricant, sweetener and edible essence. Wherein the fruit powder is at least one selected from orange powder, apple powder, grape powder, pear powder, grass toxin powder, blue toxin powder and cranberry enzyme powder; the diluent comprises at least one of konjaku flour, corn flour, soybean flour, starch, dextrin, microcrystalline cellulose and edible inorganic salt; the binder comprises at least one of starch slurry, sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, methylcellulose and ethylcellulose; the lubricant comprises at least one of magnesium stearate, talcum powder and polyethylene glycol; the sweetener comprises at least one of white sugar, glucose, fructose, xylitol, mannitol, erythritol, acesulfame potassium, sucralose and aspartame.
The donkey-hide gelatin peptide preparation provided by the specific embodiment is a solid granule preparation, the formula of the donkey-hide gelatin peptide preparation is shown in table 3, and the preparation of the solid granule preparation is obtained by adopting a conventional medicament preparation method. In Table 3, the commercial donkey-hide gelatin powder used in comparative example 1 is the raw material of donkey-hide gelatin used initially in the examples of the present application. In Table 3, the term "parts" is used only to distinguish the weight ratio relationship between the components, and is not intended to specifically limit the actual weight of the components, but may be any weight, such as 0.001mg, 0.01mg, 1mg, 10mg, 1g, 10g, 1kg, 1000t, or the like.
TABLE 3 Table 3
3. Safety experiment
Taking Kunming mice to randomly divide into 2 groups, and orally taking 50mg/mL of aqueous solution prepared by oral administration of the solid particle preparations prepared in the examples 1-14 and the comparative examples 1-6, wherein the oral dosage is 5g/kg of body weight; one group was given 0.5ml of the test substance by intraperitoneal injection 2 times daily, and the feeding was observed for 7d, and again by jugular vein injection on days 14 and 21. The body weight, diet, activity, presence or absence of symptoms of allergic reaction such as hair erection, dyspnea, and convulsion of mice were observed during the experiment.
As a result, after one week of gastric lavage, mice in the group had no significant difference in food consumption, were normal in activity, and did not show the symptom of poisoning; the mice injected into the abdominal cavity have normal diet and activity, and have no normal symptoms such as excitation anxiety, dyspnea and the like, which indicates that the test sample has no anaphylactic reaction to the mice.
4. Model and experiment of various anemia
(1) First anemia model mouse establishment and grouping experiment
The Kunming mice were injected subcutaneously into the back of the spine with a dose of 30mg/kg of 2% phenylhydrazine hydrochloride solution (CAS: 59-88-1, merck Sigma-Aldrich) once every 5d, continuously injected 3 times, blood was collected from the tip of the tail, peripheral blood images were measured, and the molding was judged to be successful by taking a hemoglobin value of less than 10.0g/L as a standard. After successful modeling, the model is randomly divided into a first model group and a first administration group. The first administration group was filled with 50mg/mL of the aqueous solution prepared from the solid particle formulations prepared in examples 1 to 14 and comparative examples 1 to 6at a dose of 20g/kg body weight, and the continuous filling was carried out for 7 days; the first model group is not processed; the tail tip of the mouse is sampled and anticoagulated, and RBC (. Times.10 12/L),HGB(g/100mL),WBC(×109/L) is detected (experimental study of the effect of the Gui Rong blood-replenishing tablet on blood deficiency syndrome animal model [ J ]. University of medical science, lesion, 2001,27 (3): 334-335.).
(2) Establishing and grouping experiments of mice with second anemia model
Taking Kunming mice, taking blood as model mice after 3.5Gy 137 Cs disposable radiation (dosage rate 1.27 Gy/min), measuring peripheral blood image, and judging modeling success by taking hemoglobin value lower than 10.0g/L as standard. Divided into a second model group and a second dosing group. The first administration group was filled with 50mg/mL of the aqueous solution prepared from the solid particle formulations prepared in examples 1 to 14 and comparative examples 1 to 6 at a dose of 20g/kg body weight, and the continuous filling was carried out for 7 days; the first model group is not processed; the mouse tail tip was collected and tested for anticoagulation RBC (. Times.10 12/L),HGB(g/100mL),WBC(×109/L).
5. Immunopotentiation experiments
(1) Moulding
The Kunming mice were harvested and injected intraperitoneally with hydrocortisone (HC, 614157, sigma-Aldrich) 25mg/kg 1 time daily for 7 consecutive days.
(2) Grouping experiments
Healthy Kunming mice were set as a blank group. Referring to the above-mentioned first anaemia model mice, in the course of the establishment and grouping experiment, 50mg/mL of the aqueous solution prepared from the solid particle preparations prepared in examples 1 to 14 and comparative examples 1 to 6 was infused in a dose of 20g/kg of body weight, and continuous administration was carried out for 10 days, to thereby obtain a third administration group.
After the completion of the experiment, each group of mice was assayed for spleen weight, and spleen weight index was calculated, spleen weight index=spleen weight/body weight×100%.
Each group of animals was continuously given by gastric lavage for 10d, each mouse was intraperitoneally injected with 0.2mL of 2% chicken erythrocyte suspension, 20 μl of each was collected from the eye 1h after the last administration, and shaking in 1mL of physiological saline, then 0.5 mL% chicken erythrocyte suspension was added, 0.5mL of the lower mouse complement was added to the ice bath (CH 50, preparation method was described in "guangzhou medicine 1999, 01.22, preparation of mouse without extracting C3 antiserum"), the reaction was stopped by incubating in a water bath at 37 ℃ for 30min, 1mL of supernatant was added to 3mL of all-purpose reagent, standing for 10min, and absorbance (OD) was read by colorimetric at 540nm wavelength.
On day 1 of administration, 0.5mL Dinitrochlorobenzene (DNCB) -acetone solution is dripped on the dehairing skin of the neck of a mouse, 2 mu L each sensitization is carried out, the continuous administration is carried out for 10 days, 0.025g/mL dinitrochlorobenzene-acetone solution is dripped on the skin of the abdomen dehairing area of the mouse 1 day before the last administration, 20 mu L each dinitrochlorobenzene-acetone solution is used for attack, 10mL/kg of Evan blue with the mass fraction of 1% is intravenously injected into the tail of the mouse after 24 hours, the mouse is killed after 30 minutes, the belly blue dyed skin is cut in a test tube, the belly blue dyed skin is soaked in 1:l acetone physiological water mixture for 24 hours, centrifugation is carried out for 10 minutes at 2000rpm, and the absorbance of the supernatant is measured at 610 nm.
Each group of animals was continuously perfused for 10d, 0.2mL of indian ink was intravenously injected per rat tail 1h after the last administration, 20 μl of each blood was taken from the posterior venous plexus of the mouse with a micropipette at 30s and 5min after the injection, and immediately blown into 2mL of 0.1% volume fraction sodium carbonate solution, and an equivalent amount of normal mouse blood was taken for zeroing, absorbance was read at 675nm of the spectrophotometer, and phagocytosis index (K) was determined according to the formula k= (lgC 1-lgC 2).
6. Statistical analysis
All test data are expressed as mean and standard deviation, data were processed using SPSS13.0 software and multiple comparisons and significance signatures were performed on each column of data.
2. Results
TABLE 4 Table 4
TABLE 5
Table 4 shows the results of the anemia modeling experiments using phenylhydrazine hydrochloride. Table 5 shows the results of the anemia modeling experiments using radiation. In tables 4 and 5, multiple comparisons were made for each column data to count significant differences therebetween.
Table 4 shows that the RBC, HGB and WBC indices of the first model group were significantly lower than those of the normal group, indicating successful modeling. Compared with the model group, after the test products provided in examples 1-14 are dosed, the indexes of RBC, HGB and WBC of the mice are all obviously improved, and particularly the test products provided in examples 12-14 show that the solid particle preparation prepared based on the donkey-hide gelatin peptide-iron chelate provided in the embodiment of the application is dosed to the mice, so that the chemical induced anemia symptoms of the mice can be improved.
In addition, in table 4, the test sample of mice administered in comparative example 1 is a solid granule preparation prepared from commercial donkey-hide gelatin powder, which has limited function of improving anemia in model mice, while the test samples of mice administered in comparative examples 2 to 4 are donkey-hide gelatin peptide mixtures obtained in the preparation process of the present application, and the effect is inferior to examples 1 to 14, although the effect is improved in anemia in model mice.
Table 5 shows that the RBC, HGB and WBC indices of the second model group were significantly lower than those of the normal group, indicating successful modeling of mice with radiation anemia model. Compared with a model group, after the test samples provided in examples 1-14 are dosed, the indexes of RBC, HGB and WBC of the mice are all obviously improved, and particularly the test samples provided in examples 12-14 show that the solid particle preparation prepared based on the donkey-hide gelatin peptide-iron chelate provided in the embodiment of the application is dosed to the mice, so that the symptoms of anemia induced by radiation of the mice can be improved. In addition, comparative examples 1 to 5 showed the same tendency as in table 4, and had limited effect of improving anemia in model mice.
TABLE 6
Table 6 lists spleen weight index, hemolysin level, DNCB induced OD value and phagocytic index for each group of mice, and each column of data was subjected to multiple comparisons to account for significant differences therebetween.
As can be seen from table 6, the spleen weight index, the hemolysin level, the DNCB induced OD value and the phagocytic index were significantly lower in the mice of the first model group than in the normal group. Compared with the model group, in the third administration group, after administration of the test products provided in examples 1-14, the indexes of RBC, HGB and WBC of mice are obviously improved, and particularly the test products provided in examples 12-14, which indicate that the solid particle preparation prepared based on the donkey-hide gelatin peptide-iron chelate provided in the embodiment of the application is administered to the mice, and can limit and improve spleen weight index, hemolysin level, DNCB-induced OD value and phagocytosis index of the mice, so that the solid particle preparation has an immune enhancement function.
The sample of the mice administered in comparative example 1 is a solid granule preparation prepared from commercial donkey-hide gelatin powder, the immunity enhancement and improvement functions of model mice are limited, and the samples of the mice administered in comparative examples 2 to 4 are donkey-hide gelatin peptide mixtures obtained in the preparation process of the application, and the effect is inferior to that of examples 1 to 14 although the sample has immunity enhancement function on model mice.
In summary, the embodiment of the application obtains 11 donkey-hide gelatin peptides with molecular weight lower than 3000 by secondary development of donkey-hide gelatin and utilizing enzymolysis, gel chromatography and preparation chromatography technologies, and prepares the donkey-hide gelatin peptide-iron chelate. The donkey-hide gelatin peptide-iron chelate not only has better stability and is suitable for high-humidity environment, but also proves that the solid granular preparation prepared from the donkey-hide gelatin peptide-iron chelate can not improve chemically induced anemia and radiation induced anemia, can also enhance the immune function of mice at the same time, and provides a wide prospect for further improving and developing products with health care functions related to donkey-hide gelatin, so as to be applied to the application fields of reinforcing vital energy, nourishing blood, preventing miscarriage and the like.
The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.
Claims (6)
1. An donkey-hide gelatin oligopeptide with an amino acid sequence shown in SEQ ID NO. 3.
2. A donkey-hide gelatin oligopeptide composition comprises polypeptide with an amino acid sequence shown as SEQ ID NO.3 and at least two donkey-hide gelatin peptides shown as SEQ ID NO. 1-2 and 4-11.
3. A donkey-hide gelatin peptide-iron chelate composition, characterized in that it is formed by chelating an iron atom or an iron ion with the donkey-hide gelatin oligopeptide according to claim 1 or the donkey-hide gelatin oligopeptide composition according to claim 2.
4. A donkey-hide gelatin peptide composition comprising the donkey-hide gelatin oligopeptide according to claim 1 or the donkey-hide gelatin oligopeptide composition according to claim 2 and a pharmaceutically acceptable auxiliary material for health care.
5. The donkey-hide gelatin peptide composition according to claim 4, wherein the pharmaceutically acceptable excipients comprise fruit powder, diluent, binder, lubricant, sweetener and flavoring essence.
6. Use of the donkey-hide gelatin oligopeptide according to claim 1, the donkey-hide gelatin oligopeptide composition according to claim 2, the donkey-hide gelatin peptide-iron chelate composition according to claim 3 or the donkey-hide gelatin peptide composition according to claim 4 for preparing a product related to tonifying qi, nourishing blood or preventing miscarriage.
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| CN102499376A (en) * | 2011-12-29 | 2012-06-20 | 山东东阿阿胶股份有限公司 | Active small-molecule donkey-hide gelatin mixture and preparation method and application thereof |
| CN107998333A (en) * | 2017-12-13 | 2018-05-08 | 国药肽谷有限公司 | A kind of preparation method of donkey-hide gelatin peptides products |
| CN114010762A (en) * | 2021-11-01 | 2022-02-08 | 杭州佰倍优生物科技有限公司 | Preparation for improving anemia symptoms and preparation method thereof |
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| CN115991741A (en) | 2023-04-21 |
| CN115991741B (en) | 2024-04-12 |
| CN114891849B (en) | 2023-04-11 |
| CN115724911A (en) | 2023-03-03 |
| CN115724911B (en) | 2023-06-06 |
| CN114891849A (en) | 2022-08-12 |
| CN115974979A (en) | 2023-04-18 |
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