A method for extracting polydeoxyribonucleotides from wheat

Abstract

This invention belongs to the field of bioextraction technology, and specifically relates to a method for extracting wheat-derived polydeoxyribonucleotides (PDRNs). The method includes steps such as CTAB lysis, alkaline lysis, potassium acetate salting-out for impurity removal, sodium chloride gradient salting-out, ethanol precipitation, and silica gel column purification. This invention improves the yield and purity of wheat PDRNs by combining dual lysis with multi-stage impurity removal. The process is mild, safe, and environmentally friendly, making it suitable for the preparation of cosmetic and medical raw materials.

Description

Technical Field

[0001] This invention belongs to the field of bio-extraction technology, and particularly relates to a method for extracting wheat-derived polydeoxyribonucleotides. Background Technology

[0002] Polydeoxyribonucleotides (PDRNs) are a class of oligodeoxynucleotide fragments with molecular weights concentrated between 50 and 1500 kDa. They can promote cell proliferation, tissue repair, and anti-inflammatory regeneration, and are widely used in medical repair, cosmetic skincare, and tissue engineering. Currently, PDRN raw materials are mainly derived from the gonads of animals such as salmon, which poses risks related to animal-derived viruses, ethical controversies, limited raw material availability, and high costs. Plant-derived DNA, on the other hand, has a wide range of sources, high safety, and no animal-derived risks, making it an ideal alternative to animal-derived PDRNs.

[0003] Wheat, as a major agricultural crop, is a high-quality raw material for preparing plant-derived PDRN due to its high DNA content, ease of acquisition, and suitability for industrialization. However, wheat tissue is rich in secondary metabolites such as polysaccharides, polyphenols, and proteins. When extracting DNA using conventional methods such as CTAB, salting out, and organic solvent extraction, severe co-precipitation of impurities occurs, making it difficult to meet product purity standards. Furthermore, the fragments tend to be large, and it is difficult to balance yield and purity, making it impossible to consistently obtain PDRN that meets application requirements. Therefore, developing and optimizing a high-yield, high-purity, and fragment-controllable wheat PDRN extraction method has significant application value. Summary of the Invention

[0004] Based on the above analysis, this application provides a method for extracting wheat-derived polydeoxyribonucleotides (PDRN). This method can solve the technical problems in existing wheat PDRN extraction processes, such as insufficient cell lysis and inadequate nucleic acid release; incomplete removal of polysaccharides and polyphenols; low PDRN purity and high impurity residue; resulting in large PDRN precipitation losses and low overall recovery rates; uneven PDRN fragment sizes, high proportion of macromolecular DNA, and insufficient enrichment of active components; and difficulty in obtaining high-purity and highly uniform wheat PDRN.

[0005] To achieve the above objectives, the technical solution of this application discloses a method for extracting wheat-derived polydeoxyribonucleotides (PDRN), comprising the following steps:

[0006] (1) Mix the wheat sample with CTAB extraction buffer containing polyvinylpyrrolidone and lyse it;

[0007] (2) Add an alkaline solution for alkaline treatment;

[0008] (3) Add potassium acetate solution to neutralize, let stand at low temperature, centrifuge, and collect the supernatant;

[0009] (4) Add sodium chloride solution to the supernatant, centrifuge, and collect the supernatant;

[0010] (5) Add ethanol to precipitate, and collect the precipitate by centrifugation;

[0011] (6) Wash, dry and redissolve the precipitate to obtain crude extract;

[0012] (7) The crude extract was purified by silica gel column chromatography, eluted, and the eluent was collected to obtain wheat-derived polydeoxyribonucleotides.

[0013] Furthermore, the CTAB extraction buffer contains 1%–3% polyvinylpyrrolidone by mass, has a lysis temperature of 55°C–75°C, and a lysis time of 30 min–2 h.

[0014] Furthermore, the alkaline solution is a sodium hydroxide solution with a concentration of 0.3 to 1.0 mol / L, and the added volume is 1 / 10 to 1 / 5 of the volume of the lysis solution obtained in step (1), and the room temperature treatment time is 3 to 10 min.

[0015] Furthermore, the concentration of the potassium acetate solution is 2-5 mol / L, the added volume is 1.0-2.0 times the volume of the lysis solution obtained in step (1), and the low-temperature standing is -20℃ to 4℃ for 10-60 min.

[0016] Furthermore, the concentration of the sodium chloride solution is 0.5 to 1.0 mol / L, and the added volume is 0.3 to 0.8 times the volume of the supernatant obtained in step (3).

[0017] Furthermore, the ethanol is anhydrous ethanol, the volume added is twice the volume of the supernatant obtained in step (4), the precipitation temperature is -20℃ to 0℃, and the precipitation time is 10 to 60 min.

[0018] Furthermore, the precipitate is washed 1 to 3 times with 70% to 80% ethanol.

[0019] Furthermore, in step (7), the elution is performed using Tris-HCl buffer solution with a pH of 7.5 to 8.5.

[0020] Beneficial effects:

[0021] (1) Double cleavage is more complete

[0022] The use of CTAB high-temperature pyrolysis coupled with short-time alkaline pyrolysis provides dual cell disruption, significantly improving the release efficiency of wheat PDRN and yielding a significantly higher yield than the CTAB method alone.

[0023] (2) Multi-stage impurity removal is more thorough

[0024] A three-step coupling process—PVPP for polyphenol removal, potassium acetate for polysaccharide precipitation, and gradient salting-out for protein removal—significantly reduces residual polysaccharides and proteins, resulting in higher PDRN purity. Silica gel column chromatography further enhances PDRN purity and integrity by assisting in the removal of polysaccharides, polyphenols, proteins, and enzyme inhibitors.

[0025] (3) Higher recovery rate

[0026] Optimizing alcohol precipitation and centrifugation parameters reduced PDRN loss and significantly improved the overall recovery rate.

[0027] (4) The process is mild, stable and safe.

[0028] It exhibits no severe denaturation, has good reproducibility, and uses readily available raw materials, making it suitable for large-scale production. It avoids the use of highly toxic organic solvents such as phenol and chloroform, ensuring safety and environmental friendliness. The product is of high quality and can be directly used as a raw material in cosmetics and medical applications. Attached Figure Description

[0029] Figure 1 The electrophoresis results are for samples from Example 1 and Comparative Example 1. Detailed Implementation

[0030] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0031] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0032] Example 1: Extraction of wheat-derived polydeoxyribonucleotides

[0033] (1) Sample pretreatment

[0034] Jimai 22 wheat grains were selected, and impurities and broken grains were removed; the grains were then cryogenically pulverized with liquid nitrogen, passed through an 80-mesh sieve, and refrigerated at 4℃ for later use.

[0035] (2) Reagent preparation

[0036] CTAB extraction solution: 2% CTAB, 100 mmol / L Tris-HCl (pH 8.0), 20 mmol / L EDTA, 1.4 mol / L NaCl, 2% PVPP.

[0037] NaOH solution: 0.5 mol / L.

[0038] Potassium acetate solution: 3 mol / L, pH 5.2.

[0039] NaCl solution: 0.68 mol / L.

[0040] Ammonium acetate solution: 7.5 mol / L.

[0041] Sodium acetate buffer: 3 mol / L, pH 5.2.

[0042] Ethanol: 75% (volume fraction).

[0043] TE buffer: 10 mmol / L Tris-HCl, 1 mmol / L EDTA, pH 8.0.

[0044] (3) Extraction steps

[0045] 1) Weigh 1.0 g of wheat germ powder, add 5 mL of CTAB solution preheated at 65℃, and lyse in a water bath at 65℃ for 1 h, mixing once every 20 min.

[0046] 2) Cool to room temperature, add 1 / 6 volume of 0.5 mol / L NaOH, mix well, and let stand at room temperature for 5 min.

[0047] 3) Add 1.5 times the volume of lysis buffer 3 mol / L potassium acetate, mix well, and let stand at -20℃ for 30 min; centrifuge at 8000 r / min and 4℃ for 20 min, and take the supernatant.

[0048] 4) Add 0.5 times the volume of the supernatant (0.68 mol / L NaCl), mix well; centrifuge at 10000 r / min for 1 h, and collect the supernatant.

[0049] 5) Add 2 times the volume of pre-cooled anhydrous ethanol, precipitate at -20℃ for 30 min; centrifuge at 12000 r / min for 10 min, and collect the precipitate.

[0050] 6) Wash the precipitate twice with 75% cold ethanol, air dry at room temperature, and reconstitute with 100 μL TE buffer to obtain the crude extract.

[0051] 7) Purification by 100 mg / 1 mL silica gel column: Activate with ethanol and pure water; loading flow rate 1.0 mL / min; elute with Tris-HCl at pH 8.0 at a flow rate of 0.5 mL / min, collect the eluent to obtain pure wheat-derived PDRN.

[0052] Comparative Example 1: Extraction of wheat-derived polydeoxyribonucleotides

[0053] Using the same wheat raw materials, liquid-to-material ratio, and equipment conditions;

[0054] Take freeze-dried wheat powder and add 650 μL of preheated CTAB extraction solution (65℃) at a ratio of 10 mg / mL. Gently invert to mix. Incubate at 65℃ for 45 min to lyse. Centrifuge the lysate at 12000 rpm for 15 min and carefully transfer 400 μL of the supernatant to a new centrifuge tube, avoiding aspiration of the lower layer residue. After cooling, add an equal volume of chloroform-isoamyl alcohol mixture and gently invert to mix. Centrifuge at 12000 rpm for 15 min and transfer the supernatant to a new centrifuge tube. Add 0.7 volumes of pre-chilled isopropanol to the supernatant, gently invert to mix, and incubate at -20℃ overnight (12-16 h). After overnight precipitation, centrifuge at 12000 rpm for 15 min, discard the supernatant, and wash the precipitate twice with 75% ethanol. After each wash, centrifuge and discard the ethanol. Air dry the precipitate at room temperature to remove residual ethanol. Finally, add an appropriate amount of TE buffer to reconstitute the DNA.

[0055] Experimental Example 1: Apparent Experiment

[0056] The DNA obtained in Example 1 and Comparative Example 1 was subjected to concentration, purity and electrophoresis tests. The results are shown in Table 1.

[0057] Table 1

[0058] Comparison items Example 1 Comparative Example 1 PDRN yield 1.76 (g / g) 0.92 (g / g) A260 / A280 2.14 2.10 A260 / A230 2.00 1.94 molecular weight range 100~3000 kDa 100~3000 kDa Appearance White powder White powder Electrophoresis Clear bands Clear bands

[0059] As shown in Table 1, the extraction method of this application can obtain wheat germ PDRN with high yield and good purity, and no toxic reagents such as chloroform, isoamyl alcohol, and isopropanol are used in the extraction process. The entire extraction process is safer and more environmentally friendly.

[0060] 1. PDRN yield

[0061] Example 1, employing CTAB + PVPP + alkaline lysis + salting out + silica gel column purification, achieved a significantly higher yield than Comparative Example 1. Comparative Example 1, lacking PVPP, alkaline lysis, salting out, and silica gel column purification, had a relatively lower yield than Example 1. This demonstrates that the extraction method provided by this invention has a significant advantage in extraction efficiency.

[0062] 2. A260 / A280 ratio (protein residue indicator)

[0063] The A260 / A280 ratio in Example 1 was 2.14, and in Comparative Example 1 it was 2.10. The A260 / A280 ratio is used to assess the degree of protein contamination in nucleic acid samples, and the theoretical value for pure DNA is 1.8-2.0. This indicates that the modified CTAB method can obtain PDRN with acceptable purity.

[0064] 3. A260 / A230 ratio (indicator of polysaccharide / phenolic residues)

[0065] The A260 / A230 ratio in Example 1 was 2.00, close to the ideal range (2.0-2.2), indicating low residual polysaccharide and phenolic impurities. The ratio in Comparative Example 1 was 1.94. This demonstrates that the modified CTAB method can obtain PDRN with acceptable purity.

[0066] 4. Molecular weight range

[0067] Example 1 contains PDRN with a molecular weight of 100-3000 kDa, which falls within the ideal molecular weight range (50-1500 kDa) and exhibits good fragment uniformity. Example 1 utilizes a silica column purification process, allowing for subsequent PDRN fractionation based on molecular weight. This meets the application needs of PDRN in various fields: 80-200 kDa PDRN is preferred for powerful tissue repair and anti-inflammation; for cosmetic filling and long-lasting anti-aging, PDRN with a molecular weight of 1500 kDa or higher is a better choice.

[0068] 5. Appearance

[0069] Example 1 is a pure white powder, indicating that impurities have been completely removed.

[0070] 6. Electrophoresis, electrophoresis comparison images as shown. Figure 1 As shown: Lanes labeled PVPP precipitated at -20℃ are for Example 1, and lanes labeled chloroform-isoamyl alcohol are for Comparative Example 1. It can be seen that the bands in Example 1 are clear, indicating that the fragments are intact and free from interference. The bands in Comparative Example 1 are weaker than those in Example 1, indicating that its extraction efficiency is slightly lower. Electrophoresis conditions: 1.0-1.5% agarose gel, 80-120 V, 2000 bp DNA Marker control.

[0071] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.

Claims

1. A method for extracting and purifying wheat-derived polydeoxyribonucleotides, characterized in that, Includes the following steps: (1) Mix the wheat sample with CTAB extraction buffer containing polyvinylpyrrolidone and lyse it; (2) Add an alkaline solution for alkaline treatment; (3) Add potassium acetate solution to neutralize, let stand at low temperature, centrifuge, and collect the supernatant; (4) Add sodium chloride solution to the supernatant, centrifuge, and collect the supernatant; (5) Add ethanol to precipitate, and collect the precipitate by centrifugation; (6) Wash, dry and redissolve the precipitate to obtain crude extract; (7) The crude extract was purified by silica gel column chromatography, eluted, and the eluent was collected to obtain wheat-derived polydeoxyribonucleotides.

2. The method according to claim 1, characterized in that, In step (1), the mass fraction of polyvinylpyrrolidone in the CTAB extraction buffer is 1% to 3%, the lysis temperature is 55℃ to 75℃, and the lysis time is 30 min to 2 h.

3. The method according to claim 1, characterized in that, In step (2), the alkaline solution is a sodium hydroxide solution with a concentration of 0.3 to 1.0 mol / L, and the added volume is 1 / 10 to 1 / 5 of the volume of the lysis solution obtained in step (1). The room temperature treatment time is 3 to 10 min.

4. The method according to claim 1, characterized in that, In step (3), the concentration of the potassium acetate solution is 2-5 mol / L, and the added volume is 1.0-2.0 times the volume of the lysis solution obtained in step (1). The low-temperature standing is -20℃ to 4℃ for 10-60 min.

5. The method according to claim 1, characterized in that, In step (4), the concentration of the sodium chloride solution is 0.5 to 1.0 mol / L, and the added volume is 0.3 to 0.8 times the volume of the supernatant obtained in step (3).

6. The method according to claim 1, characterized in that, In step (5), the ethanol is anhydrous ethanol, the volume added is twice the volume of the supernatant obtained in step (4), the precipitation temperature is -20℃ to 0℃, and the precipitation time is 10 to 60 min.

7. The method according to claim 1, characterized in that, In step (6), the precipitate is washed 1 to 3 times with 70% to 80% ethanol.

8. The method according to claim 1, characterized in that, In step (7), the elution is performed using Tris-HCl buffer solution with a pH of 7.5 to 8.5.

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