Small molecule polydeoxyribonucleotide as well as preparation and application thereof
阅读说明：本技术 一种小分子多聚脱氧核糖核苷酸及其制备与应用 (Small molecule polydeoxyribonucleotide as well as preparation and application thereof ) 是由 王超云 高原 董书萍 于 2019-11-28 设计创作，主要内容包括：本发明针提供一种分子量集中于其功效区间的三文鱼小分子多聚脱氧核糖核苷酸(Small molecule polydeoxyribonucleotide,SMPDRN)产品及其可控精准制备方法,以及其在化妆品、药品、营养食品、保健食品领域的应用。本发明中的小分子多聚脱氧核糖核苷酸的分子量为50bp～1000bp,其分子量位于50bp～500bp片段的质量占其总质量的的85％以上。本发明中的SMPDRN的分子量集中于高功效范围,针对性更强,功效更稳定,获得的产品经细胞、动物、人体实验证实在提高细胞活性、促进胶原蛋白合成、使受损皮肤再生、促进伤口愈合、紧致皮肤、增加皮肤弹性、消除皱纹、延缓皮肤衰老、抗氧化并防止色斑形成、抗炎、修复受损细胞等方面均具有显著效果,优于现有PDRN产品。(The invention provides a salmon Small molecular poly deoxyribose nucleotide (SMPDRN) product with molecular weight concentrated in an efficacy interval, a controllable and accurate preparation method thereof, and application thereof in the fields of cosmetics, medicines, nutritional foods and health-care foods. The molecular weight of the small molecular poly-deoxyribonucleotide is 50 bp-1000 bp, and the molecular weight of the small molecular poly-deoxyribonucleotide is more than 85 percent of the total mass of fragments of 50 bp-500 bp. The molecular weight of the SMPDRN is concentrated in a high-effect range, the pertinence is stronger, the effect is more stable, and the obtained product has obvious effects on improving the cell activity, promoting the synthesis of collagen, regenerating damaged skin, promoting wound healing, tightening the skin, increasing the skin elasticity, eliminating wrinkles, delaying skin aging, resisting oxidation, preventing the formation of color spots, resisting inflammation, repairing damaged cells and the like through the verification of cells, animals and human bodies, and is superior to the existing PDRN products.)
1. The small-molecule polydeoxyribonucleotide is characterized by being derived from salmon, and the molecular weight of the small-molecule polydeoxyribonucleotide is more than 85 percent of the total mass of fragments of 50 bp-500 bp.
2. The small polydeoxyribonucleotide according to claim 1, wherein the molecular weight of said small polydeoxyribonucleotide is more than 90% of the total mass of the fragments of 100bp to 200 bp.
3. The small polydeoxyribonucleotide according to claim 1 or 2, wherein the molecular weight of said small polydeoxyribonucleotide is between 50 and 1000 bp.
4. A method for preparing small molecule polydeoxyribonucleotides, which comprises the following steps:
(1) cracking salmon tissues by using an alkali cracking solution, wherein the alkali cracking solution contains 0.05-1 mol/L EDTA, 0.5-5 mol/L NaOH and 0.1-5 wt% SDS;
(2) adding Tris-HCl into the reaction system obtained in the step (1);
(3) adding HCL into the reaction system obtained in the step (2);
(4) centrifuging the reaction system obtained in the step (3), and collecting a supernatant;
(5) performing molecular disruption on the supernatant obtained in the step (4) by using a DNA disruptor;
(6) adding NH into the reaction system obtained in the step (5)4Adding absolute ethyl alcohol into the mixture, and standing the mixture for more than 30 min;
(7) and (4) centrifuging the reaction system obtained in the step (6), and reserving the precipitate to obtain the micromolecule polydeoxyribonucleotide.
5. The method according to claim 4, wherein the operating conditions of step (1) are: adding the alkaline lysis solution into salmon tissue, wherein the dosage ratio of the salmon tissue to the alkaline lysis solution is 1g (5-15) mL; crushing the tissue, reversing and mixing uniformly, and carrying out water bath at the constant temperature of 90-100 ℃ for 15-60 min.
6. The method according to claim 4, wherein the operating conditions of step (5) are: and (4) crushing the supernatant obtained in the step (4) for 5 s-5 min by using a DNA disruptor.
7. The method according to claim 4, wherein the operating conditions of step (2) are: carrying out ice-bath on the reaction system obtained in the step (1) to below 40 ℃, adding 0.5-4 mol/LTris-HCl according to the volume ratio of 1/3-1 of the total volume, and reversing and uniformly mixing;
the working conditions of the step (3) are as follows: adding 0.5-5 mol/L HCL according to the volume ratio of 1/5-1 of the total volume of the reaction system obtained in the step (2), and reversing and uniformly mixing;
the working conditions of the step (4) are as follows: centrifuging the reaction system obtained in the step (3) at the temperature of more than 0 ℃ and at the rpm of 5000-12000 for 5-20 min, and collecting a supernatant;
the working conditions of the step (6) are as follows: adding 10mol/L NH according to the volume ratio of 1/10-1/5 of the total volume of the reaction system obtained in the step (5)4Adding anhydrous ethanol pre-cooled below 0 deg.C into the AC solution, reversing, mixing, and standing below 0 deg.C for more than 30 min;
the working conditions of the step (7) are as follows: and (3) centrifuging the reaction solution obtained in the step (6) at 6000-12000 rpm above 0 ℃ for 5-20 min, pouring off anhydrous ethanol, adding 50-80 wt% of ethanol to enable the precipitate to be flooded, centrifuging at 6000-12000 rpm for 5-20 min, and keeping the precipitate.
8. The process according to claim 4, further comprising suspending the precipitate obtained in step (7) with physiological saline and then drying the suspension in a vacuum freeze-dryer.
9. Use of the small molecule polydeoxyribonucleotides according to any one of claims 1 to 3 in cosmetics, pharmaceuticals, nutraceuticals and health foods.
10. Use according to claim 9, characterized in that the application forms comprise masks, emulsions, solutions, colloidal preparations, powder injections, tablets or capsules.
11. Use according to claim 9, characterized in that it comprises use in increasing cellular activity, reducing the production of free radicals, anti-oxidant; including application in increasing skin elasticity, eliminating wrinkles, and delaying skin aging; including use in promoting wound healing; including the application in preventing the formation of black spots, chloasma and senile plaques; including anti-inflammatory and repair of damaged cells.
12. The use of claim 9, wherein the small polydeoxyribonucleotide is administered in an amount ranging from 0.1mg to 1g per dose.
Technical Field
The invention particularly relates to micromolecule polydeoxyribonucleotide as well as preparation and application thereof.
Background
Deoxyribonucleic acid (DNA) is used as an important genetic material of an organism, and plays an important role in regulating gene and protein expression, improving the cell state and maintaining the normal physiological function of the organism on the one hand; on the other hand, it is used as a raw material library to provide the required deoxyribonucleotide for the growth, development and repair of organisms. However, since the DNA chain is relatively long, large in molecular weight and charged, it is not easily absorbed into the body through the cell membrane. Researchers in pharmaceutical, medical and beauty and health care product industries have long dedicated to searching DNA biological materials with high similarity of base composition and human bodies. Through diligent efforts of researchers, it was confirmed that the similarity of the base composition of salmon DNA and human DNA reaches 98%, as shown in fig. 1.
The first time that salmon DNA was found to be effective was the Italian fisherman, they found that the periodical application of salmon spermary semen squeezed out of salmon spermary to wounds effectively inhibits wound ulceration and promotes wound healing. Polydeoxynucleotide (PDRN) is respectively extracted from germ cells of sea trout and salmon spermary by Mastelli Italy and BR PHARM respectively on the basis, and has curative effects on promoting human cell regeneration, accelerating wound healing and reducing scar formation.
However, the existing PDRN preparation technology is complex in process and complicated in steps, a large amount of organic solvents and protease are used in the purification process, and the yield is low. Meanwhile, the controllability of the preparation and the effect significance, the effect pertinence and the effect stability of related products need to be improved.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a Small Molecule Polydeoxyribonucleotides (SMPDRN) product with a molecular weight concentrated in an efficacy interval, a controllable and accurate preparation method thereof and application thereof in the fields of cosmetics, medicines, nutritional foods and health-care foods.
The specific technical scheme is as follows:
one objective of the present invention is to provide a Small Molecule Polydeoxyribonucleotides (SMPDRN) product with molecular weight concentrated in the functional region.
The salmon includes Atlantic salmon, Pacific salmon and rainbow trout.
The molecular weight range of the small molecular poly-deoxyribonucleotide (namely SMPDRN) is 10 bp-1500 bp.
Preferably, the molecular weight of the small-molecule polydeoxyribonucleotide is 50 bp-1000 bp, wherein the mass of the fragment with the molecular weight of 50 bp-500 bp accounts for more than 85% of the total mass of the small-molecule polydeoxyribonucleotide.
Furthermore, the mass of the fragment with the molecular weight of 100bp-200bp of the small-molecule polydeoxyribonucleotide accounts for more than 90 percent of the total mass of the small-molecule polydeoxyribonucleotide.
The second purpose of the invention is to provide a preparation method of salmon small molecule polydeoxyribonucleotide SMPDRN, the molecular weight of which is concentrated in the efficacy interval.
A preparation method of small molecule polydeoxyribonucleotide SMPDRN takes viscera, meat, spermary, semen and eggs of salmon as materials, and comprises the following steps:
(1) cracking salmon tissues by using an alkali cracking solution, wherein the alkali cracking solution contains 0.05-1 mol/LEDTA, 0.5-5 mol/L NaOH and 0.1-5 wt% SDS;
(2) adding Tris-HCl into the reaction system obtained in the step (1);
(3) adding HCL into the reaction system obtained in the step (2);
(4) centrifuging the reaction system obtained in the step (3), and collecting a supernatant;
(5) performing molecular disruption on the supernatant obtained in the step (4) by using a DNA disruptor;
(6) adding NH into the reaction system obtained in the step (5)4Adding absolute ethyl alcohol into the mixture, and standing the mixture for more than 30 min;
(7) and (4) centrifuging the reaction system obtained in the step (6), and reserving the precipitate to obtain the SMPDRN.
Preferably, the working conditions of the step (1) are as follows: adding the alkaline lysis solution into salmon tissue, wherein the dosage ratio of the salmon tissue to the alkaline lysis solution is 1g (5-15) mL; crushing the tissue, reversing and mixing uniformly, and carrying out water bath at the constant temperature of 90-100 ℃ for 15-60 min.
Preferably, the working conditions of the step (2) are as follows: and (2) carrying out ice-bath on the reaction system obtained in the step (1) to be below 40 ℃, adding 0.5-4 mol/L Tris-HCl according to the volume ratio of 1/3-1 of the total volume of the reaction system, and reversing and uniformly mixing.
Preferably, the working conditions of the step (3) are as follows: adding 0.5-5 mol/L HCL according to the volume ratio of 1/5-1 of the total volume of the reaction system obtained in the step (2), and reversing and uniformly mixing.
Preferably, the working conditions of the step (4) are as follows: and (4) centrifuging the reaction system obtained in the step (3) at the temperature of more than 0 ℃ and 5000-12000 rpm for 5-20 min, and collecting the supernatant.
Preferably, the working conditions of the step (5) are as follows: and (4) crushing the supernatant obtained in the step (4) for 5 s-5 min by using a DNA disruptor.
Preferably, the working conditions of the step (6) are as follows: adding 10mol/L NH according to the volume ratio of 1/10-1/5 of the total volume of the reaction system obtained in the step (5)4Adding anhydrous ethanol pre-cooled below 0 deg.C into the AC solution, reversing, mixing, and standing below 0 deg.C for more than 30 min; .
Preferably, the working conditions of the step (7) are as follows: and (3) centrifuging the reaction solution obtained in the step (6) at 6000-12000 rpm above 0 ℃ for 5-20 min, pouring off anhydrous ethanol, adding 50-80 wt% of ethanol to enable the precipitate to be flooded, centrifuging at 6000-12000 rpm for 5-20 min, and keeping the precipitate.
Preferably, the preparation method further comprises suspending the precipitate obtained in step (7) with physiological saline, and then drying in a vacuum freeze dryer.
Dissolving the precipitate obtained in the step (7) by using TE solution, and performing agarose gel electrophoresis to verify that the number of fragments with the molecular weight of 50 bp-500 bp of the SMPDRN obtained by the preparation method is more than 85 percent and is highly concentrated.
In the
The preparation method has high yield which reaches 9-14%; the obtained product has high purity, and the purity (260nm/280nm) detected by a spectrophotometry is 1.6-1.9.
The specific preparation method of the invention concentrates the molecular weight of the SMPDRN in a high-efficacy range, has stronger pertinence, and through cell, animal and human experiments, the inventor finds that the obtained product has obvious effects superior to the existing PDRN in the aspects of improving cell activity, promoting collagen synthesis, regenerating damaged skin, promoting wound healing, tightening skin, increasing skin elasticity, eliminating wrinkles, delaying skin aging, resisting oxidation, preventing color spot formation, resisting inflammation, repairing damaged cells and the like. The experimental data in the examples demonstrate the above effects.
In addition, the preparation method of the invention realizes that the molecular weight is concentrated in 50-500 bp or even 100bp-200bp, effectively avoids the generation of fragments with larger molecular weight or smaller molecular weight, and can improve the target effect and increase the biological activity of the components by relative unification.
The third purpose of the invention is to provide the application of the small molecular poly-deoxyribonucleotide in cosmetics, medicines, nutritional foods and health-care foods, wherein the application forms comprise facial masks, emulsions, solutions, colloidal preparations, powder injections, tablets or capsules.
The invention provides an application of SMPDRN in improving cell activity, reducing free radical generation and resisting oxidation
The invention provides an application of SMPDRN in increasing skin elasticity, eliminating wrinkles and delaying skin aging.
The invention provides an application of SMPDRN in promoting wound healing.
The invention provides an application of SMPDRN in preventing black spots, chloasma and senile plaques.
The invention provides an SMPDRN anti-inflammatory effect and application in repairing damaged cells.
When the SMPDRN is used for any one of the applications, the dosage range of the SMPDRN is 0.1mg to 1g per time, preferably 0.5mg to 0.1g per time, and more preferably 1mg to 20mg per time.
The invention has the following beneficial effects:
the molecular weight of the SMPDRN is concentrated in a high-effect range, the pertinence is stronger, the effect is more stable, and the obtained product has obvious effects on improving the cell activity, promoting the synthesis of collagen, regenerating damaged skin, promoting wound healing, tightening the skin, increasing the skin elasticity, eliminating wrinkles, delaying skin aging, resisting oxidation, preventing the formation of color spots, resisting inflammation, repairing damaged cells and the like through the verification of cells, animals and human bodies, and is superior to the existing PDRN products.
In addition, the preparation method of the invention realizes that the molecular weight is concentrated in 50 bp-500 bp or even 100bp-200bp, effectively avoids the generation of fragments with larger molecular weight or smaller molecular weight, has relatively simple components, can obviously improve the target effect of the molecules and enhance the biological activity.
The invention provides a preparation method with controllable molecular weight, which realizes the precise preparation of small molecular polydeoxyribonucleotide. The SMPDRN with highly concentrated molecular weight is obtained by the preparation method, and the product with highly concentrated molecular weight in a high-efficiency area and high purity is obtained by the cooperation of a special cracking process and a DNA breaking instrument. The preparation method has the advantages of simple process, high yield, low reagent cost, no use of organic solvent, contribution to industrial large-scale production and no residue and pollution discharge.
The product of the invention has obvious effect, stable effect, low sensitization and relatively simple components, provides an excellent raw material for the fields of cosmetics, medicines, nutritional foods and health-care foods, and has great potential in the aspects of anti-aging and damage repair.
Drawings
FIG. 1 is a graph comparing the similarity between salmon DNA and human DNA;
FIG. 2 is an electrophoretogram of SMPDRN obtained in example 1;
fig. 3 is an electrophoretogram of the SMPDRN obtained in example 2 and example 3;
fig. 4 is an infrared chromatogram of the SMPDRN obtained in example 2;
FIG. 5 is a graph showing the effect of SMPDRN obtained in example 2 on cell viability;
FIG. 6 is a graph of the effect of SMPDRN obtained in example 2 on apoptosis;
FIG. 7 is a graph showing the effect of SMPDRN obtained in example 2 on the antioxidant capacity of cells;
FIG. 8 is a graph of the effect of SMPDRN obtained in example 2 on wound healing in rats;
FIG. 9 is a graph of the effect of SMPDRN obtained in example 2 on wound healing in zebrafish;
FIG. 10 is the effect of SMPDRN spread nutrient solution obtained in example 8 on skin elasticity;
FIG. 11 is the effect of SMPDRN spread nutrient solution obtained in example 8 on skin moisture content;
fig. 12 is a photograph comparing the wrinkle-removing and whitening effects of the smearing type nutrient solution and the lead-in type essence solution using the SMPDRN of the present invention as an active ingredient on the forehead;
fig. 13 is a photograph comparing the effects of applying nutrient solution and leading-in essence of the invention with SMPDRN as active ingredient on wrinkle removal and whitening of neck;
fig. 14 is a photograph comparing the effects of applying nutrient solution and leading-in essence solution with SMPDRN as active ingredient on whitening back;
fig. 15 is a photograph comparing the effect of the facial mask and the lead-in essence of the invention with the SMPDRN as the active ingredient on removing acne and whitening the face;
fig. 16 is a photograph comparing the effect of the facial mask and the lead-in type essence of the invention with the SMPDRN as the active ingredient on the acne removal of the forehead;
in fig. 2: A. sample No. 1; B. sample No. 2; C. sample No. 3; D. sample No. 4;
in fig. 3: A. example 3; B. example 2; C. example 2.
Detailed Description