CN115974979A

CN115974979A - Donkey-hide gelatin peptide and application thereof in preparing health-care products related to qi tonifying, blood nourishing or miscarriage prevention

Info

Publication number: CN115974979A
Application number: CN202211696943.3A
Authority: CN
Inventors: 杨瑞金
Original assignee: Qingfeng Chain Soda Beverage Jilin Co ltd
Current assignee: Guangzhou Jiuxi Investment Co ltd
Priority date: 2022-06-07
Filing date: 2022-06-07
Publication date: 2023-04-18
Anticipated expiration: 2042-06-07
Also published as: CN115991741A; CN115724911A; CN115991741B; CN114891849A; CN114891849B; CN115974979B; CN115724911B

Abstract

The application discloses donkey-hide gelatin oligopeptide, a composition thereof and application of the donkey-hide gelatin oligopeptide in preparing health-care products related to qi tonifying, blood nourishing or miscarriage prevention. The donkey-hide gelatin oligopeptide and the composition thereof comprise at least one of polypeptides with amino acid sequences shown in SEQ ID NO. 1-11. The donkey-hide gelatin peptide-iron chelate and the solid particle preparation prepared by the method can not improve chemical induced anemia and radiation induced anemia, can also enhance the immune function of mice simultaneously, and provide wide prospects for further promoting and developing donkey-hide gelatin-related health-care functional products to be applied to the application fields of tonifying, nourishing blood, miscarriage prevention and the like.

Description

Donkey-hide gelatin peptide and application thereof in preparing health-care products related to qi tonifying, blood nourishing or miscarriage prevention

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 qi invigorating, blood nourishing or miscarriage prevention.

Background

Colla Corii Asini (Colla Corii Asini) is a traditional Chinese medicinal material, called as blood replenishing Saint medicine, and is a solid gum prepared by removing hair from dried or fresh skin of Equus asinus L, decocting, adding appropriate amount of yellow wine, semen glycines, crystal sugar, etc., and concentrating into soft extract. The main components of colla Corii Asini include protein, amino acids, trace elements, polysaccharide, chondroitin sulfate, hyaluronic acid, etc.

Donkey-hide gelatin has the effects of enriching blood, nourishing yin, moistening dryness and stopping bleeding, and is clinically applied to blood deficiency, chlorosis, dizziness, palpitation, muscle weakness, vexation, insomnia, deficient wind stirring, lung dryness, cough, fatigue, hemoptysis, hematemesis, hematuria, hematochezia, metrorrhagia and metrostaxis and pregnancy. However, it is still of practical significance to fully exploit the specific active ingredients with physiological effects and efficacies in donkey-hide gelatin.

Disclosure of Invention

In view of the above, the present 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, which comprise at least one of polypeptides with amino acid sequences shown as SEQ ID NO. 1-11.

In a second aspect, the present application discloses a donkey-hide gelatin peptide-iron chelate complex, which is formed by chelating at least one donkey-hide gelatin peptide shown in SEQ ID No. 1-11 with iron atoms or iron ions.

In a fourth aspect, the present application discloses a colla corii asini peptide preparation, which comprises at least one colla corii asini peptide shown in SEQ ID nos. 1 to 11 and an iron atom or iron ion to form a colla corii asini peptide-iron chelate through chelation, and a health-care acceptable adjuvant.

In the examples of the present application, the hygienically acceptable excipients include fruit powder, diluents, binders, lubricants, sweeteners and flavorants.

In a fifth aspect, the present application discloses a method for preparing the donkey-hide gelatin oligopeptide and the composition thereof of the first aspect, which comprises the following steps:

melting colla Corii Asini;

obtaining donkey-hide gelatin enzymolysis liquid, wherein the donkey-hide gelatin enzymolysis liquid is prepared by first enzymolysis, degreasing, second enzymolysis and third enzymolysis; wherein the first enzymolysis uses lipase, the second enzymolysis uses glycosyl peptidase, and the third enzymolysis uses papain and trypsin;

and carrying out gel chromatography and reversed-phase preparative chromatography purification on the donkey-hide gelatin enzymolysis liquid to obtain the donkey-hide gelatin oligopeptide and the composition thereof.

In the embodiment of the present application, the specific steps of the first enzymolysis include:

taking the donkey-hide gelatin molten liquid, adding lipase to enable the concentration of the donkey-hide gelatin molten liquid to be 5-15U/mL, stirring the mixture at the temperature of 40 ℃ for 90min, then inactivating enzyme in a water bath at the temperature of 100 ℃, centrifuging the mixture at 8000rpm for 30min, taking supernatant, leaching the supernatant with 95% ethanol water solution for 48h, and concentrating the supernatant to obtain extract.

In an embodiment of the present application, the degreasing specifically includes:

mixing the extract into petroleum ether, performing ultrasonic treatment for 10min under the ultrasonic treatment condition of 25 ℃ and the ultrasonic power density of 35W/L, stirring, fully and uniformly mixing, standing for 10min, and removing the petroleum ether to obtain a solid treated by the petroleum ether; adding ethyl acetate again, stirring, mixing thoroughly, ultrasonic treating for 10min at 25 deg.C with ultrasonic power density of 15W/L, and removing ethyl acetate to obtain degreased substance.

In the embodiment of the present 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 180min, 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 present application, the third enzymatic hydrolysis specifically includes:

adding the third-time enzymolysis extract into PBS buffer solution with the pH =7.5 and containing 800-1200U of papain and 100-300U of trypsin, stirring at 40 ℃ for 180min, treating in water bath at 100 ℃ for 15min for enzyme deactivation, centrifuging at 8000rpm for 30min, and taking supernatant to obtain the final enzymolysis solution.

In a sixth aspect, the present application discloses the use 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 the preparation of health 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, 11 donkey-hide gelatin peptides with the molecular weight lower than 3000 are obtained by carrying out secondary development on donkey-hide gelatin and utilizing enzymolysis, gel chromatography and preparative chromatography technologies, and thus, 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 a solid particle preparation prepared from the donkey-hide gelatin peptide-iron chelate can not improve chemical induced anemia and radiation induced anemia through animal experiments, can also enhance the immune function of mice, and provides wide prospects for further promoting and developing health-care functional products related to donkey-hide gelatin so as to be applied to the application fields of tonifying, nourishing blood, miscarriage prevention and the like.

Drawings

FIG. 1 is a gel chromatography purification elution chart of examples 1 to 2 and comparative examples 1 to 3 of the present application.

FIG. 2 is an SDS-PAGE electrophoresis of F1-F11 fractions obtained from the gel chromatography purification process of the present application.

FIG. 3 is a chromatogram of the preparation of the F1 fraction obtained during the gel chromatography purification process of the present application.

FIG. 4 is a chromatogram of the preparation of the F2 fraction obtained during the gel chromatography purification process of the present application.

FIG. 5 is a chromatogram for the preparation of the F3 fraction obtained during the gel chromatography purification process of the present application.

FIG. 6 is a chromatogram for the preparation of F5 fraction from the gel chromatography purification process of the present application.

FIG. 7 is a chromatogram of the preparation of the F6 fraction obtained during 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 is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.

Donkey-hide gelatin oligopeptide

1. Materials and methods

1. Materials of interest

Donkey-hide gelatin, product number: b13000154505, dong' a Ejiao K.K., inc.

Lipase, cat # L3001, sigma-Aldrich.

Glycosyl peptidase, brand Ludger, E-EF01: selectively releasing high mannose and part of mixed N-polysaccharide from polypeptide and protein, 1Unit/60 μ L; E-EF02: selective release of bilinear and high mannose N-glycans from polypeptides and proteins (reduction rate 40X), 0.3Unit/60 μ L; E-EF03: both trilinear and fucosylated bilinear N-glycans, 0.33Unit/60 μ L, were selectively released from polypeptides and proteins.

Papain, cat # P4762, sigma-Aldrich; trypsin, cat # Y0002311, sigma-Aldrich.

2. Enzymolysis

One specific example 1 is carried out as follows:

(1) Sieving colla Corii Asini with 60 mesh sieve, adding 20 times of distilled water, and melting in water bath at 80 deg.C for 30min to obtain molten colla Corii Asini 3L;

(2) First enzymolysis

Adding lipase into molten donkey-hide gelatin to make the final concentration of the molten donkey-hide gelatin be 10U/mL, stirring at 40 ℃ for 90min, treating in water bath at 100 ℃ for 15min to inactivate enzyme, centrifuging at 8000rpm for 30min, collecting supernatant, leaching with 95% ethanol water solution for 48h, concentrating, and concentrating for 3 times to obtain 253g of extract;

(3) Degreasing

Mixing 253g of the extract into 1.5L of petroleum ether, carrying out ultrasonic treatment for 10min under the ultrasonic treatment condition of 25 ℃ and the ultrasonic power density of 35W/L, stirring, fully and uniformly mixing, standing for 10min, and removing the petroleum ether to obtain a solid treated by the petroleum ether; and adding 1.5L of ethyl acetate again, stirring, fully mixing, performing ultrasonic treatment for 10min under the ultrasonic treatment condition of 25 ℃ and the ultrasonic power density of 15W/L, and removing the ethyl acetate to obtain 227g of degreased substance.

(4) Second enzymolysis

Taking 10g of degreased matter, adding 30mL of water, fully and uniformly mixing, adding glycosyl peptidase E-EF01 2U, E-EF02 1U and E-EF 03U so that the concentrations are respectively equal, stirring and processing for 180min at 42 ℃, processing for 15min in a water bath at 100 ℃ for enzyme deactivation, centrifuging for 30min at 8000rpm, taking supernatant, extracting for 48h by using 95% ethanol water solution, concentrating for 3 times in total to obtain 6.23g of extract;

(5) Carrying out enzymolysis for the third time

Adding 6.23g of the extract obtained by the second enzymolysis into PBS buffer (20 mL) containing 1000U of papain and 200U of trypsin and having pH =7.5, stirring at 40 deg.C for 180min, treating in water bath at 100 deg.C for 15min for inactivating enzyme, centrifuging at 8000rpm for 30min, and collecting supernatant to obtain the final enzymolysis solution.

The implementation process of a specific example 2 is as follows:

taking 10g of degreased matter prepared in the embodiment 1, adding 30mL of water, fully and uniformly mixing, adding glycosylpeptidase E-EF 01U, stirring at 42 ℃ for 180min, then treating in a water bath at 100 ℃ for 15min to inactivate enzyme, centrifuging at 8000rpm for 30min, taking supernatant, extracting by using 95% ethanol water solution for 48h, concentrating, and concentrating for 3 times to obtain 6.23g of extract; the subsequent steps were 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 PBS buffer (20 mL) of pH =7.5 containing 1000U of papain and 200U of trypsin, and after stirring at 40 ℃ for 180min, the mixture was treated in a water bath at 100 ℃ for 15min to inactivate enzymes, and after centrifugation at 8000rpm for 30min, the supernatant was collected to obtain the final enzymatic hydrolysate.

One specific comparative example 2 was carried out as follows:

taking 10g of degreased matter prepared in the embodiment 1, adding 30mL of water, fully and uniformly mixing, adding glycosylpeptidase E-EF02 4U, stirring at 42 ℃ for 180min, then treating in a water bath at 100 ℃ for 15min to inactivate enzyme, centrifuging at 8000rpm for 30min, taking supernatant, extracting by using 95% ethanol water solution for 48h, concentrating, and concentrating for 3 times to obtain 6.23g of extract; the subsequent steps were the same as in example 1.

One specific comparative example 3 was carried out as follows:

taking 10g of the degreased matter prepared in the embodiment 1, adding 30mL of water, fully and uniformly mixing, adding glycosylpeptidase E-EF 03U, stirring at 42 ℃ for 180min, treating in a water bath at 100 ℃ for 15min for enzyme deactivation, centrifuging at 8000rpm for 30min, taking supernatant, extracting with 95% ethanol water solution for 48h, concentrating, and concentrating for 3 times to obtain 6.23g of extract; the subsequent steps were the same as in example 1.

3. Purification by gel chromatography

The enzymatic hydrolysate obtained above is filtered by filter paper, and the filtrate is ultrafiltered and concentrated by hollow fiber membrane with cut-off molecular weight of 3kD, specifically, for example, hollow fiber ultrafilter cup (product number C0005552,

XL small tangential flow ultrafiltration device, nominal molecular weight 3000) to concentrate it, collect the concentrate, carry on gel chromatography separation.

Gel chromatography separation conditions: and (3) loading the 5mL of ultrafiltration concentrate to a gel chromatographic column (1.5 cm multiplied by 80 cm) of Sephadex G-50 (G50150, sigma-Aldrich), standing for 15min, eluting by using PBS (phosphate buffer solution) with pH =6 as a mobile phase, collecting chromatographic peaks at A210 light absorption positions step by step, combining collecting pipes, concentrating under reduced pressure, and freeze-drying to obtain the donkey-hide gelatin peptide freeze-dried coarse powder.

4. RP-HPLC separation and purification

And (3) dialyzing and concentrating the obtained freeze-dried coarse powder by the dialysis bag with the molecular weight cutoff of 5000, filtering by 0.22 mu m, purifying by HPLC preparative chromatography, respectively collecting sufficient eluent according to peaks, freeze-drying, dissolving in 0.15% formic acid water solution to be used as a test sample of RP-HPLC, loading the test sample on a C18 chromatographic column, collecting chromatographic peak eluent, concentrating, and freeze-drying to obtain the donkey-hide gelatin peptide freeze-dried powder.

The conditions of the preparative chromatography were: the chromatographic column is

Bio100C18N (5 μm,30mm ID), agilent HPLC1200 series system (Agilent, wald Brownian, germany), diode Array Detector (DAD).

The mobile phase is as follows: phase A0.1% trifluoroacetic acid, phase B acetonitrile; the gradient program was: 0 → 5min, linear gradient 5 → 15% phase B; 5 → 15min, linear gradient 15 → 40% B;15 → 25min,40% by weight B;25 → 30min, linear gradient 30 → 50% B; a pre-equilibration period of 20min was used between runs. The flow rate was 0.6ml/min, the column temperature was 25 ℃, the injection amount was 10. Mu.L, and the DAD wavelength was set to 214nm.

5. Sequence identification of donkey-hide gelatin peptide

An appropriate amount of the sample was dissolved in 0.1% formic acid aqueous solution and subjected to HPLC-MS analysis.

Sample pretreatment:

(1) Putting an lmg donkey-hide gelatin peptide sample into a centrifuge tube, and adding 1mL 6M Guanidine (prepared in 100mM NH4HC 03) solution with pH of 8.0 to obtain 1mg/mL sample solution; adding 20 μ L of 1M DTT into the sample solution, and reacting at 37 deg.C for 1h to reduce disulfide bond in colla Corii Asini peptide;

(2) After the reaction is finished, continuously dividing the reaction solution into two parts on average, respectively adding 25 mu L of aqueous solution containing 1M iodoacetic acid and 1M sodium hydroxide, and standing for 30min under the conditions of light shielding and room temperature;

(3) The mixture was centrifuged again at 12000rpm for 50min in a Centricon ultrafiltration tube with less than 3kDa protein isolated. Then respectively adding 200 mu L of 0.1M ammonium bicarbonate into the centrifuge tubes, and centrifuging for 30min again; repeating the operation for a plurality of times to reduce the 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 500 mu L of 0.1M ammonium bicarbonate solution for complete dissolution, the solution containing the donkey-hide gelatin peptide samples is transferred into a Trypsin test tube containing 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 in water bath at 37 ℃ for 16h, and 500 mu L of reaction liquid is respectively taken and centrifuged by a Centricon ultrafilter tube at 15000rpm for 50min.

(5) The filtrate was collected and dried under vacuum at 35 c to concentrate, which aided the decomposition and volatilization of ammonium bicarbonate and reduced the salt concentration in the sample. Drying and concentrating are continued until the sample amount reaches 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, centrifuging at 12000-14000rmp for 10min, and sampling the supernatant for analysis.

Chromatographic conditions are as follows:

HPLC detection mode: ultraviolet light; the scanning range is 50-2000 m/z; capillary exit voltage: 166.0V, skimmer coupled system Voltage: 40.0V, oct 1DC 12.00V, oct 2DC 2.70V, ampl separation width: 4.0m/z, fragmenter voltage: 1.00Vt; ion polarity: a positive ion; type of ion source: ESI (electrospray ionization); drying temperature: 325 ℃, atomizer pressure: 15.00psi, dryer flow rate: 5.00L/min. Mass to charge ratios of the polypeptides and fragments of the polypeptides 10 fragment patterns were taken after each full scan. The original file was analyzed using the de navy algorithm in Mascot 2.3 software. The relevant parameters are Enzyme = none, variable modified-canon: oxidation (M), peptides tolerance:20ppm, MS/MS tolerance:0.1u, mascot results in a filter parameter FDR ≦ 0.01.

6. Agarose gel chromatography

Collecting main chromatographic peak elution of the RP-HPLC, and carrying out SDS-PAGE electrophoresis detection.

2. Results

The elution curves of the gel chromatography purifications 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 fraction obtained in comparative example 1 was F7, the fractions obtained in comparative example 2 were F8 to F9, and the fractions obtained in comparative example 3 were F10 to F11.

As a result of SDS-PAGE of F1 to F11, peptides having a size of less than 5Ku were present in F1, F2, F3, F5 and F6 as shown in FIG. 2. Further, F1, F2, F3, F5 and F6 were prepared by RP-HPLC chromatography, and the preparation results are shown in FIGS. 3 to 7. Purification of F1 by RP-HPLC yielded four major fractions of 7.04min, 8.23min, 11.46min and 14.94 min. Purification of F2 by RP-HPLC gave two major fractions of 15.14min and 15.87 min. Purification of F3 by RP-HPLC yielded three major fractions of 18.26min, 19.36min and 20.14 min. Purification of F5 by RP-HPLC gave two major fractions of 15.11min and 15.69 min. Purification of F6 by RP-HPLC yielded three major fractions of 18.31min, 19.27min and 20.07 min.

LS-MS detection is carried out on all the fractions, search analysis is carried out through two search engines of SEQUEST and Mascot, the primary structure of the fractions is determined by reference to NCBI database comparison, and the result is 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 first order structures of F5 (15.11 min) and F5 (15.69 min), while the three fractions of F3 are the same as those of F6. And the molecular weight of the peptides shown by SEQ ID NO. 1-11 is between 1000-3000 by calculation.

Preparation of donkey-hide gelatin peptide-iron chelate

The embodiment of the application discloses a donkey-hide gelatin peptide-iron chelate which is prepared by further utilizing 11 donkey-hide gelatin peptides extracted from donkey-hide gelatin and obtained by enzymolysis, and comprises at least one donkey-hide gelatin peptide shown by SEQ ID NO. 1-11 and iron atoms or iron ions through chelation.

The preparation implementation process of the specific donkey-hide gelatin peptide-iron chelate comprises the following steps:

200mg of colla Corii Asini peptide (F1 (7.04 min)) was dissolved in 1L of 0.1wt% ascorbic acid, adjusted to an appropriate pH =5 with 10wt% NaOH or HCl aqueous solution, and FeCl was added ₂ ·4H ₂ O to make the final concentration of the donkey-hide gelatin peptide-iron chelate be 10mg/L, placing the donkey-hide gelatin peptide-iron chelate on a magnetic stirrer at 25 ℃ for chelation for 20min, centrifuging at 4500r/min for 5min, removing precipitates, carrying out reduced pressure concentration (60-65 ℃ and-0.07-0.08 MPa), and carrying out freeze drying (50 ℃ below zero and-0.01 MPa) for 24h to obtain the donkey-hide gelatin peptide-iron chelate.

And similarly, respectively preparing the donkey-hide gelatin peptide-iron chelate from the 11 donkey-hide gelatin peptides by the method, and evaluating the chelation rate in the chelation process and the primary stability of the donkey-hide gelatin peptide-iron chelate.

The method for evaluating the chelation rate comprises the following steps:

the content of iron element in the donkey-hide gelatin peptide-iron chelate is determined by adopting an atomic absorption photometry.

(1) Preparing a test sample: 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, gently shaken and uniformly mixed, and placed in the graphite digestion instrument for heating. Keeping a micro-boiling state by adopting a temperature programming method, keeping the micro-boiling state for 20min at 220 ℃, raising the temperature after the solution is clarified, continuously keeping the micro-boiling state for 30min at 280 ℃, keeping the sample solution under the dense smoke state, leading the digestion solution to be in a colorless transparent or slightly yellow state after the white smoke is dispersed, standing at room temperature, transferring the solution into a 50mL measuring flask, washing the vessel with 2% nitric acid solution, merging the washing solution into the measuring flask, diluting to a scale, shaking up, and thus obtaining the sample digestion solution.

(3) Preparing ferrous chloride solutions of 0.2, 0.4, 0.6, 0.8 and 1mg/L, and preparing standard digestion solutions with different concentrations by the above method respectively; and (3) detecting the light absorption values of the samples respectively in an atomic absorption spectrometer (American thermoelectric instruments company ICE 3500), drawing a standard curve according to the light absorption values, and calculating the content of the iron element in the sample according to the standard curve. The detection conditions are as follows: 238nm, air flow of 6.5L/min, spectral flux of 0.2nm, lamp current of 8nm, acetylene flow of 2.0L/min,

(4) Calculation of chelation rate: the chelation rate = weight of iron in the donkey-hide gelatin peptide-iron chelate/weight of donkey-hide gelatin peptide-iron chelate.

The stability evaluation method comprises the following steps:

(1) And (3) testing the sample: the 11 donkey-hide gelatin peptide-iron chelates prepared above are named as T1-T11 respectively, and are packaged by an aluminum foil composite membrane to be used as a test sample for stability investigation.

(2) High-temperature test: precisely weighing 20mg of a test article, paving the test article in a clean weighing bottle, standing the test article at a constant temperature of 60 ℃ for 10 days for sampling, measuring the total iron content, calculating the chelation rate, and observing the appearance character of the test article.

(3) High humidity test: precisely weighing 20mg of the test sample, paving the test sample in a clean weighing bottle, respectively putting the test sample in a drier with the relative humidity of 90% +/-5%, putting the drier in an incubator with the set temperature of 25 ℃, sampling on the 10 th day, measuring the total iron content, calculating the chelation rate, and observing the appearance character of the test sample.

(4) Strong light irradiation test: precisely weighing 20mg of the sample, spreading in a clean weighing bottle, placing in a lighting box or other suitable lighting device equipped with fluorescent lamp, standing under the condition of 45001x + -500 lx for 10 days, measuring the total iron content, calculating chelating rate, and observing appearance.

TABLE 2

As can be seen from Table 2, the initial iron chelation rate of the donkey-hide gelatin peptide-iron chelate prepared by the steps reaches more than 7 per mill, and the chelation rates of T7-T11 are higher. However, in the high temperature test, iron chelation rate of the alfa iron-iron chelates is reduced to different degrees, which means that the stability of the embodiment of the application at 60 ℃ is poor, and the iron chelate is damaged by high temperature. In both the high humidity test and the strong light irradiation test, the chelate rate of T1 to T11 to iron was reduced, but the chelate rate was not significantly reduced in the high temperature test. In a high-humidity environment, the chelation rate of T1-T11 to iron is almost the same as the initial iron chelation rate, which shows 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 experiments

1. Materials and methods

1. Laboratory animal

Kunming mouse, cat # hnslkjd002, silikstada, normal diet.

2. Test article

In order to carry out related experiments, the embodiment of the application also provides a donkey-hide gelatin peptide preparation which comprises a donkey-hide gelatin peptide-iron chelate formed by chelating at least one donkey-hide gelatin peptide shown by SEQ ID NO. 1-11 with iron atoms or iron ions, and auxiliary materials acceptable in health care.

Wherein, the auxiliary materials acceptable in health science comprise fruit powder, a diluent, a bonding agent, a lubricating agent, a sweetening agent and edible essence. Wherein the fruit powder is at least one of tangerine powder, apple powder, grape powder, pear powder, grass toxin powder, blue toxin powder and cranberry enzyme powder; the diluent comprises at least one of rhizoma Amorphophalli powder, semen Maydis powder, semen glycines powder, starch, dextrin, microcrystalline cellulose and edible inorganic salt; the adhesive comprises at least one of starch slurry, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, methyl cellulose and ethyl cellulose; 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 colla corii asini peptide preparation provided by the specific embodiment is a solid particle preparation, the formula of which is shown in table 3, and the solid particle preparation is prepared by adopting a conventional medicament preparation method. In table 3, the commercially available donkey-hide gelatin powder used in comparative example 1 is the starting donkey-hide gelatin material used in the examples of the present application. In table 3, the term "part" is used only for distinguishing the weight ratio relationship between the components, and is not used to specifically limit the actual weight of each component, and may be any weight, such as 0.001mg, 0.01mg, 1mg, 10mg, 1g, 10g, 1kg, 1000kg or 1000t, etc.

TABLE 3

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3. Safety test

Dividing Kunming mice into 2 groups randomly, and orally taking 50mg/mL aqueous solution prepared by the solid particle preparations prepared in the above examples 1-14 and comparative examples 1-6 for oral administration for intragastric administration, wherein the oral dose is 5g/kg body weight; one group was given intraperitoneal injection of test article 0.5mL 2 times daily, and feeding was observed for 7 days, again by jugular injection on days 14 and 21. The mice were observed for weight, diet, activity, presence or absence of piloerection, dyspnea, convulsion, and other symptoms of anaphylaxis during the experiment.

Results after one week of intragastric administration of the mice in the oral group, the mice have no obvious difference in food intake, have normal activities and do not show the symptom of poisoning; the mice injected into the abdominal cavity have normal diet and activity, do not have excitation, dyspnea and the like, and indicate that the test article does not have anaphylactic reaction to the mice.

4. Model and experiment of various anemia

(1) First anemia model mouse establishment and grouping experiment

Kunming mice were injected with 30mg/kg spinal subcutaneous 2% phenylhydrazine hydrochloride solution (CAS: 59-88-1, merck Sigma-Aldrich) every 5d for 3 times, blood was collected from the tail tip, peripheral hemogram was measured, and success of molding was judged using a hemoglobin value lower than 10.0g/L as a standard. After the molding is successful, the model is randomly divided into a first model group and a first administration group. The first administration group was gavaged with a 50mg/mL aqueous solution prepared from the solid granule preparations prepared in examples 1 to 14 and comparative examples 1 to 6 at a dose of 20g/kg body weight, and the gavage was continued for 7 days; the first model group is not processed; blood sampling from mouse tail tip, anticoagulant detection (Experimental study of Guirong blood tonifying tablet on blood deficiency animal model action [ J)]University of Bai-Cai-En medical science, 2001,27 (3): 334-335.) RBC (x 10) ¹² /L)，HGB(g/100mL)，WBC(×10 ⁹ /L)。

(2) Establishment and grouping experiment of second anemia model mouse

Preparing Kunming mouse according to 3.5Gy ¹³⁷ Taking the model mouse after Cs disposable radiation (dosage rate of 1.27 Gy/min), collecting blood at the tail tip, measuring peripheral hemogram, and judging the success of molding by taking the hemoglobin value lower than 10.0g/L as a standard. Divided into a second model group and a second administration group. The first administration group was gavaged with a 50mg/mL aqueous solution prepared from the solid granule preparations prepared in examples 1 to 14 and comparative examples 1 to 6 at a dose of 20g/kg body weight, and the gavage was continued for 7 days; the first model group is not processed; collecting blood from mouse tail tip, performing anticoagulation detection on RBC (x 10) ¹² /L)，HGB(g/100mL)，WBC(×10 ⁹ /L)。

5. Immunity enhancement assay

(1) Molding die

Kunming mice were injected intraperitoneally with hydrocortisone (HC, 614157, sigma-Aldrich) at 25mg/kg 1 time daily for 7 consecutive days.

(2) Grouping experiment

Healthy Kunming mice were set as a blank group. In the course of the experiment for establishing and grouping the first anemia model mouse as described above, a 50mg/mL aqueous solution prepared from the solid particle preparations obtained in examples 1 to 14 and comparative examples 1 to 6 was gavaged at a dose of 20g/kg body weight and continuously administered for 10 days to serve as a third administration group.

After completion of the experiment, each group of mice measured the spleen weight, and the spleen weight index was calculated, the spleen weight index = spleen weight/body weight × 100%.

Each group of animals was continuously administered by gavage for 10 days, and each mouse was intraperitoneally injected with 0.2mL of 2% chicken red blood cell suspension on the 3 rd day of administration, 20 μ L of blood was collected from the eye 1h after the last administration, shaken in 1mL of physiological saline, and then 0.5mL of 5% chicken red blood cell suspension was added, and the mouse complement (CH 50, preparation method refer to "Guangzhou medicine 1999, 01.22 published on the description, mouse does not extract C3 antiserum") was added to 0.5mL of the suspension, incubated in a water bath at 37 ℃ for 30min to terminate the reaction, 1mL of supernatant was added to 3mL of Dushi reagent, and left to stand for 10min, and colorimetric at 540nm wavelength and absorbance (OD) was read.

On the 1 st day of administration, each group of animals was sensitized by dropping 0.5mL of Dinitrochlorobenzene (DNCB) -acetone solution 2. Mu.L per one on the depilated skin of the neck of the mouse, continuously administered for 10 days, challenged by dropping 0.025g/mL of dinitrochlorobenzene-acetone solution 20. Mu.L per one on the skin of the abdominal depilated area of the mouse 1 day before the last administration, after 24 hours, the mouse was intravenously injected with 10mL/kg of Evans blue with a mass fraction of 1% per mouse tail, after 30 minutes, the abdominal blue-stained skin was cut into pieces in a test tube, soaked in 1:l acetone physiological aqueous mixture for 24h, centrifuged at 2000rpm for 10 minutes, and the supernatant was measured for absorbance at 610 nm.

Each group of animals was continuously gavaged for 10d, 0.2mL of indian ink was injected into each rat tail vein 1h after the last administration, 20 μ L of each animal was taken from the orbital posterior venous plexus of the mouse with a micropipette 30s and 5min after the injection, immediately insufflated into 2mL of a sodium carbonate solution with a volume fraction of 0.1%, the blood of another equivalent normal mouse was zeroed, the absorbance was read at 675nm of a spectrophotometer, and the phagocytosis index (K) was determined according to the K = (lgC 1-lgC 2) formula.

6. Statistical analysis

All test data are expressed as mean and standard deviation, data were processed using SPSS13.0 software, and multiple comparisons and marked for significant differences for each column of data.

2. Results

TABLE 4

TABLE 5

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Table 4 lists the results associated with the anaemia modelling experiment using phenylhydrazine hydrochloride. Table 5 lists the relevant results of the anaemia modelling experiment using radiation. In tables 4 and 5, multiple comparisons were made for each column of data to count for significant differences therebetween.

Table 4 shows that the RBC, HGB and WBC indices of the first model group are significantly lower than those of the normal group, indicating successful molding. Compared with a model group, after the test samples provided by the embodiments 1 to 14 are administrated, the mouse RBC, HGB and WBC indexes are all obviously improved, and especially the test samples provided by the embodiments 12 to 14 prove that the solid particle preparation prepared on the basis of the donkey-hide gelatin peptide-iron chelate provided by the embodiment of the application, which is administrated to the mouse, can improve the symptom of chemically induced anemia of the mouse.

In table 4, the samples administered to the mice in comparative example 1 were solid particle preparations prepared from commercially available donkey-hide gelatin powder, which had limited function of improving anemia in model mice, while the samples administered to the mice in comparative examples 2 to 4 were all donkey-hide gelatin peptide mixtures obtained in the preparation process of the present application, which had an effect of improving anemia in model mice, but the effect was inferior to that of examples 1 to 14.

Table 5 shows that the RBC, HGB and WBC indices of the second model group are significantly lower than those of the normal group, indicating that the model mouse model of radiation anemia was successful. Compared with a model group, after the test samples provided by the embodiments 1 to 14 are administered, the mouse RBC, HGB and WBC indexes are all remarkably improved, and particularly, the test samples provided by the embodiments 12 to 14 show that the solid particle preparation prepared on the basis of the donkey-hide gelatin peptide-iron chelate provided by the embodiment can improve the symptom of the radiation-induced anemia of the mouse when the test sample is administered to the mouse. In addition, comparative examples 1 to 5 showed the same tendency as in table 4, and had a limited effect of improving anemia in model mice.

TABLE 6

Table 6 lists spleen weight index, hemolysin level, DNCB-induced OD value and phagocytosis index of each group of mice, and each data was subjected to multiple comparisons to count significant differences therebetween.

As can be seen from table 6, the spleen weight index, hemolysin level, DNCB-induced OD value and phagocytosis index of the mice of the first model group were significantly lower than those of the normal group. Compared with the model group, in the third administration group, after the test products provided by examples 1 to 14 are administered, the mouse RBC, HGB and WBC indexes are all remarkably improved, especially the test products provided by examples 12 to 14 show that the solid particle preparation prepared on the basis of the donkey-hide gelatin peptide-iron chelate provided by the embodiment of the application, which is administered to the mouse, can limit the improvement of the spleen weight index, hemolysin level, DNCB induced OD value and phagocytosis index of the mouse, and the solid particle preparation has the function of immune enhancement.

The test sample of the mouse to be administrated in the comparative example 1 is a solid particle preparation prepared from commercial donkey-hide gelatin powder, the immunity enhancement and improvement function of the test sample to the model mouse is limited, and the test samples of the mice to be administrated in the comparative examples 2 to 4 are donkey-hide gelatin peptide mixtures obtained in the preparation process of the embodiment, although the test sample has the immunity enhancement effect to the model mouse, the effect is inferior to that of the examples 1 to 14.

In summary, the embodiment of the present application obtains 11 kinds of donkey-hide gelatin peptides with molecular weight less than 3000 by performing secondary development on donkey-hide gelatin and using enzymolysis, gel chromatography and preparative chromatography techniques, 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 a high-humidity environment, but also proves that a solid particle preparation prepared from the donkey-hide gelatin peptide-iron chelate can not improve chemical induced anemia and radiation induced anemia, can simultaneously enhance the immune function of mice, and provides wide prospects for further promoting and developing health-care functional products related to donkey-hide gelatin so as to be applied to the application fields of tonifying, nourishing blood, miscarriage prevention and the like.

The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.

Claims

1. A colla Corii Asini oligopeptide is 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. 1-2,4-11.

3. A donkey-hide gelatin peptide-iron chelate composition, which is formed by chelating the donkey-hide gelatin oligopeptide according to claim 1 or the donkey-hide gelatin oligopeptide composition according to claim 2 with iron atoms or iron ions.

4. A colla Corii Asini peptide composition comprising the colla Corii Asini oligopeptide of claim 1 or the colla Corii Asini peptide-iron chelate of claim 2, and a pharmaceutically acceptable adjuvant.

5. The colla corii asini peptide composition according to claim 3, wherein the nutraceutical acceptable excipients include fruit powder, diluents, binders, lubricants, sweeteners and flavorants.

6. Use of the donkey-hide gelatin oligopeptide of claim 1, the donkey-hide gelatin oligopeptide composition of claim 2, the donkey-hide gelatin peptide-iron chelate composition of claim 3 or the donkey-hide gelatin peptide composition of claim 4 in preparation of relevant products for tonifying qi, nourishing blood or preventing miscarriage.