Preparation method of polydeoxyribonucleotide natural macromolecular polysaccharide composite solution

Abstract

The invention relates to the field of compounding of high-molecular polysaccharide materials, in particular to a preparation method of a polydeoxyribonucleotide natural high-molecular polysaccharide composite solution, which comprises the following steps: dissolving polydeoxyribonucleotide to obtain a solution 1; dissolving natural macromolecular polysaccharide to obtain a solution 2; mixing the solution 1 and the solution 2 under a water bath condition, uniformly mixing, performing gradient programmed cooling at a cooling rate of less than 2 DEG C / min, and cooling to room temperature to obtain a polydeoxyribonucleotide natural polymer polysaccharide composite solution with clear appearance and uniform layers; according to the invention, the occurrence of quality phenomena such as turbidity, non-uniform layering, non-uniform content during product filling, unqualified clarity and the like of the composite solution can be avoided; the polydeoxyribonucleotide natural high-molecular polysaccharide composite solution disclosed by the invention is relatively good in product quality.

Description

Technical Field

[0001] The invention relates to the field of high molecular polysaccharide material composites, and in particular to a method for preparing a polydeoxyribonucleotide natural high molecular polysaccharide composite solution. Background Art

[0002] Polysaccharides are widely present in nature and are natural macromolecules with diverse functions. Polysaccharides extracted from natural plants, animals and fungi have multiple biological activities such as antioxidant and anti-tumor activities, 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, high polymerization degrees, and many side chains have hydroxyl groups, which are 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 the solubility in water. Some polysaccharides are even insoluble in water.

[0003] In general, different polymer sizes of polysaccharides produce different physicochemical properties and may lead to different therapeutic effects. Studies have shown that polysaccharide molecules with lower molecular weight have a greater effect on accelerating epidermal renewal, while molecules with higher molecular weight are more effective in supporting fibroblasts. For example, low molecular weight HA stimulates keratinocytes to produce CD44, leading to epidermal hyperplasia and differentiation, while medium and high molecular weight HA are more effective in improving skin hydration.

[0004] It is therefore inferred that polydeoxyribonucleotides of different molecular weights will also affect their physiological effects in the skin. Polydeoxyribonucleotides are PDRN / PN polymers, and the units that make up the polymer are deoxyribonucleotides. PDRN is strictly used to describe short-chain and medium-length 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 broken down locally by endogenous nucleases. Several clinical studies have confirmed the effectiveness of PDRN in skin regeneration and the clinical benefits of PN. Polydeoxyribonucleotides are known as hydrophilic polymers that are able to absorb water. From a biological point of view, the entire surface of the DNA molecule is covered with water, which 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), thereby showing superior properties in restoring skin water balance. In addition, another benefit of PN over PDRN is that it stays in the skin for a longer time, which means that PN will promote a longer skin stimulation effect, with potential enhanced benefits for patients.

[0005] In comparison, PN has high molecular weight and hydrophilicity, and is more suitable for injection into the skin to restore lost volume. However, due to these two reasons, PN is not suitable for preparing solutions with too high concentrations, nor is it suitable for compounding with other polysaccharide materials, and is prone to turbidity and uneven layers. Patent CN118845531A discloses a composite solution, preparation method and application of PDRN-sodium hyaluronate, which chooses to dissolve polyribonucleotides under water bath conditions to increase their solubility, but turbidity will also occur at room temperature. Summary of the invention

[0006] In order to solve the technical problems of unqualified clarity and uneven layers of a composite solution containing polyribonucleotides, a method for preparing a polydeoxyribonucleotide-natural polymer polysaccharide composite solution is provided. The invention separately dissolves polydeoxyribonucleotides and natural polymer polysaccharides, mixes them under water bath conditions, and then slowly cools down to obtain a polydeoxyribonucleotide-natural polymer polysaccharide composite solution with a clear appearance and uniform layers.

[0007] In order to achieve the above objectives, the present invention is implemented by the following technical solutions: A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: S1, dissolving polydeoxyribonucleotide to obtain solution 1; S2, dissolving natural high molecular weight polysaccharide to obtain solution 2; S3, mixing the solution 1 and the solution 2 under water bath conditions, after mixing evenly, performing gradient program cooling at a cooling rate of less than 2°C / min, and obtaining a polydeoxyribonucleotide natural polymer polysaccharide composite solution with a clear appearance and uniform layers after cooling to room temperature. By controlling the cooling rate and slowly cooling, the polydeoxyribonucleotide retains its own double-stranded structure to a great extent to maintain better drug efficacy. The scheme of the present invention can avoid the occurrence of quality phenomena such as turbidity, uneven layers, uneven content during product filling, and unqualified clarity in the composite solution.

[0008] Furthermore, the cooling rate in S3 is in the range of 0.18°C / min-1°C / min.

[0009] Further, the water bath condition in S3 is 40°C-80°C; Furthermore, the mixing time of the solution 1 and the solution 2 under the water bath condition is 0.5h-4h.

[0010] Furthermore, the mass concentration of the solution 1 is 1g / L-40g / L; the mass concentration of the solution 2 is 1g / L-20g / L; the solution 1 and the solution 2 are mixed in any volume ratio.

[0011] Furthermore, the natural high molecular weight polysaccharide is selected from one or more of polydeoxyribonucleotides, hyaluronic acid, cellulose, and chitosan; and the solvents in the solution 1 and the solution 2 are both water for injection.

[0012] Furthermore, the molecular weight of the natural high molecular weight polysaccharide is 200kDa-2000kDa; the molecular weight of the polydeoxyribonucleotide is 400kDa-2000kDa.

[0013] Beneficial technical effects: The present invention can solve the problem of turbidity and uneven layers of the polydeoxyribonucleotide solution by dissolving the polydeoxyribonucleotide and the natural high molecular weight polysaccharide separately, mixing them in a water bath and then slowly cooling them, especially for PN with high molecular weight and high concentration; this is because the cooling speed is too fast, which easily leads to turbidity and precipitation, and the uniformity and clarity of the product are easily unqualified during filling, which makes it difficult to restore the double-stranded structure of the polydeoxyribonucleotide after high-temperature sterilization and it is difficult to exert the original efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The process flow chart of the polydeoxyribonucleotide natural polymer polysaccharide composite solution of the present invention; Figure 2 The following are the physical appearance photos of Examples 1 to 6 and Comparative Examples 1 to 2; Figure 3 The light transmittance test results of Examples 1-6 and Comparative Examples 1-2 are shown. DETAILED DESCRIPTION

[0015] The technical solutions in the embodiments of the present invention will be described clearly and completely below in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] Unless otherwise specifically stated, the numerical value set forth in these embodiments does not limit the scope of the present invention. The technology and method known to those of ordinary skill in the relevant art may not be discussed in detail, but in appropriate cases, the technology and method should be considered as a part of the specification. In all examples shown and discussed here, any specific value should be interpreted as being merely exemplary, rather than as a limitation. Therefore, other examples of exemplary embodiments may have different values.

[0017] The experimental methods in the following examples without specifying specific conditions are usually measured in accordance with national standards; if there is no corresponding national standard, the method is carried out in accordance with the general standard requirements or general methods.

[0018] The molecular weights below are weight average molecular weights.

[0019] Example 1 A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: 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; 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; S3. Mix equal volumes of solution 1 and solution 2 in a 40°C water bath and stir for 2 hours; then cool the temperature to 20°C (taking 60 minutes) at a rate of 10°C every 30 minutes (cooling rate of about 0.33°C / min) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0020] In this case, the composite solution was filled into a glass prefilled syringe and the appearance of the composite solution was observed under natural light. The actual appearance photo is as follows: Figure 2 As shown, the composite solution is clear and transparent with uniform layers at room temperature.

[0021] Example 2 A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: 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; 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; S3. Mix equal volumes of solution 1 and solution 2 in a 60°C water bath and stir for 2 hours; then cool the temperature to 20°C (taking 120 minutes) at a rate of 10°C every 30 minutes (cooling rate of about 0.33°C / min) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0022] In this case, the composite solution was filled into a glass prefilled syringe and the appearance of the composite solution was observed under natural light. The actual appearance photo is as follows: Figure 2 As shown, the composite solution is clear and transparent with uniform layers at room temperature.

[0023] Example 3 A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: 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; 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; S3. Mix equal volumes of solution 1 and solution 2 in a water bath at 80°C and stir for 2 hours; then cool the temperature down to 20°C at a rate of 10°C every 40 minutes (cooling rate 0.25°C / min) (taking 240 minutes) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0024] In this case, the composite solution was filled into a glass prefilled syringe and the appearance of the composite solution was observed under natural light. The actual appearance photo is as follows: Figure 2 As shown, the composite solution is clear and transparent with uniform layers at room temperature.

[0025] Example 4 A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: 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; 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; S3. Mix equal volumes of solution 1 and solution 2 in a 60°C water bath and stir for 2 hours; then cool the temperature down to 20°C (taking 120 minutes) at a rate of 10°C every 30 minutes (cooling rate of about 0.33°C / min) to obtain a polydeoxyribonucleotide-cellulose composite solution.

[0026] In this case, the composite solution was filled into a glass prefilled syringe and the appearance of the composite solution was observed under natural light. The actual appearance photo is as follows: Figure 2 As shown, the composite solution is slightly whitish at room temperature because cellulose is insoluble in water, but it is still clear and transparent with uniform layers.

[0027] Example 5 A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: 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; 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; S3. Mix equal volumes of solution 1 and solution 2 in a 60°C water bath and stir for 2 hours; then cool the temperature down to 20°C (taking 120 minutes) at a rate of 10°C every 30 minutes (cooling rate of about 0.33°C / min) to obtain a polydeoxyribonucleotide-chitosan complex solution.

[0028] In this case, the composite solution was filled into a glass prefilled syringe and the appearance of the composite solution was observed under natural light. The actual appearance photo is as follows: Figure 2 As shown, it can be seen that the composite solution is clear and transparent with uniform layers.

[0029] Example 6 A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: 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; 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; S3. Mix equal volumes of solution 1 and solution 2 in a 60°C water bath and stir for 45 minutes; then cool the temperature to 20°C (taking 120 minutes) at a rate of 10°C every 30 minutes (cooling rate of about 0.33°C / min) to obtain a polydeoxyribonucleotide complex solution.

[0030] In this case, the composite solution was filled into a glass prefilled syringe and the appearance of the composite solution was observed under natural light. The actual appearance photo is as follows: Figure 2 As shown, the composite solution is clear and transparent with uniform layers at room temperature.

[0031] Example 7 A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: 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; 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; S3. Mix equal volumes of solution 1 and solution 2 in a 60°C water bath and stir for 2 hours; then cool the temperature to 20°C (taking 120 minutes) at a rate of 10°C every 30 minutes (cooling rate of about 0.33°C / min) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0032] In this case, the composite solution was filled into a glass prefilled syringe. The appearance of the composite solution was observed under natural light, and it was found that the composite solution was clear and transparent with uniform layers at room temperature.

[0033] Example 8 A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: 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; 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; S3. Mix equal volumes of solution 1 and solution 2 in a 60°C water bath and stir for 4 hours; then cool the temperature to 20°C (taking 120 minutes) at a rate of 10°C every 30 minutes (cooling rate of about 0.33°C / min) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0034] In this case, the composite solution was filled into a glass prefilled syringe. The appearance of the composite solution was observed under natural light, and it was found that the composite solution was clear and transparent with uniform layers at room temperature.

[0035] Comparative Example 1 A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: 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; 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; S3. Mix equal volumes of solution 1 and solution 2 in a 60°C water bath and stir for 2 hours; then cool the temperature to 20°C at a rate of 10°C per 10 minutes (cooling rate 1°C / min) (taking 40 minutes) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0036] In this case, the composite solution was filled into a glass prefilled syringe and the appearance of the composite solution was observed under natural light. The actual appearance photo is as follows: Figure 2 As shown, it can be seen that the composite solution produces turbidity at room temperature.

[0037] Comparative Example 2 A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide composite solution comprises the following steps: 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; 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; S3. Mix equal volumes of solution 1 and solution 2 in a 60°C water bath and stir for 2 hours; then place in an ice water bath and cool the temperature to 20°C at a rate of 20°C per 10 minutes (cooling rate 2°C / min) (taking 20 minutes) to obtain a polydeoxyribonucleotide-sodium hyaluronate composite solution.

[0038] In this case, the composite solution was filled into a glass prefilled syringe and the appearance of the composite solution was observed under natural light. The actual appearance photo is as follows: Figure 2 As shown, it can be seen that the composite solution produces turbidity at room temperature, and the turbidity problem is more serious than that of Comparative Example 1.

[0039] Test Case 1. Light transmittance test The composite solutions of each embodiment and comparative example were diluted ten-fold with physiological saline, and then the physiological saline was used as a blank control to detect the transmittance of the composite solutions of each embodiment and comparative example after being diluted ten-fold in the wavelength range of 400nm~800nm. The results are shown in FIG. Figure 3 And Table 1.

[0040] Table 1 Transmittance of the composite solution of the embodiment and the comparative example after dilution ten times

[0041] It can be seen from Examples 1-3 that the water bath mixing temperature has a certain effect on the transmittance of the composite solution. The higher the temperature, the higher the transmittance of the composite solution. However, when the temperature is higher than 60°C, the transmittance will not be significantly improved. Combining Examples 2, 4, 5 and 6, it can be seen that the natural polymer polysaccharide is best water-soluble. Combining Example 2, Comparative Example 1 and Comparative Example 2, it can be seen that slow cooling can make the polydeoxyribonucleotide natural polymer polysaccharide composite solution clear in appearance and uniform in layers.

[0042] 2. Color enhancement effect detection The color enhancement effect refers to the increase in light absorption caused by DNA denaturation, that is, the effect of enhanced ultraviolet absorption of DNA solution after structural change. The composite solutions of Example 1, Comparative Example 1 and Comparative Example 2 were steam sterilized at 121°C for 15 minutes, and the color enhancement effects of each example and comparative example before and after sterilization were detected. The detection process is as follows: Take samples of each example and comparative example before and after sterilization, dilute the samples with injection water and 0.1M sodium hydroxide to a DNA concentration of about 50μg / mL, and detect the absorbance value of each dilution at 260nm. The color enhancement effect of each example and comparative example was calculated according to the following formula, and the results are shown in Table 2.

[0043] Hyperchromic effect = (OD260 NaOH - OD260 H2O / OD260 H2O )×100%(where OD260 NaOH It indicates the UV absorption value of the diluted solution with sodium hydroxide at 260nm, OD260 H2O It represents the ultraviolet absorption value of the diluent diluted with water for injection at 260nm).

[0044] Table 2 Color enhancement effect test results

[0045] As shown in Table 2, the color enhancement effect of the composite solution of Example 1 before and after sterilization does not change significantly, while the color enhancement effect of Comparative Examples 1 and 2 before and after sterilization changes significantly. This shows that slowly cooling the polydeoxyribonucleotide and its composite solution can maintain the integrity of the natural polymer structure of the polydeoxyribonucleotide and exert the original efficacy.

[0046] In addition, the polydeoxyribonucleotide-sodium hyaluronate composite solution of the present invention has good effects of removing scars, removing acne, improving fine lines, etc.

[0047] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A method for preparing a polydeoxyribonucleotide natural polymer polysaccharide complex solution, characterized in that: The steps include: S1, dissolving polydeoxyribonucleotide to obtain solution 1; S2, dissolving natural high molecular weight polysaccharide to obtain solution 2; S3, mixing the solution 1 and the solution 2 under water bath conditions, and after mixing evenly, performing gradient program cooling at a cooling rate of less than 2°C / min, and obtaining a polydeoxyribonucleotide natural polymer polysaccharide composite solution with a clear appearance and uniform layers after cooling to room temperature.

2. The method for preparing a polydeoxyribonucleotide natural polymer polysaccharide complex solution according to claim 1, characterized in that: The cooling rate in S3 is in the range of 0.18°C / min-1°C / min.

3. The method for preparing a polydeoxyribonucleotide natural polymer polysaccharide complex solution according to claim 2, characterized in that: The water bath condition in S3 is 40°C-80°C.

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

5. The method for preparing a polydeoxyribonucleotide natural polymer polysaccharide complex solution according to claim 2, characterized in that: 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.

6. The method for preparing a polydeoxyribonucleotide natural polymer polysaccharide complex solution according to claim 2, characterized in that: The natural high molecular weight polysaccharide is selected from one or more of polydeoxyribonucleotides, hyaluronic acid, cellulose and chitosan.

7. The method for preparing a polydeoxyribonucleotide natural polymer polysaccharide complex solution according to claim 2, characterized in that: The molecular weight of the natural high molecular weight polysaccharide is 200kDa-2000kDa; the molecular weight of the polydeoxyribonucleotide is 400kDa-2000kDa.

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