Ejiao characteristic peptide paste, preparation method thereof and application thereof in postpartum restoration

Abstract

The present invention belongs to the technical field of donkey-hide gelatin polypeptides, and specifically relates to donkey-hide gelatin characteristic peptide ointments, a preparation method thereof, and their application in postpartum repair. The present invention isolates donkey-hide gelatin characteristic peptides P1 and P5 from donkey-hide gelatin, and mixes these two characteristic peptides P1 and P5 with concentrated solutions of Angelica sinensis, Codonopsis pilosula, Prunus persica, Pueraria lobata, and Alpinia officinarum fermented with Aspergillus oryzae CICC 2053 and Candida utilis AS2.281 bacterial solutions, as well as crucian carp soup to prepare an ointment, which is of great significance in aspects such as postpartum uterine wound repair, relieving pregnant women's mental state to prevent postpartum depression, improving immunity to promote milk secretion, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of donkey-hide gelatin polypeptides, and particularly relates to donkey-hide gelatin characteristic peptide ointments, a preparation method thereof, and their application in postpartum repair. Background Art

[0002] Donkey-hide gelatin, also known as ass hide glue, is a traditional Chinese medicine made from donkey hide through processes such as boiling and distillation, and has the effects of enriching blood, nourishing yin and tonifying the kidney, moistening the intestine and relieving constipation, etc. Donkey-hide gelatin has a long history in China and is regarded as a precious tonic, and is widely used in the fields of traditional Chinese medicine treatment and health care. Donkey-hide gelatin characteristic peptides refer to a class of polypeptide compounds with biological activity and functionality present in donkey-hide gelatin. These characteristic peptides have characteristics such as diversity, biological activity, and safety, and have a positive impact on human health. In postpartum repair, the application of donkey-hide gelatin characteristic peptides has been widely studied and applied. For example, there will be certain incisions and laceration wounds after childbirth, and donkey-hide gelatin characteristic peptides have the effect of promoting wound healing. Research shows that donkey-hide gelatin characteristic peptides can promote cell proliferation, stimulate the synthesis of collagen and elastic fibers, thereby promoting wound healing and recovery; postpartum pregnant women are prone to problems such as anemia, and donkey-hide gelatin characteristic peptides have the effect of enriching blood, which can increase the hemoglobin level and accelerate blood circulation; postpartum pregnant women are prone to problems such as kidney deficiency and yin deficiency, and donkey-hide gelatin characteristic peptides have the effect of nourishing yin and tonifying the kidney, which can regulate endocrine, promote metabolism and detoxification, thereby helping the parturient to recover physical strength and mental state; postpartum pregnant women are prone to problems such as depression and anxiety, and donkey-hide gelatin characteristic peptides can relieve these symptoms and promote physical and mental health. Research shows that donkey-hide gelatin characteristic peptides can regulate the secretion of neurotransmitters and enhance the nerve function of the brain, thereby improving the postpartum mood state; the immune system of postpartum pregnant women is affected to a certain extent, and donkey-hide gelatin characteristic peptides have the effect of enhancing immunity, which can improve the body's immunity and resistance and prevent the occurrence of infections and diseases. Thus, it can be seen that the application of donkey-hide gelatin characteristic peptides in postpartum repair is of great significance.

[0003] The applicant's prior application 2023111319005 disclosed P1 - P5, but did not identify all of their properties. The present invention aims to further research and develop the properties. Summary of the Invention

[0004] For the above reasons, the present invention extracts two donkey-hide gelatin characteristic peptides from donkey-hide gelatin and uses them for postpartum repair.

[0005] In the first aspect, the present invention provides a preparation method of donkey-hide gelatin characteristic peptide ointment, which specifically includes the following steps:

[0006] S1. Preparation of crude donkey-hide gelatin peptide powder:

[0007] (1) Obtain the donkey-hide gelatin solution; mix the commercially available donkey-hide gelatin powder with a 40-fold amount (40 mL: 1 g) of a solution containing 1% ammonium bicarbonate, sonicate for 30 min, and then place in a water bath at 80 °C for 30 min to obtain the donkey-hide gelatin solution.

[0008] (2) Centrifuge and take the supernatant, extract successively with cyclohexane and dichloromethane, remove the cyclohexane layer and the dichloromethane layer respectively to obtain the extracted solution; wherein, the amount of cyclohexane used is 1.5 times the volume of the supernatant, and the amount of dichloromethane used is 2 times the volume of the solution after removing the cyclohexane layer.

[0009] (3) Centrifuge, ultrafilter, centrifuge, concentrate and lyophilize the extracted solution to obtain the crude donkey-hide gelatin powder.

[0010] (4) Digest the crude donkey-hide gelatin powder with pepsin and collect the first digestion solution. The steps of digesting the crude donkey-hide gelatin powder with pepsin include: preparing a sample solution with a pH of 2 by mixing the crude donkey-hide gelatin powder with a 1% phosphoric acid solution, adding pepsin at 100,000 U / g of the crude donkey-hide gelatin powder, placing in a water bath at 42 °C for 2 h, inactivating the enzyme at 100 °C for 15 min, and collecting the first digestion solution.

[0011] (5) Digest the first digestion solution with trypsin and collect the second digestion solution. The steps of digesting the first digestion solution with trypsin include: adjusting the pH of the first digestion solution to 8 with 2% sodium bicarbonate, adding trypsin at 20,000 U / g of the crude donkey-hide gelatin powder, placing in a water bath at 42 °C for 3 h, inactivating the enzyme at 100 °C for 15 min, and collecting the second digestion solution.

[0012] (6) Centrifuge, ultrafilter, centrifuge, concentrate and lyophilize the second digestion solution to obtain the crude donkey-hide gelatin peptide powder; wherein, after centrifuging the second digestion solution, ultrafiltration is carried out using a 3 kDa ultrafiltration tube.

[0013] S2. Purify the donkey-hide gelatin peptide powder obtained in S1 by gel chromatography, name each elution peak as P1, P2, P3, P4, P5 through the chromatogram, collect P1 and P5 and freeze-dry them into powder, which are the donkey-hide gelatin characteristic peptides P1 and P5.

[0014] Among them, the conditions for the gel chromatography purification include: using Sephadex G-15 as the packing material, using deionized water as the eluent, the sample loading volume is 5 mL, the flow rate is 1.0 mL / min, and the wavelength of the ultraviolet detector is 220 nm.

[0015] S3. Preparation of Aspergillus oryzae CICC 2053 bacterial solution: Prepare for use a bacterial solution of Aspergillus oryzae CICC 2053 with a concentration of 1.8×10 9 ~3.6×10 9 cfu / ml; Preparation of Candida utilis AS2.281 bacterial solution: Prepare a bacterial solution of Candida utilis AS2.281 with a concentration of 3.5×10 9 ~4.6×109 cfu / ml of Candida utilis AS2.281 bacterial solution for use;

[0016] S4, pretreatment of angelica, codonopsis, peach kernel, kudzu root and galangal: angelica, codonopsis, peach kernel, kudzu root and galangal are crushed in the same mass ratio and mixed evenly, mixed with water in a mass ratio of 1-2:2-3, and then added with Aspergillus oryzae CICC 2053 solution and Candida utilis AS2.281 solution prepared in step S2, and fermented in a cylinder, wherein the Chinese medicine mixture: Aspergillus oryzae CICC2053 solution: Candida utilis AS2.281 solution are in a mass ratio of 1-3:0.5-1.5:0.5-2, aerobic fermentation, temperature is 32-35°C, and fermentation time is 12-24 hours; after fermentation is completed, boil, evaporate and concentrate, cool naturally, and set aside; In this step, Aspergillus oryzae CICC 2053 solution and Candida utilis AS2.281 solution are used to ferment Chinese medicine, the purpose is to enhance the effect of Chinese medicine and obtain more beneficial substances in Chinese medicine;

[0017] S5. Preparation of crucian carp, wolfberry and red date soup: clean the fresh crucian carp, put it into an oil pan and fry until both sides are golden brown, remove the core of the red dates, put them into a casserole, add 500-800 ml of boiling water, add wolfberries, cook over high heat for 10 minutes, then cook over low heat for 15-20 minutes, after that, remove the crucian carp, red dates and wolfberries, remove the fish bones, and mash the crucian carp meat, red dates and wolfberries into a meat paste for later use;

[0018] S6. The donkey-hide gelatin characteristic peptides P1 and P5 prepared in step S2 are mixed in a mass ratio of 1:1 and mixed with the concentrated solution in step S4 and the crucian carp soup and meat paste in step S5 in proportion to obtain donkey-hide gelatin peptide paste for postpartum repair. The donkey-hide gelatin peptide paste is vacuum-packed in units of 5 g.

[0019] Preferably, the concentrations of the phosphoric acid solution and the sodium bicarbonate solution used in step S1 of the present invention are 1% and 2%, respectively.

[0020] Preferably, the concentration of Aspergillus oryzae CICC 2053 bacterial solution prepared in step S3 of the present invention is 2.5×10 9 cfu / ml.

[0021] Preferably, the concentration of the Candida utilis AS2.281 bacterial solution prepared in step S3 of the present invention is 3.5×10 9 cfu / ml

[0022] Preferably: in step S4 of the present invention, angelica sinensis, codonopsis pilosula, peach kernel, kudzu root and galangal are crushed in the same mass ratio and mixed evenly, and then mixed with water in a mass ratio of 1:2.

[0023] Preferably, in step S4 of the present invention, heating is stopped when the fermentation liquid is concentrated to half of its mass.

[0024] Preferably, in step S6 of the present invention, the mass ratio of donkey-hide gelatin characteristic peptide: Chinese medicine concentrated liquid: crucian carp soup: meat paste is 3:2:3:2.

[0025] In a second aspect, the present invention provides a donkey-hide gelatin characteristic peptide prepared by the preparation method of the donkey-hide gelatin characteristic peptide paste described above and its application in postpartum repair.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. Between the donkey-hide gelatin characteristic peptides P1 and P5 prepared by the present invention, they can synergistically promote cell proliferation, stimulate the synthesis of collagen and elastic fibers, thereby promoting wound healing and recovery, and contributing to the repair of wounds such as the postpartum uterus.

[0028] 2. The present invention uses Aspergillus oryzae CICC 2053 bacterial liquid and Candida utilis AS2.281 bacterial liquid to ferment Chinese medicine, aiming to enhance the effect of Chinese medicine, obtain more beneficial substances in Chinese medicine, and improve the postpartum repair performance.

[0029] 3. The donkey-hide gelatin peptide paste prepared by the present invention has the effect of nourishing yin and tonifying the kidney, can regulate endocrine, promote metabolism and detoxification, thereby helping parturients to recover physical strength and mental state.

[0030] 4. The donkey-hide gelatin peptide paste prepared by the present invention helps to enhance immunity, prevent postpartum depression, and promote milk secretion.

[0031] 5. The donkey-hide gelatin characteristic peptide prepared by the present invention can regulate the secretion of neurotransmitters, enhance the nerve function of the brain, thereby improving the postpartum emotional state.

[0032] 6. The donkey-hide gelatin composite polypeptide prepared by the present invention can be used to prepare donkey-hide gelatin polypeptide paste, oral liquid, decoction, tablets, etc., which are convenient to use. Description of the Drawings

[0033] Figure 1 It is the Sephadex G-15 chromatography map of P1 to P5.

[0034] Figure 2 It is the secondary MS diagram of P1.

[0035] Figure 3 It is the secondary MS diagram of P5.

[0036] Figure 4 It is the HE staining map of the uterine section of the normal group.

[0037] Figure 5 It is the HE staining map of the uterine section of the model group.

[0038] Figure 6 TNF-α level graphs in sera of rats in each group

[0039] Figure 7 TGF-β1 level graphs in sera of rats in each group

[0040] Figure 8 Apoptosis rate graphs of uteri of rats in the normal group, model group, Example 1-2 groups and Comparative Example 1-2 Detailed implementation manners

[0041] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, without conflict, the following examples and the features in the examples can be combined with each other.

[0042] Example 1

[0043] S1 Preparation of crude ejiao peptide

[0044] (1) The ejiao powder used in the experiment was purchased from Dong'e Ejiao Co., Ltd. Add 40 times the amount (40 mL: 1 g) of a solution containing 1% ammonium bicarbonate, ultrasonic for 30 min, and then water bath at 80 °C for 30 min to obtain an ejiao solution.

[0045] (2) Centrifuge at 8000 rpm for 20 min, transfer the supernatant to a separating funnel, add cyclohexane (the dosage is 1.5 times the volume of the supernatant), shake for 5 min, then discard the cyclohexane layer. Add dichloromethane (2 times the volume of the solution after removing the cyclohexane layer), shake and extract for 5 min, discard the dichloromethane layer, and filter the extracted aqueous layer through a 0.45 μm microporous membrane to obtain the extracted solution.

[0046] (3) Subject the extracted solution to a water bath at 90 °C for 30 min, centrifuge at 8000 rpm for 20 min, transfer the supernatant to a 10 kDa ultrafiltration tube, centrifuge at 3000 rpm for 40 min, collect the filtrate, concentrate under reduced pressure, and then freeze-dry to obtain crude ejiao powder.

[0047] (4) Prepare a sample solution with a pH of 2 by using 1% phosphoric acid solution for the crude ejiao powder, add pepsin at 100,000 / g of the crude ejiao powder, water bath at 42 °C for 2 h, inactivate the enzyme at 100 °C for 15 min, and collect the first enzymolysis solution.

[0048] (5) Adjust the pH of the first enzymolysis solution to 8 with 2% sodium bicarbonate, add trypsin at 20,000 / g of the crude ejiao powder, water bath at 42 °C for 3 h, inactivate the enzyme at 100 °C for 15 min, and collect the second enzymolysis solution.

[0049] (5) Centrifuge the second enzymatic hydrolysate at 8000 rpm for 20 min, take the supernatant, add it to a 3 kDa ultrafiltration tube, centrifuge at 3000 rpm for 40 min, collect the filtrate, concentrate it under reduced pressure, and then freeze-dry it to obtain crude donkey-hide gelatin peptides.

[0050] S2. Prepare the crude donkey-hide gelatin peptides obtained in S1 into a solution with a mass concentration of 100 mg / mL using deionized water. After filtering through a 0.45 μm aqueous filter, separate and purify it again using a gel chromatography column. The pretreated Sephadex G-15 (G15120, Sigma-Aldrich) is filled into a glass chromatography column with dimensions of 2.6 cm × 60 cm. Use deionized water as the eluent, with a sample loading volume of 5 mL, a flow rate of 1.0 mL / min, and the wavelength of the ultraviolet detector set at 220 nm. As Figure 1 shown, name each elution peak as P1, P2, P3, P4, P5 through the chromatogram. Collect P1 and P5 and freeze-dry them into powders, which are the donkey-hide gelatin characteristic peptides P1 and P5;

[0051] The method for preparing donkey-hide gelatin characteristic peptides P1 and P5 in steps S1 and S2 refers to the relevant content of donkey-hide gelatin characteristic peptides P1 and P5 recorded in Example 1 of the Chinese invention patent: Donkey-hide Gelatin Characteristic Peptides, Preparation Method, Composition and Their Application in Premarital Conditioning Ointment (Application No. 2023111319005, Publication Date: November 7, 2023).

[0052] S3. Preparation of Aspergillus oryzae CICC 2053 bacterial solution: Prepare a bacterial solution of Aspergillus oryzae CICC 2053 with a concentration of 1.8×10 9 cfu / ml for use; Preparation of Candida utilis AS2.281 bacterial solution: Prepare a bacterial solution of Candida utilis AS2.281 with a concentration of 3.5×10 9 cfu / ml for standby; Among them, Aspergillus oryzae CICC 2053, with the catalog number CICC 2053, is purchased from Beijing Yuwei Technology Co., Ltd.; Candida utilis AS2.281, with the product number B311818, is purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0053] S4. Pretreatment of Angelica sinensis, Codonopsis pilosula, Prunus persica, Pueraria lobata, and Alpinia officinarum: Crush Angelica sinensis, Codonopsis pilosula, Prunus persica, Pueraria lobata, and Alpinia officinarum according to the same mass ratio and mix them evenly. Then mix them with water at a mass ratio of 1:2, and add the Aspergillus oryzae CICC 2053 bacterial liquid and Candida utilis AS2.281 bacterial liquid prepared in step S2 into a vat for fermentation. Among them, the mass ratio of the traditional Chinese medicine mixture: Aspergillus oryzae CICC 2053 bacterial liquid: Candida utilis AS2.281 bacterial liquid is 1:1.5:2. Conduct aerobic fermentation at a temperature of 33°C for 12 hours. After fermentation, boil to evaporate and concentrate, and let it cool naturally for later use. The purpose of using the Aspergillus oryzae CICC 2053 bacterial liquid and Candida utilis AS2.281 bacterial liquid to ferment the traditional Chinese medicine in this step is to enhance the effect of the traditional Chinese medicine and obtain more beneficial substances in the traditional Chinese medicine.

[0054] S5. Preparation of crucian carp, wolfberry, and red date soup: Clean the fresh crucian carp, fry it in an oil pan until both sides are golden brown. Remove the pits from the red dates, put them into a casserole, add 500 ml of boiling water, add wolfberries, boil over high heat for 10 minutes, and then simmer over low heat for 15 - 20 minutes. After completion, remove the crucian carp, red dates, and wolfberries, remove the fish bones, and make the crucian carp meat, red dates, and wolfberries into a meat paste for later use.

[0055] S6. Mix the prepared Colla Corii Asini characteristic peptides P1 and P5 in step S2 according to a mass ratio of 1:1, and mix them with the concentrated liquid in step S4, the crucian carp soup in step S5, and the meat paste in proportion to obtain the Colla Corii Asini peptide paste for postpartum repair. The Colla Corii Asini peptide paste is vacuum-packed in units of 5 g.

[0056] Comparative example 1: Except for only collecting the Colla Corii Asini characteristic peptide P1 in step S2, all other steps are the same as those in Example 1.

[0057] Comparative example 2: Except for only collecting the Colla Corii Asini characteristic peptide P5 in step S2, all other steps are the same as those in Example 1.

[0058] LC-MS / MS analysis of Colla Corii Asini characteristic peptides

[0059] (1) LC conditions

[0060] Mobile phase: A is 0.1% formic acid solution, B is 0.1% formic acid - acetonitrile solution. Injection volume is 2 μL, sample loading flow rate is 500 nL / min, and separation flow rate is 300 nL / min. Gradient elution conditions: 0 - 105 min, 95% - 70% A, 5% - 30% B; 105 - 110 min, 70% - 10% A, 30% - 90% B; 110 - 112 min, 10% A, 90% B; 112 - 113 min, 10% - 95% A, 90% - 5% B; 95% A, 5% B is maintained for 7 min.

[0061] (2) MS conditions

[0062] The spray voltage of the ion source was 2.2 kV, the temperature of the heated capillary was 320 °C, and data acquisition was switched between MS and MS / MS. Full-scan MS: scanning range m / z 400 - 1600, scanning resolution 120,000 (at m / z 200), maximum ion injection time 50 ms, and automatic gain control (AGC) was set to 1.0×106. MS / MS: scanning resolution 15,000, scanning range m / z 110 - 2000, minimum ion intensity 50,000, maximum ion injection time 100 ms, AGC was set to 1.0×105, and the parent ion selection was set to 1.6 Da.

[0063] (3) Qualitative analysis by searching the library using Proteome Discoverer software

[0064] The raw mass spectrometry files (raw files) were searched using the Sequest HT search engine in Proteome Discoverer (version 2.4.0.305) software. Search parameters: no fixed modifications, variable modifications were methionine oxidation and protein N-terminal acetylation; primary mass spectrometry accuracy was 1.0×10 -5 , secondary mass spectrometry accuracy was 0.02 Da; the Uniprot database was of the species Equus asinus (Mnemonic: EQUAS); the false positive rates of peptides and proteins were ≤1%; the peptides contained at least 6 amino acids.

[0065] (4) Amino acid sequence results of the characteristic peptides of Ejiao

[0066] Mass spectrometry analysis and library search qualitative analysis were performed on the above-obtained Ejiao characteristic peptides P1 - P8. The MS analysis results showed that the number of amino acids in the detected peptides was between 4 and 15, the molecular mass distribution was between 661.4 and 2851.4 Da, and they were derived from 6 proteins in total.

[0067] Among them, the amino acid sequence of P1 is YKTTKTSRLPIIDVAPLDVGAP, as shown in SEQ ID NO.1, with the reference sequence GenBank: ACI29947.1, Capra hircus. The amino acid sequence of P2 is DVWKPVPCQICVCDNGNVLCD, with the reference sequence [Ovis aries] GenBank: KAG5203341.1. The amino acid sequence of P3 is DDANVVRDRDLEVDTTLK, as shown in SEQ ID NO.3, with the reference sequence [Ovis aries] GenBank: KAG5203341.1. The amino acid sequence of P4 is DRGDAGPKGADGAPGKDGVR, as shown in SEQ ID NO.4, with the reference sequence [Ovis aries] GenBank: KAG5203341.1. The amino acid sequence of P5 is GEGSNPADVAIQLTFLR, with the reference sequence CO1A1 protein, partial [Zapornia atra] GenBank: NXT83303.1.

[0068] Figure 1 It is the Sephadex G-15 chromatography map of P1 - P5.

[0069] Figure 2 It is the secondary MS map of P1.

[0070] Figure 3 It is the secondary MS map of P5.

[0071] Example 2

[0072] Preparation of crude donkey-hide gelatin peptide powder;

[0073] (1) The donkey-hide gelatin powder used in the experiment was purchased from Dong'e E-Jiao Co., Ltd. Add 40 times the amount (40 mL: 1 g) of a solution containing 1% ammonium bicarbonate, sonicate for 30 min, and then heat in a water bath at 80 °C for 30 min to obtain a donkey-hide gelatin solution.

[0074] (2) Centrifuge at 8000 rpm for 20 min, transfer the supernatant to a separatory funnel, add cyclohexane (the amount used is 1.5 times the volume of the supernatant), shake for 5 min, then discard the cyclohexane layer. Add dichloromethane (2 times the volume of the solution after removing the cyclohexane layer), shake and extract for 5 min, discard the chloroform layer, and filter the extracted aqueous layer through a 0.45 μm microporous filter membrane to obtain the extracted solution.

[0075] (3) Heat the extracted solution in a water bath at 90 °C for 30 min, centrifuge at 8000 rpm for 20 min, transfer the supernatant to a 10 kDa ultrafiltration tube, centrifuge at 3000 rpm for 40 min, collect the filtrate, concentrate under reduced pressure, and then freeze-dry to obtain the crude donkey-hide gelatin powder.

[0076] (4) Prepare a sample solution with a pH of 2 by dissolving the crude donkey-hide gelatin powder in a 1% phosphoric acid solution. Add pepsin at a dosage of 100,000 units per gram of the crude donkey-hide gelatin powder, and incubate in a water bath at 42 °C for 2 h. Then inactivate the enzyme at 100 °C for 15 min, and collect the first enzymatic hydrolysate.

[0077] (5) Adjust the pH of the first enzymatic hydrolysate to 8 with 2% sodium bicarbonate. Add pancreatin at a dosage of 20,000 units per gram of the crude donkey-hide gelatin powder, and incubate in a water bath at 42 °C for 3 h. Then inactivate the enzyme at 100 °C for 15 min, and collect the second enzymatic hydrolysate.

[0078] (5) Centrifuge the second enzymatic hydrolysate at 8000 rpm for 20 min. Take the supernatant and add it to a 3 kDa ultrafiltration tube, then centrifuge at 3000 rpm for 40 min. Collect the filtrate, concentrate it under reduced pressure, and then freeze-dry to obtain the crude donkey-hide gelatin peptide powder.

[0079] S2. Prepare a solution of the crude donkey-hide gelatin peptide powder with a mass concentration of 100 mg / mL using deionized water. After filtering through a 0.45 μm aqueous filter, separate and purify it again using a gel chromatography column. The pretreated Sephadex G-15 (G15120, Sigma-Aldrich) is filled into a glass chromatography column with a size of 2.6 cm × 60 cm. Use deionized water as the eluent, with a sample loading volume of 5 mL and a flow rate of 1.0 mL / min. The wavelength of the ultraviolet detector is 220 nm. As Figure 1 shown in the figure above, name each elution peak as P1, P2, P3, P4, and P5 through the chromatogram. Collect P1 and P5 and freeze-dry them into powders, which are the donkey-hide gelatin characteristic peptides P1 and P5;

[0080] The method for preparing the donkey-hide gelatin characteristic peptides P1 and P5 in steps S1 and S2 refers to the relevant content of the donkey-hide gelatin characteristic peptides P1 and P5 described in Example 1 of the Chinese invention patent: Donkey-hide Gelatin Characteristic Peptides, Preparation Method, Composition and Their Application in Pre-pregnancy Conditioning Ointment (Application No. 2023111319005, Publication Date: November 7, 2023).

[0081] S3. Preparation of Aspergillus oryzae CICC 2053 bacterial solution: Prepare a bacterial solution of Aspergillus oryzae CICC 2053 with a concentration of 2.5×10 9 cfu / ml for use; Preparation of Candida utilis AS2.281 bacterial solution: Prepare a bacterial solution of Candida utilis AS2.281 with a concentration of 3.5×10 9 cfu / ml for standby; Among them, Aspergillus oryzae CICC 2053, product number CICC 2053, is purchased from Beijing Yuwei Technology Co., Ltd.; Candida utilis AS2.281, product number B311818, is purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0082] S4, pretreatment of angelica, codonopsis, peach kernel, kudzu root and galangal: angelica, codonopsis, peach kernel, kudzu root and galangal are crushed in the same mass ratio and mixed evenly, mixed with water in a mass ratio of 2:3, and then added with Aspergillus oryzae CICC 2053 bacterial solution and Candida utilis AS2.281 bacterial solution prepared in step S2 for fermentation in a cylinder, wherein the Chinese medicine mixture: Aspergillus oryzae CICC 2053 bacterial solution: Candida utilis AS2.281 bacterial solution are in a mass ratio of 2:1.5:2, aerobic fermentation, temperature is 34°C, and fermentation time is 18 hours; after fermentation is completed, boil, evaporate and concentrate, cool naturally, and set aside; In this step, Aspergillus oryzae CICC 2053 bacterial solution and Candida utilis AS2.281 bacterial solution are used to ferment Chinese medicine, the purpose of which is to enhance the effect of Chinese medicine and obtain more beneficial substances in Chinese medicine;

[0083] S5. Preparation of crucian carp, wolfberry and red date soup: clean the fresh crucian carp, put it into an oil pan and fry until both sides are golden brown, remove the core of the red dates, put them into a casserole, add 800 ml of boiling water, add wolfberries, cook over high heat for 10 minutes, then cook over low heat for 15-20 minutes, after that, remove the crucian carp, red dates and wolfberries, remove the fish bones, and mash the crucian carp meat, red dates and wolfberries into a meat paste for later use;

[0084] S6. The donkey-hide gelatin characteristic peptides P1 and P5 prepared in step S2 are mixed with the concentrated solution in step S4 and the crucian carp soup and meat paste in step S5 in a mass ratio of 1:1 to obtain donkey-hide gelatin peptide paste for postpartum repair. The donkey-hide gelatin peptide paste is vacuum-packed in units of 5 g.

[0085] Comparative Example 3: Except that Aspergillus oryzae CICC 2053 culture liquid is not used for fermentation in step S4, the remaining steps are the same as those in Example 2.

[0086] Comparative Example 4: Except that the Candida utilis AS2.281 bacterial solution was not used for fermentation in step S4, the remaining steps were the same as those in Example 2.

[0087] Comparative Example 5: Except that the Aspergillus oryzae CICC 2053 bacterial solution was replaced with Aspergillus oryzae CICC 2057 (purchased from Beijing Yuwei Technology Co., Ltd.; product number CICC 2057) for fermentation in step S4, the remaining steps were the same as those in Example 2.

[0088] Comparative Example 6: In addition to replacing the bacterial solution of Candida utilis AS2.281 in step S4 with Candida utilis AS2.120 (purchased from Shanghai Xuanke Biotechnology Co., Ltd.; product number: XK-SH-12661)

[0089] Animal experiments

[0090] SPF-grade SD rats (120 females and 60 males), all 10 weeks old, with body weights of (210 ± 25) g, were provided by Spf (Beijing) Biotechnology Co., Ltd. The animal production license is SCXK (Jing) 2016-0002. Experiments were conducted in accordance with the "Regulations on the Administration of Laboratory Animals". The rats were housed in an animal room at room temperature with a humidity of about 55%, simulating the biological cycle of animals with alternating light and dark (12 h / 12 h), and were adaptively fed with sterile diet for 2 weeks. During the experiment, all rats had free access to water and food. Female and male rats were caged together at a ratio of 2:1. The next morning, vaginal plugs were observed and vaginal examinations were performed. The day when sperm were found in the vaginal plug was considered the first day of pregnancy. All pregnant rats were singly caged in the same environment until the first day after parturition. On the first day after parturition, 50 female rats (after parturition) were randomly divided into 10 groups as the first experimental group, namely the normal group, the model group, the example groups (two groups of Example 1 and Example 2), and the comparative example groups (six groups of Comparative Example 1-6); each group of 5 rats was caged together. Except for the normal group, the remaining rats were modeled. Escherichia coli was diluted with NaCl solution to 1×10 7 cfu / mL, and 0.2 mL was inoculated into the uterus. One rat in each group was sacrificed on the second day after injection, and uterine sections were stained with HE for observation. Compared with the normal group ( Figure 5 ), the model group ( Figure 4 ) had abnormal uterine cavity morphology, thinner endometrium, irregular arrangement of muscle cells, large-area hyaline changes in blood vessels and inflammatory infiltration, and poor uterine involution, indicating successful modeling.

[0091] After successful modeling, the example groups were fed with the Ejiao peptide paste prepared in Example 1 and Example 2 respectively, and the comparative example groups were fed with the Ejiao peptide paste prepared in Comparative Example 1-6 respectively, once in the morning and once in the evening every day, and each time was fed at a dose of 4 g / Kg. The rats in the normal group and the model group were intragastrically administered an equal volume of 0.9% normal saline. After continuous feeding for 7 days, 3 rats in each group were randomly sacrificed for detecting indexes.

[0092] At the same time, 50 of the female rats on the 1st day after remaining parturition were randomly divided into 10 groups as the second experimental large group, namely the normal group, the model group, the example groups (two groups of Example 1 and Example 2), and the comparative example groups (six groups of Comparative Example 1-6), with 5 rats in each cage. Starting from the 3rd day after the mother rats gave birth, except for the normal group, the remaining groups were intragastrically administered bromocriptine at a dose of 1.6 mg / kg daily for 10 days to establish a rat model of postpartum hypogalactia. During the modeling, according to the human-animal dose conversion method, the example groups (two groups of Example 1 and Example 2) and the comparative example groups (six groups of Comparative Example 1-6) were respectively given the Ejiao characteristic peptide paste prepared in Example 1, Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 4, Comparative Example 5, and Comparative Example 6 at a dose of 1.2 g / 100 g·d for feeding, and the normal group and the hypogalactia model group (model group) were given an equal amount of normal saline. After 10 days of gavage, the gavage was stopped, and the milk secretion of the rats was recorded on the 3rd, 6th, and 9th days respectively.

[0093] Serum specimen collection

[0094] The rats in the first experimental large group were sacrificed, and blood specimens were collected from the carotid arteries. After centrifugation at 3000 r / min for 15 min, the supernatant (serum) was taken and stored at -20°C, and the serum indexes were detected uniformly.

[0095] Detection of cytokines TNF-a and TGF-B by serum ELISA method

[0096] The frozen serum specimens of the normal group, the model group, the Example 1-2 groups, and the Comparative Example 1-6 groups were placed in a 4°C refrigerator overnight, and then rewarmed to room temperature. The contents of TNF-α and TGF-β in the rat serum were detected by the double antibody sandwich ELISA method. The results are shown in Table 1, Figure 6 and Figure 7 .

[0097] Uterine cell apoptosis rate test

[0098] After incubating the uterine tissue sections of the normal group, the model group, the Example 1-2 groups, and the Comparative Example 1-2 groups with proteinase K at room temperature, they were immersed in the TUNEL reaction solution and rinsed 3 times with PBS. After rinsing with hydrogen peroxide to quench the enzyme activity, combined avidin peroxidase and diaminobenzidine were covered, and apoptotic cell nuclei showing brownish yellow were observed under an optical high-power microscope, and 5 fields of view were randomly selected. The HE staining map of the uterine section of the normal group is shown in Figure 4 ; the HE staining map of the uterine section of the model group is shown in Figure 5 ;

[0099] The cell apoptosis rate was calculated, and the results are shown in Figure 8 .

[0100] Cell apoptosis rate = number of apoptotic cells / total number of cells × 100%

[0101] Table 1

[0102]

[0103]

[0104] The serum TNF-α and TGF-β1 levels of each group of rats were measured and compared. Due to incomplete uterine recovery, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) can cause an increase in the release of TNF-α, and the increased release of TNF-α promotes the phosphorylation of NF-κB, further activating NF-κB and leading to a vicious cycle, exacerbating the uterine inflammatory response. TGF-β1 is considered to be one of the most important profibrotic factors and a key initiating factor in the occurrence and development of fibrosis. It can stimulate an increase in the number of epithelial cells in the endometrial tissue and induce their transformation into myofibroblasts. According to Table 1 (the corresponding bar chart is as Figure 6 and Figure 7 shown), from the results of Examples 1-2 and Comparative Examples 1-2, when the donkey-hide gelatin characteristic peptides P1 and P5 are added simultaneously, they can effectively synergistically inhibit the increase in TNF-α and TGF-β1 levels, synergistically promote cell proliferation, stimulate the synthesis of collagen and elastic fibers, reduce the uterine cell apoptosis rate, thereby promoting wound healing and recovery, and contributing to the repair of postpartum uterine wounds and other wounds. At the same time, according to the results of Examples 1-2 and Comparative Examples 3-4, the synergistic fermentation of Angelica sinensis, Codonopsis pilosula, Prunus persica, Pueraria lobata, and Alpinia officinarum by Aspergillus oryzae CICC 2053 and Candida utilis AS2.281 can enhance the effect of traditional Chinese medicine, obtain more beneficial substances in traditional Chinese medicine to help uterine repair, and according to the results of Comparative Examples 5-6, the fermentation of Angelica sinensis, Codonopsis pilosula, Prunus persica, Pueraria lobata, and Alpinia officinarum by Aspergillus oryzae CICC 2053 and Candida utilis AS2.281 has specificity. According to Figure 8 it can be seen that P1 and P5 can synergistically promote cell proliferation, stimulate the synthesis of collagen and elastic fibers, reduce the uterine cell apoptosis rate, thereby promoting wound healing and recovery, and contributing to the repair of postpartum uterine wounds and other wounds.

[0105] The milk production of the remaining rats in the normal group, example group, and comparative example group of the second large experimental group was recorded, and the results are shown in Table 2.

[0106] Table 2

[0107]

[0108]

[0109] According to the results of Comparative Examples 1-2 in Table 2, it is found that there is a synergistic effect between P1 and P5, which has an unexpected effect of increasing milk production. At the same time, due to the more beneficial substances obtained by the co-fermentation of Aspergillus oryzae CICC 2053 and Candida utilis AS2.281 on Angelica sinensis, Codonopsis pilosula, Prunus persica, Pueraria lobata and Alpinia officinarum, the milk production can also be increased to a certain extent.

[0110] The above-described embodiments only represent the specific implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation to the protection scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention should be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A donkey-hide gelatin characteristic peptide paste, characterized in that: P1 and P5 are mixed with concentrated solutions of angelica, codonopsis, peach kernel, kudzu root and galangal fermented with Aspergillus oryzae CICC 2053 and Candida utilis AS2.281, and crucian carp soup to prepare a paste, wherein the amino acid sequence of P1 is YKTTKTSRLPIIDVAPLDVGAP, as shown in SEQ ID NO.1; the amino acid sequence of P5 is GEGSNPADVAIQLTFLR, as shown in SEQ ID NO.

5.

2. The method for preparing the donkey-hide gelatin characteristic peptide paste according to claim 1, characterized in that: The specific preparation method is as follows: S1. Preparation of coarse donkey-hide gelatin powder: (1) Obtaining a donkey-hide gelatin solution; mixing commercially available donkey-hide gelatin powder with a 40-fold amount of a 1% ammonium bicarbonate solution, wherein the 40-fold amount is 40 mL:1 g, ultrasonically mixing for 30 minutes, and then placing in a water bath at 80° C. for 30 minutes to obtain the donkey-hide gelatin solution; (2) extracting the supernatant by centrifugation, extracting with cyclohexane and dichloromethane in sequence, removing the cyclohexane layer and the dichloromethane layer respectively, and obtaining an extracted solution; wherein the amount of the cyclohexane used is 1.5 times the volume of the supernatant, and the amount of the dichloromethane used is 2 times the volume of the solution after removing the cyclohexane layer; (3) subjecting the extracted solution to centrifugation, ultrafiltration, centrifugation, concentration and freeze-drying to obtain coarse donkey-hide gelatin powder; (4) hydrolyzing the coarse donkey-hide gelatin powder with pepsin and collecting a first enzymatic hydrolysis solution; the step of hydrolyzing the coarse donkey-hide gelatin powder with pepsin comprises: preparing a sample solution with a pH of 2 using a 1% phosphoric acid solution, adding 100,000 pepsin per gram of coarse donkey-hide gelatin powder, bathing the solution in a 42°C water bath for 2 hours, inactivating the enzyme at 100°C for 15 minutes, and collecting a first enzymatic hydrolysis solution; (5) hydrolyzing the first enzymatic solution with pancreatic enzymes, and collecting a second enzymatic solution; the step of hydrolyzing the first enzymatic solution with pancreatic enzymes comprises: adjusting the pH of the first enzymatic solution to 8 with 2% sodium bicarbonate, adding 20,000 / g of donkey-hide gelatin coarse powder of pancreatic enzymes, bathing the solution in 42°C water for 3 hours, inactivating the enzyme at 100°C for 15 minutes, and collecting the second enzymatic solution; (6) subjecting the second enzymatic hydrolysate to centrifugation, ultrafiltration, centrifugation, concentration and freeze-drying to obtain coarse donkey-hide gelatin peptide powder; wherein, after the second enzymatic hydrolysate is centrifuged, ultrafiltration is performed using a 3 kDa ultrafiltration tube; S2, purifying the crude donkey-hide gelatin peptide powder obtained in S1 by gel chromatography, naming each elution peak as P1, P2, P3, P4, and P5 according to the spectrum, collecting P1 and P5 and freeze-drying them into powder, namely the donkey-hide gelatin characteristic peptides P1 and P5; S3. Preparation of Aspergillus oryzae CICC 2053 bacterial solution: the concentration was 1.8×10 9 ~3.6×10 9 cfu / ml of Aspergillus oryzae CICC 2053 bacterial solution for standby use; preparation of Candida utilis AS2.281 bacterial solution: the preparation concentration is 3.5×10 9 ~4.6×10 9 cfu / ml of Candida utilis AS2.281 bacterial solution for use; S4, pretreatment of angelica, codonopsis, peach kernel, kudzu root and galangal: angelica, codonopsis, peach kernel, kudzu root and galangal are crushed and mixed uniformly in the same mass ratio, and then mixed with water in a mass ratio of 1-2:2-3, and then the Aspergillus oryzae CICC 2053 solution and Candida utilis AS2.281 solution prepared in step S3 are added and fermented in a cylinder, wherein the mass ratio of the Chinese medicine mixture: Aspergillus oryzae CICC2053 solution: Candida utilis AS2.281 solution is 1-3:0.5-1.5:0.5-2, and aerobic fermentation is carried out at a temperature of 32-35° C. and a fermentation time of 12-24 hours; after the fermentation is completed, the fermented product is boiled, evaporated and concentrated to obtain a concentrated solution, which is naturally cooled and set aside; S5. Preparation of crucian carp, wolfberry and red date soup: clean the fresh crucian carp, put it into an oil pan and fry until both sides are golden brown, remove the core of the red dates, put them into a casserole, add 500-800 ml of boiling water, add wolfberry, crucian carp, red dates and wolfberry in a mass ratio of 35:1:0.5, cook over high heat for 10 minutes, then cook over low heat for 15-20 minutes, remove the crucian carp, red dates and wolfberry after cooking, collect the crucian carp soup, remove the fish bones, and beat the crucian carp meat, red dates and wolfberry into meat paste for later use; S6. The donkey-hide gelatin characteristic peptides P1 and P5 prepared in step S2 are mixed in a mass ratio of 1:1 and mixed with the concentrated solution in step S4 and the crucian carp soup and meat paste in step S5 in proportion to obtain donkey-hide gelatin peptide paste for postpartum repair. The donkey-hide gelatin peptide paste is vacuum-packed in units of 5 g.

3. The method for preparing the donkey-hide gelatin characteristic peptide ointment according to claim 2, characterized in that: The concentrations of the phosphoric acid solution and the sodium bicarbonate solution used in step S1 are 1% and 2%, respectively.

4. The method for preparing the donkey-hide gelatin characteristic peptide ointment according to claim 2, characterized in that: The concentration of the Aspergillus oryzae CICC 2053 bacterial solution prepared in step S3 is 2.5×10 9 cfu / ml.

5. The method for preparing the donkey-hide gelatin characteristic peptide ointment according to claim 2, characterized in that: The concentration of the prepared bacterial solution of Candida utilis AS2.281 in step S3 is 3.5×10 9 cfu / ml.

6. The method for preparing the donkey-hide gelatin characteristic peptide paste according to claim 5, characterized in that: In the step S4, angelica sinensis, codonopsis pilosula, peach kernel, kudzu root and galangal are crushed in the same mass ratio, mixed evenly, and mixed with water in a mass ratio of 1:

2.

7. The method for preparing the donkey-hide gelatin characteristic peptide ointment according to claim 2, characterized in that: In step S4, heating is stopped when the fermentation liquid is concentrated to half of its mass.

8. The method for preparing the donkey-hide gelatin characteristic peptide ointment according to claim 2, characterized in that: In the step S6, the mass ratio of donkey-hide gelatin characteristic peptide: traditional Chinese medicine concentrate: crucian carp soup: meat paste is 3:2:3:

2.

9. The donkey-hide gelatin characteristic peptide ointment prepared by the preparation method of the donkey-hide gelatin characteristic peptide ointment according to any one of claims 2 to 8.

10. Use of the donkey-hide gelatin characteristic peptide paste according to claim 9 for preparing postpartum repair products.

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