High-stability water-in-oil PDRN emulsion and preparation method thereof

Abstract

The present application relates to a kind of high stability water-in-oil PDRN emulsion and its preparation method, belong to the field of biotechnology;The preparation method includes the following steps: (1) PDRN enzymolysis: PDRN is added in DNaseI, supplement metal ion activates enzyme, when fragment enrichment shows <800bp, terminate enzymolysis reaction, obtain crude enzymolysis solution;(2) moderate molecular weight screening: pressurized ultrafiltration crude enzymolysis solution with ultrafiltration membrane, and screen out the target fragment of the permeate of molecular weight in 120-500bp;(3) water phase system optimization: 1,3-propanediol and ubiquinol are added in the permeate;(4) semi-continuous feeding gradient emulsification.The present application is by precise control PDRN molecular weight to take into account stability and solubility, optimize water phase component to realize system viscosity and transparency double standard, cooperate gradient emulsification to guarantee water-in-oil system stability, significantly improve product comprehensive quality and application value, with outstanding technical advantages.

Description

Technical Field

[0001] This invention relates to a highly stable water-in-oil PDRN emulsion and its preparation method, belonging to the field of biotechnology. Background Technology

[0002] Polydeoxyribonucleotide (PDRN) is a biomolecule with important biological activities and has shown broad application prospects in many fields such as biomedicine, cosmetics, and regenerative medicine. It has excellent properties such as promoting cell proliferation, repairing damaged tissues, anti-inflammatory and antioxidant effects, and has therefore attracted widespread attention in the industry.

[0003] However, in the actual preparation and application of PDRN, existing technical solutions still face many key technical bottlenecks that urgently need to be addressed, severely limiting their industrialization and the improvement of their application effects. Specific problems are as follows:

[0004] First, PDRN exhibits extremely poor system stability. As PDRN is a biological macromolecule carrying a large number of negative charges in its molecular structure, this characteristic easily leads to stratification in traditional emulsion systems. Even after homogenization, the system's transparency remains poor, failing to meet the appearance and stability requirements of many applications. Furthermore, PDRN's slight solubility in water makes it prone to precipitation in aqueous solutions, further exacerbating system instability and affecting product shelf life and reliability.

[0005] Secondly, PDRN has a low transdermal absorption efficiency. PDRN has a wide molecular weight distribution and is insoluble in lipids. The skin barrier function has a strict selectivity for the penetration of substances. These characteristics make it difficult for PDRN to effectively penetrate the skin barrier and enter the target tissue to exert its effects, which greatly limits its application in cosmetics, topical drugs and other fields.

[0006] Finally, the traditional preparation process of PDRN has significant drawbacks. Existing preparation methods generally suffer from high energy consumption, which is inconsistent with the current trend of green production. At the same time, the process has poor repeatability, resulting in large fluctuations in product quality and making it difficult to achieve large-scale, standardized industrial production. This further restricts the market promotion and industrial development of PDRN-related products.

[0007] In summary, given the problems of poor system stability, low transdermal absorption efficiency, and complex preparation process of PDRN in the existing technology, there is an urgent need to develop a technical solution that can solve the above defects, so as to improve the application performance of PDRN and promote the healthy development of its related industries. This has become a technical problem that needs to be solved by those skilled in the art. Summary of the Invention

[0008] The purpose of this invention is to provide a highly stable water-in-oil PDRN emulsion and its preparation method to solve the technical problems existing in the prior art as described above.

[0009] The technical solution provided by this invention is as follows:

[0010] One objective of this invention is to provide a method for preparing a highly stable water-in-oil PDRN emulsion, comprising the following steps:

[0011] (1) PDRN enzymatic hydrolysis: DNaseI is added to PDRN, and then metal ion activating enzyme is added. The reaction is carried out at pH 6.0-8.0 and temperature 37-40℃ for 0.5-2h. Samples are taken every 10min to analyze the distribution of nucleic acid fragments. When the fragment enrichment shows <800bp, the enzymatic hydrolysis reaction is terminated to obtain crude enzymatic hydrolysate.

[0012] (2) Screening for appropriate molecular weight: Ultrafiltration of crude enzyme digest under pressure using an ultrafiltration membrane to screen permeate the target fragment with a molecular weight of 120-500bp;

[0013] (3) Optimization of aqueous system: 1,3-propanediol and panthenol were added to the permeate screened in step (2);

[0014] (4) Semi-continuous gradient emulsification: The optimized aqueous phase system is dripped into the oil phase system in a semi-continuous gradient feeding manner.

[0015] Based on the above technical solution, the present invention can be further improved as follows:

[0016] Further, in step (1), the ratio of DNaseI to PDRN is (1-10U):(200-500mg), and the metal ion activating enzyme is Mg. 2+ Activate enzymes.

[0017] Furthermore, the Mg 2+ The amount of activating enzyme added is 0.1-4 mmol / L.

[0018] Furthermore, the Mg 2+ The amount of activating enzyme added was 3 mmol / L.

[0019] Further, in step (1), the termination reaction is achieved by heating to 65°C and adding 0.5 mol / L EDTA solution to terminate the reaction.

[0020] Further, the specific steps for screening the appropriate molecular weight in step (2) are as follows: First, use an ultrafiltration membrane with a molecular weight cutoff of 500 bp to perform pressure ultrafiltration on the crude enzymatic hydrolysate, retaining fragments with a molecular weight greater than 500 bp and collecting the permeate rich in small molecular fragments of ≤500 bp; Second, use an ultrafiltration membrane with a molecular weight cutoff of 120 bp to perform secondary ultrafiltration on the permeate collected in the first step, retaining the target fragments with a molecular weight between 120-500 bp and discarding the permeate with a molecular weight less than 120 bp.

[0021] Further, in step (3), the mass percentage of 1,3-propanediol is 1-3%, and the mass percentage of panthenol is 2-10%.

[0022] Furthermore, the specific steps of the semi-continuous feeding gradient emulsification in step (4) are as follows: the aqueous phase liquid is dripped into the oil phase in a semi-continuous feeding manner; the emulsification process is maintained at a constant temperature of 80°C throughout, and the aqueous phase flow rate, cumulative addition amount and stirring speed are adjusted in time segments according to the following parameters:

[0023] 0-20min: Control the flow rate of the aqueous phase to be 15mL / min. The volume of the aqueous phase added during this stage accounts for 15% of the total volume of the aqueous phase. The stirring speed is 1500rpm.

[0024] 21-40 min: Adjust the flow rate of the aqueous phase to 10 mL / min. After this stage of addition is completed, the cumulative volume of the aqueous phase accounts for 28% of the total volume of the aqueous phase used. Increase the stirring speed to 3000 rpm.

[0025] 41 min until the emulsification process ends: reduce the flow rate of the aqueous phase to 6 mL / min, add the remaining aqueous phase dropwise, adjust the stirring speed to 5000 rpm and maintain it until the end.

[0026] The second objective of this invention is to provide a highly stable water-in-oil PDRN emulsion, which is prepared using the preparation method described above.

[0027] The technical solution provided by this invention has the following advantages compared with the prior art:

[0028] 1. This invention adopts a "segmented enzymatic hydrolysis + real-time monitoring" strategy, which directionally regulates the molecular weight of PDRN within the <800bp range through enzymatic hydrolysis. This specific molecular weight range is the core process parameter, which can stabilize the molecular structure of PDRN to ensure its stability on the one hand, and significantly improve the solubility of PDRN (by about 30%) on the other hand, inhibiting the precipitation of PDRN at the molecular level and solving the problem that solubility and stability are difficult to balance in traditional processes.

[0029] 2. This invention reduces the amount of 1,3-propanediol to 1-3% and adds panthenol (2-10%). Panthenol imparts suitable viscosity to the homogenized solution, ensures the dispersion stability of the system, and can precisely control the refractive index of the water-in-oil system, ultimately achieving a clear and transparent system with a transmittance of >90%, thus improving the appearance and performance of the product.

[0030] 3. This invention innovatively adopts a semi-continuous feeding gradient emulsification process, which is different from the traditional constant shear force emulsification method. Through the synergistic operation of semi-continuous feeding of the aqueous phase and continuous increase of shear force, the aqueous phase can be more fully and uniformly dispersed in the oil phase, effectively refining the dispersed particle size, improving the emulsification stability of the water-in-oil system, significantly reducing the probability of system stratification and demulsification, ensuring the consistency of product quality between batches, and improving the product qualification rate. Attached Figure Description

[0031] Figure 1 This is a diagram showing the nucleic acid analysis results after PDRN enzymatic digestion according to the present invention.

[0032] Figure 2 The images show the agarose gel electrophoresis results after PDRN molecular weight screening in Examples 1 and 2 of this invention.

[0033] Figure 3 This is the result of improved solubility of PDRN molecular weight after enzymatic enrichment.

[0034] Figure 4 The image shows a comparison of the improved transmittance of the water-in-oil systems in Examples 1 and 2 of this invention after the introduction of panthenol.

[0035] Figure 5 The figure shows the particle size stability test results of the PDRN emulsion under normal temperature storage and accelerated testing in Example 1 of the present invention. Detailed Implementation

[0036] The principles and features of the present invention are described below with reference to examples. The examples are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0037] PDRN comes from Ruijiming (Shandong) Biotechnology Co., Ltd.;

[0038] DNaseI was purchased from Shanghai Yuanye Biotechnology Co., Ltd., LOT: 15Y749669.

[0039] Example 1

[0040] A method for preparing a highly stable water-in-oil PDRN emulsion includes the following steps:

[0041] (1) PDRN enzymatic hydrolysis: DNase I was added to PDRN (the ratio of DNase I to PDRN was 1U:300mg), and then 3mmol / L Mg was added. 2+ The enzyme was activated and reacted at pH 7.5 and 37℃ for 1 hour. Samples were taken every 10 minutes. After sampling, the sample was heated to 65℃ and 0.5 mol / L EDTA solution was added to terminate the reaction, resulting in an enzyme hydrolysate. Finally, multiple crude enzyme hydrolysates were analyzed. When the fragment enrichment showed less than 800 kDa, the enzyme hydrolysis conditions of that hydrolysate were recorded and used as the final conditions for preparing the enzyme hydrolysate.

[0042] (2) Screening for appropriate molecular weight: First, use an ultrafiltration membrane with a molecular weight cutoff of 500 bp (about 150 kDa) to perform pressure ultrafiltration on the crude enzymatic hydrolysate to retain fragments with a molecular weight greater than 500 bp and collect the permeate rich in small molecular fragments of ≤500 bp; Second, use an ultrafiltration membrane with a molecular weight cutoff of 120 bp (about 90 kDa) to perform secondary ultrafiltration on the permeate collected in the first step to retain the target fragments with a molecular weight between 120-500 bp and discard the permeate with a molecular weight less than 120 bp.

[0043] (3) Optimization of aqueous system: 3% by mass of 1,3-propanediol and 5% by mass of panthenol were added to the permeate screened in step (2);

[0044] (4) Oil phase preparation: Add caprylic / capric triglyceride to the oil phase storage tank, start stirring at 120 rpm / min; add sorbitan oleate, mix the two for 10 min; after mixing, reduce the speed to 80 rpm / min, slowly add sorbitan isostearate, mix for 15 min, then raise the temperature to 80℃, and maintain the temperature and stirring speed throughout the process.

[0045] (5) Semi-continuous feeding gradient emulsification: The aqueous phase storage tank is connected by a 5mm diameter dropper, and the aqueous phase liquid is stably dripped into the oil phase in a bead-like manner to achieve semi-continuous feeding; the emulsification process is maintained at a constant temperature of 80℃ throughout, and the aqueous phase flow rate, cumulative dosage and stirring speed are adjusted in time segments. The specific parameters are as follows:

[0046] ① 0-20 min: Control the flow rate of the aqueous phase to be 15 mL / min. The volume of the aqueous phase added during this stage accounts for 15% of the total volume of the aqueous phase. The stirring speed is set to 1500 rpm.

[0047] ② 21-40 min: Adjust the flow rate of the aqueous phase to 10 mL / min. After this stage of dripping is completed, the cumulative volume of the aqueous phase accounts for 28% of the total volume of the aqueous phase. Increase the stirring speed to 3000 rpm.

[0048] ③ 41 min until the end of the emulsification process: reduce the flow rate of the aqueous phase to 6 mL / min, slowly add the remaining aqueous phase, adjust the stirring speed to 5000 rpm and maintain it until the end;

[0049] (6) Stirring, cooling and filling to obtain a highly stable water-in-oil PDRN emulsion.

[0050] Example 2

[0051] The difference from Example 1 is that in step (3), 1,3-propanediol and panthenol with a mass percentage of 1% and 2% respectively are added.

[0052] Example 3

[0053] The difference from Example 1 is that in step (3), 2% by mass of 1,3-propanediol and 10% by mass of panthenol are added.

[0054] Comparative Example 1

[0055] The difference from Example 1 is that in step (5), the aqueous phase is added to the oil phase in one step, and then homogenization is performed.

[0056] Comparative Example 2

[0057] The difference from Example 1 is that in step (3), only 5% panthenol by mass is added.

[0058] Comparative Example 3

[0059] The difference from Example 1 is that in step (3), 5% by mass of 1,3-propanediol and 5% by mass of panthenol are added.

[0060] Comparative Example 4

[0061] The difference from Example 1 is that in step (3), 1,3-propanediol is replaced with the same mass of 1,2-propanediol.

[0062] test:

[0063] The PDRN emulsions of Examples 1-3 and Comparative Examples 1-4 were subjected to stability and transmittance tests. The test results are shown in Table 1.

[0064] 1. Stability Testing: The stability of the water-in-oil PDRN emulsion was tested at low temperature (4℃) and high temperature (50℃, 60% relative humidity). Sealed HDEP bottles (10mL / bottle) were used as the test packaging, and a comprehensive pharmaceutical stability test chamber was used as the testing instrument. Samples were dispensed into HDEP bottles at a rate of 20g / bottle. The water-oil equilibrium stability of the samples was observed at 0, 5, 10, 20, and 30 days. The absorbance at 400nm was measured, and the particle size difference of the PDRN emulsion at 0 and 30 days was also measured.

[0065] 2. Transmittance test: The transmittance of the sample was measured at 400nm using an ultraviolet spectrophotometer. The oil phase was used as a reference for zeroing. The transmittance of the stable sample at this wavelength was measured.

[0066] Table 1 Test Results

[0067]

[0068] As shown in Table 1, in Examples 1-3, the amounts of panthenol and 1,3-propanediol were within the specified range. The emulsions maintained stable water-oil equilibrium for 30 days under both low temperature (4°C) and high temperature (50°C, 60% relative humidity) conditions, which was superior to Comparative Examples 1-4. The transmittance at 400 nm was greater than 90% in all examples, significantly better than that of Comparative Examples 1-4. This indicates that the water-in-oil PDRN emulsion prepared by this invention has excellent stability and transmittance.

[0069] Figure 1 This is a graph showing the nucleic acid analysis results after PDRN enzymatic digestion according to the present invention. The results show that the molecular weight of PDRN after enzymatic digestion is less than 800 bp.

[0070] Figure 2 The images show the agarose gel electrophoresis results of the second-step ultrafiltration of PDRN in Examples 1 and 2 of this invention. The results show that after two ultrafiltrations, the molecular weight of PDRN can be controlled between 120-500 bp.

[0071] Figure 3 The results show the improved solubility of PDRN after enzymatic enrichment. The results indicate that after enzymatic hydrolysis and two-step ultrafiltration, the PDRN aqueous solution changed from a gel state to a solution state, and the PDRN solubility increased from 5.8% to 7.5%. Even after an approximately 30% increase in solubility, the PDRN remained in a solution state, facilitating subsequent emulsion preparation in this invention.

[0072] Figure 4 This is a comparison chart showing the improvement in transmittance of the water-in-oil systems in Examples 1 and 2 of this invention after the introduction of panthenol. The results show that the transmittance of the system changed significantly after the introduction of panthenol, with the measured transmittance >90%.

[0073] Figure 5 The figure shows the particle size stability test results of PDRN emulsion under ambient temperature storage and accelerated testing in Example 1 of the present invention. The results show that the particle size of the water-in-oil system did not change significantly after 30 days under accelerated testing conditions of 50±2℃ and 60±5% relative humidity.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a highly stable water-in-oil PDRN emulsion, characterized in that, Includes the following steps: (1) PDRN enzymatic hydrolysis: DNaseI is added to PDRN, and then metal ion activating enzyme is added. The reaction is carried out at pH 6.0-8.0 and temperature 37-40℃ for 0.5-2h. Samples are taken every 10-15min to analyze the distribution of nucleic acid fragments. When the fragment enrichment shows <800bp, the enzymatic hydrolysis reaction is terminated to obtain crude enzymatic hydrolysate. (2) Screening for appropriate molecular weight: Ultrafiltration of crude enzyme digest under pressure using an ultrafiltration membrane to screen permeate the target fragment with a molecular weight of 120-500bp; (3) Optimization of aqueous system: 1,3-propanediol and panthenol are added to the permeate screened in step (2); the mass percentage of 1,3-propanediol is 1-3% and the mass percentage of panthenol is 2-10%. (4) Oil phase preparation: Add caprylic / capric triglyceride to the oil phase storage tank, start stirring at 120 rpm / min; add sorbitan oleate, mix the two for 10 min; after mixing, reduce the speed to 80 rpm / min, slowly add sorbitan isostearate, mix for 15 min, then raise the temperature to 80℃, and maintain the temperature and stirring speed throughout the process. (5) Semi-continuous feeding gradient emulsification: The aqueous phase liquid is dripped into the oil phase in a semi-continuous feeding manner; the emulsification process is maintained at a constant temperature of 80℃ throughout, and the flow rate of the aqueous phase, the cumulative dosage and the stirring speed are adjusted in time segments. The specific parameters are as follows: 0-20min: Control the flow rate of the aqueous phase to be 15mL / min. The volume of the aqueous phase added during this stage accounts for 15% of the total volume of the aqueous phase. The stirring speed is 1500rpm. 21-40 min: Adjust the flow rate of the aqueous phase to 10 mL / min. After this stage of addition is completed, the cumulative volume of the aqueous phase accounts for 28% of the total volume of the aqueous phase used. Increase the stirring speed to 3000 rpm. 41 min until the emulsification process ends: reduce the flow rate of the aqueous phase to 6 mL / min, add the remaining aqueous phase dropwise, adjust the stirring speed to 5000 rpm and maintain it until the end.

2. The method for preparing a highly stable water-in-oil PDRN emulsion according to claim 1, characterized in that, In step (1), the ratio of DNaseI to PDRN is (1-10U):(200-500mg), and the metal ion activating enzyme is Mg. 2+ Activate enzymes.

3. The method for preparing a highly stable water-in-oil PDRN emulsion according to claim 2, characterized in that, The Mg 2 + The amount of activating enzyme added is 0.1-4 mmol / L.

4. The method for preparing a highly stable water-in-oil PDRN emulsion according to claim 1, characterized in that, In step (1), the termination reaction is achieved by heating to 65°C and adding 0.5 mol / L EDTA solution.

5. The method for preparing a highly stable water-in-oil PDRN emulsion according to claim 1, characterized in that, The specific steps for screening the appropriate molecular weight in step (2) are as follows: First, use an ultrafiltration membrane with a molecular weight cutoff of 500 bp to perform pressure ultrafiltration on the crude enzymatic hydrolysate, retaining fragments with a molecular weight greater than 500 bp and collecting the permeate rich in small molecular fragments of ≤500 bp; Second, use an ultrafiltration membrane with a molecular weight cutoff of 120 bp to perform secondary ultrafiltration on the permeate collected in the first step, retaining the target fragments with a molecular weight between 120-500 bp and discarding the permeate with a molecular weight less than 120 bp.

6. A highly stable water-in-oil PDRN emulsion, characterized in that, It is prepared by the preparation method described in any one of claims 1-5.

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