Anti-cracking cream containing sheep placenta cell extract and preparation process of anti-cracking cream

Abstract

The invention belongs to the technical field of biology, and discloses anti-cracking cream containing sheep placenta cell extract and a preparation process of the anti-cracking cream. Through an optimized raw material treatment process, a composite enzymolysis technology, a formula synergistic interaction and a quality control system, active small molecule peptides in sheep placentas are efficiently extracted, and a stable and uniform emulsion product is prepared by adopting a proper emulsification process, so that the skin antioxidant, moisturizing and anti-cracking repairing effects are remarkably improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to an anti-cracking cream containing sheep placenta cell extract and a preparation process thereof. Background Art

[0002] There are many types of anti-cracking creams on the market, but they all have problems such as poor penetration of active ingredients, insufficient antioxidant and moisturizing properties. As a natural biomaterial, sheep placenta contains a rich amount of small molecule active peptides in its cell extracts, which can exert various biological activities such as antioxidant and cell repair. However, the preparation process of sheep placenta peptides still has the following bottlenecks:

[0003] (1) High requirements for raw material collection and sterility: Sheep placenta must be collected within a short period of time after delivery and operated under sterile conditions to prevent microbial contamination and enzymatic degradation, which places strict requirements on collection, transportation and preliminary processing.

[0004] (2) Complex extraction process and difficulty in maintaining activity: Sheep placenta contains a variety of bioactive ingredients, but the extraction and refining process is extremely complex, requiring multiple steps such as mechanical crushing, low-temperature freeze-thaw, centrifugation, ultrafiltration, and chromatographic separation. Each step may destroy the active ingredients, resulting in a decrease in the activity of the final product. In order to retain the active ingredients to the greatest extent, the entire process must be carried out under low-temperature and sterile conditions, which places extremely high demands on equipment and process control.

[0005] (3) Difficulty in large-scale production and high costs: Due to the complex process and stringent environmental requirements, most of the current production can only be completed under laboratory conditions, making it difficult to achieve industrial mass production, resulting in high costs. Even if the extraction is successful, the stability of the active substances is poor. Purified sheep placenta extract can only be stored for a few months at most under extremely harsh conditions, making it unsuitable for long-distance transportation or large-scale circulation.

[0006] (4) Insufficient market recognition and scientific basis: Although sheep placenta extract is often promoted as having anti-aging and regeneration effects, there is currently a lack of unanimously recognized scientific evidence to support these effects. Consumers and the medical community are cautious about this, which further affects the prospects for large-scale promotion. Summary of the Invention

[0007] In order to overcome the shortcomings of the existing technology, the purpose of the present invention is to provide an anti-cracking ointment containing small molecule peptides extracted from sheep placenta cells and its preparation process. High-purity small molecule peptides are obtained through the steps of separation, pretreatment, freeze-thaw, enzymatic hydrolysis, ultrafiltration and other steps of sheep placenta, and an oil-water emulsification process is used to prepare a stable and delicate emulsion structure, finally forming an anti-cracking ointment product with excellent skin penetration, antioxidant and anti-cracking and repair effects.

[0008] To achieve the above objectives, the present invention provides the following technical solutions:

[0009] 1. Preparation process of sheep placenta cell extract

[0010] Mechanical separation: fresh placenta collected from healthy ewes within 1 hour after delivery was selected, and the surface amniotic membrane and fascia tissue were peeled off with a scalpel;

[0011] Chemical sterilization: Soak the placenta in 0.1% peracetic acid solution for 10 minutes to inactivate surface microorganisms;

[0012] Low-temperature homogenization: Cut the placenta into 1 cm³ pieces and add PBS buffer (pH 7.4) containing 1 mM PMSF and 1 mM EDTA at a weight ratio of 1:5 in a pre-cooled sterile homogenizer. Homogenize at 8000 rpm for 3-5 minutes to fully release the cellular contents.

[0013] Freeze-thaw cycle membrane disruption technology: freeze the homogenate at -80℃ for 30 minutes, thaw in ice water bath for 30 minutes, repeat 5 times to completely destroy the cell membrane and organelle membrane structure;

[0014] Composite enzymatic hydrolysis system: Trypsin and papain are compounded, and the total enzyme addition amount is 0.5%~2.0% of the protein amount;

[0015] Dynamic pH control: The pH of the reaction system is controlled at 7.0-8.0, and the pH is adjusted every 30 minutes through online monitoring with an error of ≤±0.1;

[0016] Gradient temperature reaction: the enzymatic hydrolysis temperature is 35.5~37°C to explore the optimal enzymatic hydrolysis efficiency;

[0017] Inactivation of hydrolase: After the enzymatic hydrolysis is completed, heat at 95℃ for 10 minutes to inactivate the enzyme.

[0018] Small molecule peptide purification: Ultrafiltration (5 kDa cut-off membrane) combined with dialysis (1 kDa dialysis membrane) removes large molecular impurities and salts to obtain small molecule peptide extracts with a molecular weight of less than 5 kDa.

[0019] 2. Quality Control

[0020] Extraction rate calculation: The protein concentration of the stock solution was determined by BCA method, and the extraction rate was ≥70%;

[0021] Antioxidant activity standard: ABTS free radical scavenging rate ≥ 73%;

[0022] 3. Formula and preparation process of anti-cracking cream

[0023] Core formula design: The oil phase ingredients are jojoba oil (5%), polysorbate (3%), vitamin E (1%), and the water phase ingredients are hyaluronic acid (0.2%), sheep placenta small molecule peptide extract (0.8%~1.6%), potassium sorbate (0.1%;

[0024] The key steps of the preparation process are as follows: the oil phase is heated to 40°C for melt mixing, the water phase is heated to the same temperature to avoid demulsification caused by temperature difference, the water phase is slowly injected into the oil phase at a flow rate of 0.5 mL / s, and pre-emulsification is performed by mechanical stirring at 500 rpm. The pre-emulsion is treated with a high-pressure homogenizer (40 MPa, 3 cycles) to form a stable emulsion with a particle size of ≤200 nm.

[0025] 4. Efficacy Verification System

[0026] Antioxidant activity verification: The free radical scavenging rate was determined by ABTS method, and the equivalent antioxidant capacity was calculated by comparing with the Trolox standard.

[0027] In vitro efficacy testing: Using a Franz diffusion cell and fresh pig ear skin as a model, the cumulative permeation over 24 hours was measured, with the highest value reaching 124.18 μg / cm².

[0028] Human efficacy test: Randomized double-blind controlled trial, experimental group (containing 1.4% small molecule peptide) vs. control group (vitamin E lotion); test period 30 days, test indicators include skin moisture value, elasticity and firmness.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention provides an anti-cracking cream containing sheep placenta cell extract and a preparation process thereof, which comprises:

[0031] 1. Raw material processing technology: Through the three-step method of "mechanical separation + double sterilization + low-temperature homogenization", the active ingredients of the placenta are retained to the maximum extent;

[0032] 2. Composite enzymatic hydrolysis technology: A gradient combination of trypsin and papain, combined with dynamic pH control, increases the extraction rate by 15% to 20%;

[0033] 3. Synergistic formula: Small molecule peptides, hyaluronic acid, and jojoba oil form a synergistic system of "repair-moisturizing-penetration";

[0034] 4. Quality Control System: Establish a triple-index standard for extraction rate, antioxidant activity, and skin penetration.

[0035] It significantly improves the skin's antioxidant, moisturizing and crack-proof repair effects, outperforms existing technologies in performance, and has broad prospects for clinical application and market promotion. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with specific embodiments. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0037] Example 1

[0038] Sheep placenta separation and pretreatment:

[0039] Fresh placentas were collected from healthy ewes within 1 hour after the lambs were born. The amniotic membrane on the surface of the placenta was mechanically separated and rinsed with physiological saline containing kanamycin (100 U / mL) to remove blood stains. The samples were sterilized by soaking in 0.1% peracetic acid for 10 minutes and then rinsed with sterile water until no residue was left. The samples were immediately transported on ice and stored at 4°C for no more than 24 hours. Under sterile conditions, the outer membrane and fascia on the surface of the placenta were carefully peeled off with a scalpel. The processed placenta was cut into approximately 1 cm 3 Place the small pieces into a pre-cooled sterile tissue homogenizer, add 1× PBS buffer (containing 1 mM PMSF and 1 mM EDTA to inhibit protease activity) 5 times the weight of the placenta, and grind thoroughly for about 3-5 minutes to release the cells.

[0040] Table 1 Solution formula

[0041]

[0042] Example 2

[0043] Extraction of small molecule peptides:

[0044] The homogenized mixture was aliquoted into sterile centrifuge tubes and frozen at -80°C for 30 minutes. Immediately thaw in an ice-water bath for 30 minutes. Repeat the freeze-thaw cycle five times to disrupt the cell membrane and fully release the contents. The frozen-thawed samples were centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was collected and filtered through a 0.45 μm sterile filter to remove residual cell debris and large particulate matter. This supernatant was used as the stock solution for subsequent small peptide extraction. The protein concentration of the stock solution was determined by spectrophotometry and adjusted to 10 mg / mL.

[0045] For the protease, trypsin or papain should be weighed and dissolved in an appropriate amount of PBS. Add the enzyme to the pretreatment solution and mix gently. The enzyme concentration should be 1% to 3% of the protein concentration. Place the mixed reaction system in a thermostatic shaker at approximately 100 rpm for 2 to 4 hours. Measure the pH every 30 minutes during the reaction and adjust it with 0.1 M HCl or NaOH to maintain a stable pH. After the reaction, quickly heat the sample to 95°C for 10 minutes to completely inactivate the enzyme. Then, quickly cool the sample to room temperature in an ice bath.

[0046] Table 2 Conditions for extracting the stock solution of small molecule peptides by enzymatic hydrolysis

[0047]

[0048] The cooled sample was centrifuged at 10,000 g for 10 minutes at 4°C. The supernatant was collected and loaded into an ultrafiltration device. Ultrafiltration was performed using a 5 kDa cutoff membrane at a transmembrane pressure of 1–2 bar to collect small peptides <5 kDa. The sample was then dialyzed for 12 hours using a 1 kDa cutoff dialysis membrane to further remove salt and small molecule impurities.

[0049] Example 3

[0050] Extraction yield verification and quality control:

[0051] The BCA method was used to determine the peptide concentration in each group of samples. Reagent A and reagent B in the BCA protein concentration assay kit were mixed at a volume ratio of 50:1. In a 96-well plate, 25 µL of standard (bovine serum albumin BSA) or sample solution and 200 µL of BCA working solution were added to each well, mixed gently, and placed in a 37°C constant temperature incubator for 30 minutes. After incubation, the absorbance of each well at a wavelength of 562 nm was measured using a microplate reader. The absorbance of each standard and sample was recorded, and a standard curve was drawn with the BSA standard concentration as the horizontal axis and the absorbance as the vertical axis. The average absorbance value corresponding to the sample was substituted into the standard curve equation to calculate its protein concentration. Calculate the yield of small molecule peptide extraction:

[0052]

[0053] Table 3 Extraction yield of small molecule peptides

[0054]

[0055] The antioxidant effect of the small peptides was verified using the ABTS assay. The concentration of all peptides was adjusted to 0.5 mg / mL. Weigh 3.84 g of ABTS powder and dissolve it in 1000 mL of distilled water to prepare a 7 mM ABTS solution. Weigh 0.66 g of potassium persulfate and dissolve it in 1000 mL of distilled water to prepare a 2.45 mM potassium persulfate solution. Mix 50 mL of the ABTS solution with an equal volume of the potassium persulfate solution, then incubate in the dark at room temperature for 12 hours to fully generate ABTS⁺ free radicals. Dilute the ABTS⁺ solution with 1× PBS to an absorbance of approximately 0.70 ± 0.02 at 734 nm. Weigh 2 mg of Trolox and dissolve it in 50 μL of DMSO to prepare a stock solution at a concentration of 40 mg / mL. Then, dilute the solution with saline to prepare Trolox standard solutions at concentrations of 0, 50, 100, 150, 200, 250, and 300 μg / mL. In a 96-well plate, add 10 μL of the standard or sample solution (a blank control is 10 μL of PBS for background subtraction) and 190 μL of ABTS⁺ solution to each well. Mix thoroughly and incubate at room temperature in the dark for 6 minutes to allow the antioxidant components in the sample to fully react with the ABTS⁺ free radicals. Repeat each sample at least three times, and calculate the average and standard deviation. Measure the absorbance of each well at a wavelength of 734 nm using a microplate reader, and calculate the antioxidant inhibition rate of each sample:

[0056]

[0057] Among them, 𝐴0 is the absorbance of the blank control well, 𝐴 sample = is the absorbance of the sample well. Draw a standard curve based on the concentration of the Trolox standard solution and the corresponding inhibition rate. Substitute the inhibition rate of the sample into the standard curve equation to calculate the equivalent Trolox concentration.

[0058] Table 4 Antioxidant effect of small molecule peptides

[0059]

[0060] Example 4

[0061] Preparation of anti-cracking cream:

[0062] Oil phase: jojoba oil 5%, emulsifier polysorbate 3%, stabilizer vitamin E 1%. Use a magnetic stirrer to heat to about 40°C to fully mix the oil phase.

[0063] Aqueous phase: moisturizer hyaluronic acid 0.2%, sheep placenta small molecule peptide extract 0.8%~1.6%, mild preservative potassium sorbate 0.1%, dissolved in deionized water, maintain the aqueous phase temperature at about 40°C for subsequent mixing with the oil phase.

[0064] Slowly add the aqueous phase to the oil phase to form a preliminary emulsion under mechanical stirring. Perform a secondary homogenization process using a homogenizer at below 40°C to ensure a fine, uniform emulsion structure. Gradually cool the emulsion to room temperature while gently stirring to prevent stratification. Finally, recheck the pH and adjust it to 5.5-6.5 using citric acid or sodium hydroxide. Under aseptic conditions, fill the finished emulsion into sterile containers and seal them.

[0065] Example 5

[0066] Skin penetration ability of anti-cracking cream:

[0067] Fresh pig ear skin was used as a model to test the release and skin penetration of small molecule peptides in a Franz diffusion cell. The skin was then removed of excess fat and impurities, rinsed with saline, and cut into 1.5 cm diameter circular pieces that fit within the receptor chamber of the Franz diffusion cell. The prepared skin membrane was mounted on the middle partition of the diffusion cell, with the stratum corneum facing the donor chamber and the inner layer facing the receptor chamber, ensuring a tight fit without air bubbles. The receptor solution consisted of 10% ethanol in 1× PBS, pre-filtered (0.22 μm), degassed, and heated to 35°C. After the receptor solution was added, the diffusion cell was incubated at a constant temperature for 30 minutes to allow the membrane to fully hydrate. A 0.5 g sample of the anti-cracking ointment was evenly applied to the donor chamber on the outside of the membrane, ensuring even coverage of the entire membrane area. A timer was started, and magnetic stirring in the receptor chamber was simultaneously activated to ensure uniform concentration of the receptor solution. 0.5 mL samples were collected from the receptor chamber at 1, 2, 4, 8, 12, and 24 hours. The concentration of small molecule peptides was measured for the receptor fluid samples at each time point, and the cumulative permeation amount of small molecule peptides at each time point was calculated based on the detection data.

[0068] Table 5 Skin penetration ability of small molecule peptides

[0069]

[0070] Example 6

[0071] Determination of anti-cracking performance of anti-cracking paste:

[0072] Ten adults aged 25 to 50 with dry, crack-prone skin were recruited. Individuals with a history of skin diseases or allergies were excluded. All participants had their skin moisture, elasticity, and firmness measured on the back of their hands using a corneometer before the start of the trial. They were randomly assigned to a test group (this product) or a control group (vitamin E lotion). Each participant cleaned their hands and applied the same amount of product to their entire hands three times daily. Skin indicators were retested on the mornings of days 2, 5, 10, and 30 before product application, and the data were recorded.

[0073] Table 6 Average values of skin indicators after using anti-cracking cream

[0074]

[0075] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A process for extracting small molecule peptides from sheep placenta, characterized in that: The following steps are involved: (1) Raw material pretreatment: sterilization, adding PBS buffer, and homogenization to obtain a homogenous solution; (2) Enzymatic extraction: The homogenate solution is frozen and thawed repeatedly to break the membrane, and a composite protease is added for enzymatic hydrolysis. After the reaction, the enzymatic solution is inactivated; (3) Small molecule peptide purification: After centrifugation, the supernatant passes through an ultrafiltration membrane and a dialysis membrane in sequence to collect small molecule peptide solutions with a molecular weight of less than 5 kDa.

2. The extraction process according to claim 1, characterized in that The sterilization treatment in step (1) is specifically as follows: first rinse with physiological saline containing 100 U / mL of kanamycin, then soak in 0.1% peracetic acid for 10 minutes, and rinse with sterile water until no residue is left.

3. The extraction process according to claim 1, characterized in that The composite protease in step (2) is trypsin and papain, which are compounded in a mass ratio of 1:1 to 3:1, and the total amount of enzyme added is 0.5% to 2.0% of the substrate protein mass.

4. The extraction process according to claim 1, characterized in that The enzymatic hydrolysis conditions in step (2) are pH 7.0-8.0, temperature 35.5-37.0° C., and enzymatic hydrolysis time 2-4 hours.

5. The sheep placenta small molecule peptide prepared according to the process of any one of claims 1 to 4, characterized in that: Its molecular weight is less than 5kDa, and its ABTS free radical scavenging rate is ≥73%.

6. An anti-cracking paste, characterized in that: The invention comprises the sheep placenta small molecule peptide according to claim 5, wherein the amount of the small molecule peptide added to the anti-cracking cream is 0.8% to 1.6% (w / w), and is used in conjunction with the following components: Oil phase: jojoba oil 3%-5%, emulsifier 2%-4%, vitamin E 0.5%-1.0%; Aqueous phase: hyaluronic acid 0.1% to 0.3%, potassium sorbate 0.05% to 0.2%, and the balance is deionized water.

7. The anti-cracking paste according to claim 6, characterized in that: The mixing temperature of the oil phase and the water phase is 35-40° C., the homogenization pressure is 30-50 MPa, and the pH in the final product is 5.5-6.

5.

8. The anti-cracking paste according to claim 7, characterized in that: The anti-cracking cream is applied externally to the skin surface to achieve the effects of improving skin moisturizing, anti-cracking and anti-oxidation.

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