Preparation method of a polydeoxyribonucleotide natural polymer polysaccharide composite solution

Abstract

The present invention relates to the field of composite of polymer polysaccharide materials, and particularly relates to a preparation method of a polydeoxyribonucleotide natural polymer polysaccharide composite solution, which comprises the following steps: dissolving polydeoxyribonucleotide to obtain solution 1; dissolving natural polymer polysaccharide to obtain solution 2; mixing solution 1 and solution 2 under water bath conditions, and after mixing evenly, performing gradient programmed cooling at a cooling rate of less than 2 °C / min, and obtaining a polydeoxyribonucleotide natural polymer polysaccharide composite solution with clear appearance and uniform layers after cooling to room temperature; the present invention can avoid quality phenomena such as turbidity of the composite solution, uneven layers, uneven content during product filling, and unqualified clarity; the product quality of the polydeoxyribonucleotide natural polymer polysaccharide composite solution of the present invention is relatively good.

Description

Technical Field

[0001] The present invention relates to the field of polymer polysaccharide material composites, and particularly to a method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution. Background Art

[0002] Polysaccharides are widely present in nature and are natural macromolecules with diverse functions. Polysaccharides extracted from natural animals, plants, and fungi have various biological activities such as antioxidant and anti-tumor effects, and can be used as additives in the food industry, biomedicine, and other fields. They are natural substances with extremely wide applications. Generally speaking, polysaccharides are composed of multiple monosaccharide molecules connected by glycosidic bonds. They have long molecular chains, large degrees of polymerization, and most of the branches carry hydroxyl groups, making it easy to form intermolecular hydrogen bonds. This special structure results in poor solubility in water. The higher the molecular weight of the polysaccharide, the worse its solubility in water, and some polysaccharides are even insoluble in water.

[0003] Generally, different polymer sizes of polysaccharides produce different physicochemical properties and may lead to different therapeutic effects. Some studies have pointed out that polysaccharide molecules with lower molecular weights have a greater impact on accelerating epidermal renewal, while molecules with higher molecular weights are more effective in supporting fibroblasts. For example, low-molecular-weight HA stimulates keratinocytes to produce CD44, resulting in epidermal hyperplasia and differentiation, while medium- and high-molecular-weight HA are more effective in improving skin hydration.

[0004] Therefore, it is inferred that different molecular weights of polydeoxyribonucleotides will also affect their physiological effects in the skin. Polydeoxyribonucleotides are PDRN / PN polymers, and the units composing the polymers are deoxyribonucleotides. PDRN is strictly used to specifically describe short-chain and medium-long-chain deoxyribonucleotides, while PN is used to refer to longer-chain deoxyribonucleotides. All polydeoxyribonucleotides may potentially be able to stimulate skin regeneration because they are naturally locally decomposed by endogenous nucleases. Some clinical studies have confirmed the effectiveness of PDRN in skin regeneration and the clinical benefits of PN. Polydeoxyribonucleotides are called hydrophilic polymers and can absorb water. From a biological perspective, the entire surface of the DNA molecule is covered by water, and water interacts with phosphate groups through hydrogen bonds. Longer DNA fragments (i.e., PN) will have a higher hydration capacity than shorter polymers (i.e., PDRN), thus showing superior properties in restoring skin water balance. In addition, another advantage of PN compared to PDRN is that it has a longer residence time in the skin, which means that PN will promote a longer-lasting skin stimulation effect, potentially enhancing the benefits for patients.

[0005] In contrast, PN has a high molecular weight and hydrophilicity, making it more suitable for injection into the skin to restore lost volume. However, due to these two characteristics, PN is not suitable for formulating solutions with too high a concentration, nor for compounding with other polysaccharide materials, as it is prone to phenomena such as turbidity and uneven layers. A composite solution of PDRN-sodium hyaluronate, its preparation method and application disclosed in Patent CN118845531A dissolve polyribonucleotide under water bath conditions to increase its solubility, but turbidity also occurs at room temperature. Summary of the Invention

[0006] In order to solve the technical problems of unqualified clarity and uneven layers of the composite solution containing polyribonucleotide, a preparation method of a polydeoxyribonucleotide natural high molecular polysaccharide composite solution is provided. In the present invention, polydeoxyribonucleotide and natural high molecular polysaccharide are separately dissolved, mixed under water bath conditions, and then a polydeoxyribonucleotide natural high molecular polysaccharide composite solution with clear appearance and uniform layers is obtained by slowly cooling.

[0007] To achieve the above object, the present invention is realized through the following technical solutions:

[0008] A preparation method of a polydeoxyribonucleotide natural high molecular polysaccharide composite solution, comprising the following steps:

[0009] S1. Dissolve polydeoxyribonucleotide to obtain Solution 1;

[0010] S2. Dissolve natural high molecular polysaccharide to obtain Solution 2;

[0011] S3. Mix Solution 1 and Solution 2 under water bath conditions. After mixing evenly, perform gradient program cooling at a cooling rate of less than 2°C / min. After cooling to room temperature, a polydeoxyribonucleotide natural high molecular polysaccharide composite solution with clear appearance and uniform layers is obtained. By controlling the cooling rate to cool slowly, polydeoxyribonucleotide can retain its double-stranded structure to a great extent to maintain better drug efficacy. The solution of the present invention can avoid quality phenomena such as turbidity, uneven layers, uneven content during product filling, and unqualified clarity of the composite solution.

[0012] Further, the cooling rate in S3 is in the range of 0.18°C / min - 1°C / min.

[0013] Further, the water bath conditions in S3 are 40°C - 80°C;

[0014] Further, the mixing time of Solution 1 and Solution 2 under the water bath conditions is 0.5h - 4h.

[0015] Further, the mass concentration of the solution 1 is 1 g / L - 40 g / L; the mass concentration of the solution 2 is 1 g / L - 20 g / L; the solution 1 and the solution 2 are mixed in any volume ratio.

[0016] Further, the natural polymer polysaccharide is selected from one or more of polydeoxyribonucleotide, hyaluronic acid, cellulose, and chitosan; the solvents in the solution 1 and the solution 2 are both injection water.

[0017] Further, the molecular weight of the natural polymer polysaccharide is 200 kDa - 2000 kDa; the molecular weight of the polydeoxyribonucleotide is 400 kDa - 2000 kDa.

[0018] Beneficial technical effects:

[0019] By separately dissolving polydeoxyribonucleotide and natural polymer polysaccharide, mixing them under water bath and then slowly cooling, the present invention can solve the problems of turbidity and uneven layer in the polydeoxyribonucleotide solution, especially for high molecular weight and high concentration of PN; this is because if the cooling rate is too fast, turbidity and precipitation are likely to occur, and the uniformity and clarity of the product are likely to be unqualified during filling, which leads to the difficulty in restoring the double-stranded structure of polydeoxyribonucleotide after high-temperature sterilization and the difficulty in exerting the original drug effect. Description of the drawings

[0020] Figure 1 is the process flow chart of the polydeoxyribonucleotide natural polymer polysaccharide composite solution of the present invention;

[0021] Figure 2 are the physical appearance photos of Examples 1 - 6 and Comparative Examples 1 - 2;

[0022] Figure 3 are the light transmittance detection results of Examples 1 - 6 and Comparative Examples 1 - 2. Specific embodiments

[0023] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. The description of at least one exemplary embodiment is actually only illustrative and in no way restrictive of the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0024] Unless otherwise specifically stated, the numerical values set forth in these embodiments do not limit the scope of the present invention. Technologies and methods known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, such technologies and methods should be regarded as part of the specification. In all examples shown and discussed herein, any specific values should be construed as merely exemplary and not as limiting. Accordingly, other examples of the exemplary embodiments may have different values.

[0025] For the experimental methods without specific conditions noted in the following examples, they are generally determined according to national standards; if there are no corresponding national standards, they are carried out according to general standard requirements or general methods.

[0026] The following molecular weight is the weight-average molecular weight.

[0027] Example 1

[0028] A method for preparing a polydeoxyribonucleotide natural high-molecular polysaccharide composite solution, comprising the following steps:

[0029] S1. Accurately weigh 2.0 g of polydeoxyribonucleotide (molecular weight 430 kDa), add 100 mL of injection water, and stir at room temperature for 6 h to obtain Solution 1;

[0030] S2. Accurately weigh 2.0 g of sodium hyaluronate (molecular weight 1200 kDa), add 100 mL of injection water, and stir at room temperature for 12 h to obtain Solution 2;

[0031] S3. Under the condition of a 40°C water bath, mix equal volumes of Solution 1 and Solution 2, and stir and mix for 2 h; then cool to 20°C (taking 60 min) at a rate of decreasing 10°C every 30 min (cooling rate about 0.33°C / min) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0032] The composite solution in this case is filled into a glass pre-filled syringe, and the appearance of the composite solution is observed under natural light. The physical appearance photo is as Figure 2 shown, and it can be seen that the composite solution is clear and transparent and has a uniform layer at room temperature.

[0033] Example 2

[0034] A method for preparing a polydeoxyribonucleotide natural high-molecular polysaccharide composite solution, comprising the following steps:

[0035] S1. Accurately weigh 2.0 g of polydeoxyribonucleotide (molecular weight 430 kDa), add 100 mL of injection water, and stir at room temperature for 6 h to obtain Solution 1;

[0036] S2. Accurately weigh 2.0 g of sodium hyaluronate (molecular weight 1200 kDa), add 100 mL of water for injection, and stir at room temperature for 12 h to obtain Solution 2;

[0037] S3. Under the condition of a 60 °C water bath, mix equal volumes of Solution 1 and Solution 2, and stir for 2 h; then cool to 20 °C at a rate of 10 °C drop every 30 min (cooling rate about 0.33 °C / min) (taking 120 min) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0038] The composite solution in this case was filled into a glass pre-filled syringe, and the appearance of the composite solution was observed under natural light. The physical appearance photo is as Figure 2 shown. It can be seen that the composite solution is clear and transparent at room temperature and has a uniform layer.

[0039] Example 3

[0040] A preparation method of a polydeoxyribonucleotide-natural high molecular polysaccharide composite solution, comprising the following steps:

[0041] S1. Accurately weigh 2.0 g of polydeoxyribonucleotide (molecular weight 1700 kDa), add 100 mL of water for injection, and stir at room temperature for 6 h to obtain Solution 1;

[0042] S2. Accurately weigh 2.0 g of sodium hyaluronate (molecular weight 1200 kDa), add 100 mL of water for injection, and stir at room temperature for 12 h to obtain Solution 2;

[0043] S3. Under the condition of an 80 °C water bath, mix equal volumes of Solution 1 and Solution 2, and stir for 2 h; then cool to 20 °C at a rate of 10 °C drop every 40 min (cooling rate 0.25 °C / min) (taking 240 min) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0044] The composite solution in this case was filled into a glass pre-filled syringe, and the appearance of the composite solution was observed under natural light. The physical appearance photo is as Figure 2 shown. It can be seen that the composite solution is clear and transparent at room temperature and has a uniform layer.

[0045] Example 4

[0046] A preparation method of a polydeoxyribonucleotide-natural high molecular polysaccharide composite solution, comprising the following steps:

[0047] S1. Accurately weigh 2.0 g of polydeoxyribonucleotide (molecular weight 430 kDa), add 100 mL of water for injection, and stir at room temperature for 6 h to obtain Solution 1;

[0048] S2. Accurately weigh 2.0 g of cellulose (molecular weight 2000 kDa), add 100 mL of water for injection, and stir at room temperature for 12 h to obtain Solution 2;

[0049] S3. Under the condition of a 60 °C water bath, mix equal volumes of Solution 1 and Solution 2, and stir for 2 h; then cool to 20 °C at a rate of 10 °C drop every 30 min (cooling rate is about 0.33 °C / min, taking 120 min) to obtain a polydeoxyribonucleotide-cellulose composite solution.

[0050] The composite solution in this case is filled into a glass pre-filled syringe, and the appearance of the composite solution is observed under natural light. The physical appearance photo is as Figure 2 shown. It can be seen that since cellulose is insoluble in water, the composite solution is slightly whitish at room temperature, but still clear, transparent, and has uniform layers.

[0051] Example 5

[0052] A method for preparing a polydeoxyribonucleotide-natural high molecular polysaccharide composite solution, comprising the following steps:

[0053] S1. Accurately weigh 2.0 g of polydeoxyribonucleotide (molecular weight 430 kDa), add 100 mL of water for injection, and stir at room temperature for 6 h to obtain Solution 1;

[0054] S2. Accurately weigh 2.0 g of chitosan (molecular weight 200 kDa), add 100 mL of water for injection, and stir at room temperature for 12 h to obtain Solution 2;

[0055] S3. Under the condition of a 60 °C water bath, mix equal volumes of Solution 1 and Solution 2, and stir for 2 h; then cool to 20 °C at a rate of 10 °C drop every 30 min (cooling rate is about 0.33 °C / min, taking 120 min) to obtain a polydeoxyribonucleotide-chitosan composite solution.

[0056] The composite solution in this case is filled into a glass pre-filled syringe, and the appearance of the composite solution is observed under natural light. The physical appearance photo is as Figure 2 shown. It can be seen that the composite solution is clear, transparent, and has uniform layers.

[0057] Example 6

[0058] A method for preparing a polydeoxyribonucleotide-natural high molecular polysaccharide composite solution, comprising the following steps:

[0059] S1. Accurately weigh 2.0 g of polydeoxyribonucleotide (molecular weight 430 kDa), add 100 mL of water for injection, and stir at room temperature for 6 h to obtain Solution 1;

[0060] S2. Accurately weigh 2.0 g of polydeoxyribonucleotide (molecular weight 1700 kDa), add 100 mL of water for injection, and stir at room temperature for 6 h to obtain Solution 2;

[0061] S3. Under the condition of a 60°C water bath, mix equal volumes of Solution 1 and Solution 2, and stir for 45 min; then cool to 20°C at a rate of 10°C decrease every 30 min (cooling rate is about 0.33°C / min, taking 120 min) to obtain a polydeoxyribonucleotide composite solution.

[0062] The composite solution in this case is filled into a glass pre-filled syringe, and the appearance of the composite solution is observed under natural light. The physical appearance photo is as Figure 2 shown. It can be seen that the composite solution is clear, transparent, and has uniform layers at room temperature.

[0063] Example 7

[0064] A method for preparing a polydeoxyribonucleotide natural high-molecular polysaccharide composite solution includes the following steps:

[0065] S1. Accurately weigh 4.0 g of polydeoxyribonucleotide (molecular weight 430 kDa), add 100 mL of water for injection, and stir at room temperature for 6 h to obtain Solution 1;

[0066] S2. Accurately weigh 2.0 g of sodium hyaluronate (molecular weight 1200 kDa), add 100 mL of water for injection, and stir at room temperature for 12 h to obtain Solution 2;

[0067] S3. Under the condition of a 60°C water bath, mix equal volumes of Solution 1 and Solution 2, and stir for 2 h; then cool to 20°C at a rate of 10°C decrease every 30 min (cooling rate is about 0.33°C / min, taking 120 min) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0068] The composite solution in this case is filled into a glass pre-filled syringe, and the appearance of the composite solution is observed under natural light. It can be seen that the composite solution is clear, transparent, and has uniform layers at room temperature.

[0069] Example 8

[0070] A method for preparing a polydeoxyribonucleotide natural high-molecular polysaccharide composite solution includes the following steps:

[0071] S1. Accurately weigh 0.5 g of polydeoxyribonucleotide (molecular weight 600 kDa), add 100 mL of water for injection, and stir at room temperature for 6 h to obtain Solution 1;

[0072] S2. Accurately weigh 0.2 g of sodium hyaluronate (molecular weight 1570 kDa), add 100 mL of water for injection, and stir at room temperature for 12 h to obtain Solution 2;

[0073] S3. Under the condition of a 60 °C water bath, mix equal volumes of Solution 1 and Solution 2, and stir and mix for 4 h; then cool to 20 °C at a rate of 10 °C drop per 30 min (cooling rate about 0.33 °C / min) (taking 120 min) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0074] The composite solution in this case is filled into a glass pre-filled syringe. Observe the appearance of the composite solution under natural light. It can be seen that the composite solution is clear, transparent, and has uniform layers at room temperature.

[0075] Comparative Example 1

[0076] A preparation method of a polydeoxyribonucleotide-natural polymer polysaccharide composite solution includes the following steps:

[0077] S1. Accurately weigh 2.0 g of polydeoxyribonucleotide (molecular weight 430 kDa), add 100 mL of water for injection, and stir at room temperature for 6 h to obtain Solution 1;

[0078] S2. Accurately weigh 2.0 g of sodium hyaluronate (molecular weight 1200 kDa), add 100 mL of water for injection, and stir at room temperature for 12 h to obtain Solution 2;

[0079] S3. Under the condition of a 60 °C water bath, mix equal volumes of Solution 1 and Solution 2, and stir and mix for 2 h; then cool to 20 °C at a rate of 10 °C drop per 10 min (cooling rate 1 °C / min) (taking 40 min) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0080] The composite solution in this case is filled into a glass pre-filled syringe. Observe the appearance of the composite solution under natural light. The physical appearance photo is as Figure 2 shown, and it can be seen that the composite solution becomes turbid at room temperature.

[0081] Comparative Example 2

[0082] A preparation method of a polydeoxyribonucleotide-natural polymer polysaccharide composite solution includes the following steps:

[0083] S1. Accurately weigh 2.0 g of polydeoxyribonucleotide (molecular weight 430 kDa), add 100 mL of water for injection, and stir at room temperature for 6 h to obtain Solution 1;

[0084] S2. Accurately weigh 2.0 g of sodium hyaluronate (molecular weight 1200 kDa), add 100 mL of water for injection, and stir at room temperature for 12 h to obtain Solution 2;

[0085] S3. Under the condition of a 60 °C water bath, mix equal volumes of Solution 1 and Solution 2, and stir for 2 h; then place it in an ice-water bath and cool it to 20 °C at a rate of 20 °C drop every 10 min (cooling rate 2 °C / min) (it takes 20 min), to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0086] The composite solution of this case is filled into a glass pre-filled syringe, and the appearance of the composite solution is observed under natural light. The physical appearance photo is as Figure 2 shown. It can be seen that the composite solution becomes turbid at room temperature, and the turbidity problem is more serious than that of Comparative Example 1.

[0087] Test Example

[0088] 1. Transmittance test

[0089] Dilute the composite solutions of each example and comparative example tenfold with normal saline, and then use normal saline as a blank control to detect the transmittance of the composite solutions of each example and comparative example after tenfold dilution in the wavelength range of 400 nm to 800 nm. The results are shown in Figure 3 and Table 1.

[0090] Table 1 Transmittance of the composite solutions of examples and comparative examples after tenfold dilution

[0091]

[0092] It can be seen from Examples 1-3 that the water bath mixing temperature has a certain influence on the transmittance of the composite solution. The higher the temperature, the higher the transmittance of the composite solution, but when the temperature is higher than 60 °C, the transmittance will not increase significantly. Combining Examples 2, 4, 5 and 6, it can be seen that water-soluble polysaccharides are the best among natural high molecular polysaccharides. Combining Examples 2, Comparative Example 1 and Comparative Example 2, it can be seen that slow cooling can make the appearance of the polydeoxyribonucleotide natural high molecular polysaccharide composite solution clear and the layers uniform.

[0093] 2. Hyperchromic effect detection

[0094] The hyperchromic effect refers to the increase in light absorption caused by DNA denaturation, that is, the effect of enhanced ultraviolet absorption of the DNA solution after structural change. The composite solutions of Example 1, Comparative Example 1 and Comparative Example 2 were sterilized by steam at 121 °C for 15 min, and the hyperchromic effects of each example and comparative example before and after sterilization were detected respectively. The detection process is as follows: Samples of each example and comparative example before and after sterilization were taken, and diluted to a DNA concentration of about 50 μg / mL with water for injection and 0.1 M sodium hydroxide respectively, and the absorbance values of each dilution at 260 nm were detected. The hyperchromic effects of each example and comparative example were calculated according to the following formula, and the results are shown in Table 2.

[0095] Hyperchromic effect = (OD260 NaOH - OD260 H2O / OD260 H2O ) × 100% (where OD260 NaOH represents the ultraviolet absorption value at 260 nm of the dilution solution diluted with sodium hydroxide, and OD260 H2O represents the ultraviolet absorption value at 260 nm of the dilution solution diluted with water for injection).

[0096] Table 2 Detection results of hyperchromic effect

[0097]

[0098] As can be seen from Table 2, the change in the hyperchromic effect of the composite solution of Example 1 before and after sterilization is not significant, while the change in the hyperchromic effect of Comparative Example 1 and Comparative Example 2 before and after sterilization is extremely significant. This shows that slow cooling of polydeoxyribonucleotide and its composite solution can maintain the integrity of the natural polymer structure of polydeoxyribonucleotide and exert its original drug effect.

[0099] In addition, the polydeoxyribonucleotide-sodium hyaluronate composite solution of the present invention has good effects on removing scars, treating acne, improving fine lines, etc.

[0100] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, makes equivalent substitutions or changes, and should be covered by the protection scope of the present invention.

Claims

1. A preparation method of a polydeoxyribonucleotide natural polymer polysaccharide composite solution, characterized in that, It includes the following steps: S1. Dissolve polydeoxyribonucleotide in water to obtain Solution 1; S2. Dissolve natural high molecular polysaccharide in water to obtain Solution 2; The natural high molecular polysaccharide is selected from one or more of hyaluronic acid, cellulose, and chitosan; S3. Mix Solution 1 and Solution 2 under a water bath condition, the water bath condition is 40°C - 80°C. After mixing evenly, perform gradient program cooling at a cooling rate within the range of 0.18°C / min - 1°C / min. After cooling to room temperature, a polydeoxyribonucleotide natural high molecular polysaccharide composite solution with a clear appearance and uniform layers is obtained.

2. The preparation method of a polydeoxyribonucleotide natural polymer polysaccharide composite solution according to claim 1, characterized in that, The mixing time of Solution 1 and Solution 2 under the water bath condition is 0.5 h - 4 h.

3. The preparation method of a polydeoxyribonucleotide natural polymer polysaccharide composite solution according to claim 1, wherein The mass concentration of Solution 1 is 1 g / L - 40 g / L; the mass concentration of Solution 2 is 1 g / L - 20 g / L.

4. The preparation method of a polydeoxyribonucleotide natural high molecular polysaccharide composite solution according to claim 1, wherein The molecular weight of the natural high molecular polysaccharide is 200 kDa - 2000 kDa; the molecular weight of the polydeoxyribonucleotide is 400 kDa - 2000 kDa.

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