A method for extracting oligonucleotides from fresh animal medicine and its application

Abstract

The invention discloses a method for extracting oligonucleotides from fresh animal medicines and the oligonucleotides obtained by the method. The extraction method comprises: enzymolyzing the fresh medicines with a mixed enzyme of trypsin and proteinase K to obtain enzymolyzates; then grading by ultrafiltration to collect the part with a molecular weight cutoff of 3kDa to 30kDa; finally, subjecting the ultrafiltrate to anion exchange column chromatography, washing away unbound impurities and eluting with an eluent to obtain the oligonucleotides from the fresh animal medicines. The extraction method is based on the pharmacology and medicinal components of the fresh medicines, and adopts the method of enzymolysis, ultrafiltration and ion exchange chromatography to retain and enrich the medicinal active components as much as possible, and well avoids the oligonucleotides from being degraded by nucleases.

Description

Technical Field

[0001] The invention relates to a method for extracting oligonucleotides from fresh animal medicines and the oligonucleotides obtained by the method, belonging to the technical field of traditional Chinese medicine component extraction. Background Art

[0002] In the Shennong Herbal Classic, 67 kinds of animal medicines are recorded. Animal Chinese medicines have been used throughout the entire process of the development of traditional Chinese medicine. They are still widely used in clinical practice and have good development prospects. In recent years, the research on fresh animal medicines has attracted widespread attention from both Chinese and Western medicine circles. At present, the commonly used medicinal materials in clinical practice are fresh medicinal materials that are dried in the sun or in the shade, and then processed into decoction pieces, or directly extracted and processed into Chinese patent medicines. This process of fresh-to-dry usually causes great changes in the medicinal active ingredients in the fresh medicinal materials, resulting in the failure to achieve the original medicinal purpose. Only by protecting all its active ingredients can its physiological functions be effectively exerted. However, the existing processing process greatly limits the retention of medicinal active ingredients in medicinal materials. For example, after boiling in water for several hours to produce a decoction and the processing process requires high temperature treatment, the oligonucleotides in the fresh medicine will be degraded during the preparation process, and thus cannot be retained, resulting in a waste of resources and an obstacle to the application of drug ingredients. Summary of the invention

[0003] In order to overcome the shortcomings of the prior art, the first purpose of the present invention is to provide a method for extracting oligonucleotides from fresh animal medicines. Based on the pharmacological and medicinal component characteristics of the fresh medicines, enzymatic hydrolysis, ultrafiltration and ion exchange chromatography are used to retain the medicinal active ingredients as much as possible and to avoid the degradation of oligonucleotides.

[0004] The second object of the present invention is to provide an oligonucleotide obtained by the oligonucleotide extraction method of the fresh herb.

[0005] The first object of the present invention can be achieved by adopting the following technical scheme: a method for extracting oligonucleotides from fresh animal medicines, wherein the enzymatic hydrolysis step is as follows: taking a living animal medicine, freezing it at a temperature of -20°C to -40°C for 4 to 8 hours, adding 2 to 5 times water to homogenize it, and then adding trypsin and proteinase K for enzymatic hydrolysis to obtain an enzymatic solution; the ratio of the enzyme activities of the trypsin and proteinase K is 4:1 to 10:1; the amount of the protease is 500 to 1000 U of protease per 1 gram of fresh medicine; the temperature of the enzymatic hydrolysis is 45-65°C; the living animal medicine is at least one of leeches, cockroaches, earthworms, earthworms, scorpions, centipedes, geckos, seahorses, geckos, white flower snakes, black-necked snakes and horseflies.

[0006] Furthermore, the enzymatic hydrolysis step is: taking a living animal medicinal material, freezing it at a temperature of -30°C to -40°C for 6 hours, adding 2 to 5 times water to homogenize, then adding trypsin and proteinase K, adjusting the pH to 7.5 to 8.5, and performing enzymatic hydrolysis at a temperature of 50-65°C; after the enzymatic hydrolysis is completed, heating to 80-90°C and keeping warm for 30 to 60 minutes to obtain an enzymatic solution; the ratio of the enzyme activity of the trypsin and proteinase K is 5:1 to 8:1; the dosage of the protease is 750U of protease per 1 gram of fresh medicine.

[0007] Furthermore, the enzymatic hydrolysis time is 2-6h.

[0008] Furthermore, after the enzymatic hydrolysis is completed, the mixture is heated to 85° C. and kept warm.

[0009] Furthermore, the extraction method further comprises an ultrafiltration step: after the enzymatic hydrolysate is microfiltered, ultrafiltration is performed using an ultrafiltration membrane to collect an ultrafiltrate having a relative molecular mass of 3KDa to 30KDa.

[0010] Furthermore, the pH of the enzymatic hydrolysate is adjusted to 6.5-7.5, and then the enzymatic hydrolysate is filtered through a 0.5-2 μm microporous filter membrane to obtain a microfiltrate.

[0011] Further, the pH of the microfiltrate is adjusted to 6.5-7.5, and then passed through 30KDa and 3KDa ultrafiltration membranes respectively, ultrafiltrate with a relative molecular mass of 3KDa-30KDa is collected, and the pH is adjusted to 8.5-9.5.

[0012] Furthermore, the oligonucleotide extraction method further comprises a chromatography purification step: the ultrafiltrate is chromatographed through an anion exchange column, washed with a balance solution, and then eluted with an eluent to obtain the oligonucleotides in the animal fresh medicine.

[0013] Furthermore, the equilibrium solution is a Tris-hydrochloric acid buffer or a diethanolamine-hydrochloric acid buffer with a pH of 8.5 to 9.5.

[0014] Furthermore, the eluent is a 0.25-0.5 mol / L NaCl solution.

[0015] Furthermore, the chromatography medium in the anion exchange column chromatography is at least one of anion exchange resins with styrene-divinylbenzene copolymer, polymethacrylic acid, ethyl cellulose, cross-linked dextran and agarose as matrices.

[0016] The second object of the present invention can be achieved by adopting the following technical scheme: an oligonucleotide is obtained by a method for extracting oligonucleotides from fresh animal medicines.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The method for extracting oligonucleotides from fresh animal medicines of the present invention is based on the pharmacology and medicinal components of fresh medicines, and adopts enzymatic hydrolysis, ultrafiltration and ion exchange chromatography to retain the medicinal active ingredients as much as possible and well avoid the degradation of oligonucleotides;

[0019] 2. The method for extracting oligonucleotides from fresh animal medicines of the present invention changes the previous application mode of using raw powder of animal Chinese medicines as medicine after traditional processing methods, and also improves the value of oligonucleotides in the medicinal field, providing new ideas for the extraction, processing and application of fresh medicines. DETAILED DESCRIPTION

[0020] Below, in conjunction with specific implementation modes, the present invention is further described:

[0021] A method for extracting oligonucleotides from fresh animal medicines, comprising:

[0022] Enzyme hydrolysis step: take living animal medicinal materials, freeze them for 4 to 8 hours (-20°C to -40°C), add 2 to 5 times water to homogenize, then add trypsin and proteinase K to perform enzymolysis to obtain an enzymolysis solution; the ratio of the enzyme activity of the trypsin and proteinase K is 4:1 to 10:1; the amount of the protease is 500 to 1000 U of trypsin and proteinase K per 1 gram of fresh medicine; the temperature of the enzymolysis is 40-65°C;

[0023] Ultrafiltration step: filter the enzymatic hydrolyzate with a microporous filter membrane to obtain a microfiltrate; then pass it through a 30KDa and a 3KDa ultrafiltration membrane respectively, collect the ultrafiltrate with a relative molecular mass of 3KDa to 30KDa, and adjust the pH to 8.5 to 9.5;

[0024] Purification steps: the ultrafiltrate is passed through anion exchange column chromatography, washed with a balance solution, and then eluted with an eluent to obtain the oligonucleotides in the animal fresh medicine.

[0025] In fact, the above extraction method can be used for insect medicines, including but not limited to leeches. For example, it can also be used to extract fresh medicines of earthworms, earthworms, scorpions, centipedes, geckos, geckos, cockroaches, white flower snakes, black-necked snakes, horseflies, etc.

[0026] The oligonucleotides in the present invention are not provided in the form of boiled extracts obtained from plants, such as decoctions, because oligonucleotides are easily degraded at high temperatures and in the presence of nucleases. Therefore, it can be explained from this specific embodiment that extraction from fresh products breaks through the traditional model of drug extraction.

[0027] Embodiment 1:

[0028] A method for extracting oligonucleotides from fresh leech medicine, comprising:

[0029] The enzymolysis step comprises taking 1 kg of fresh leeches, freezing them for 6 hours (-40° C.), adding 3 times of water to homogenize, then adding 750 U of trypsin and proteinase K per 1 gram of fresh medicine, wherein the ratio of the enzyme activity of trypsin and proteinase K is 6:1, adjusting the pH value to 8.0 with a 20 wt % sodium hydroxide solution (the pH value is adjusted to 8.0 every 30 minutes during the enzymolysis process), performing enzymolysis at a temperature of 55° C., and after the enzymolysis is completed, heating to 80-90° C. and keeping warm for 30-60 minutes to obtain an enzymolysis solution.

[0030] Ultrafiltration step: Filter the enzymatic hydrolyzate with a 1 μm microporous filter membrane to obtain a microfiltrate; then pass through 30KDa and 3KDa ultrafiltration membranes respectively to collect ultrafiltrate with a relative molecular mass of 3KDa to 30KDa. Adjust the pH to 9 with 20wt% sodium hydroxide to obtain a loading solution;

[0031] Purification steps: Add 200 mL of the sample solution into the chromatographic mobile phase solvent bottle, and equilibrate the chromatographic column with 2 column volumes (one column volume is 180 mL) of diethanolamine-hydrochloric acid buffer until the baseline and conductivity are stable, with a flow rate of 5 mL / min.

[0032] The sample was loaded through the channel (load flow rate 3.5 mL / min) and passed through the Q Sepharose agarose strong anion exchange column. After loading, 3 column volumes of diethanolamine-hydrochloric acid buffer were used to wash away the unbound part at a flow rate of 5 mL / min.

[0033] Elution steps: isocratic elution, first eluting with 3 column volumes of 0.25M NaCl, flow rate 5mL / min; then eluting with 3 column volumes of 0.5M NaCl, flow rate 5mL / min; collecting with an automatic collector, the wavelength of the UV detector is 260nm.

[0034] Desalting and freeze-drying steps: the eluate is desalted by electrodialysis, concentrated under reduced pressure, and freeze-dried by a freeze dryer to obtain fresh drug oligonucleotides.

[0035] For those skilled in the art, various other corresponding changes and modifications can be made according to the technical solutions and concepts described above, and all of these changes and modifications should fall within the protection scope of the claims of the present invention.

Claims

1. A method for extracting oligonucleotides from fresh animal medicines, characterized in that: The extraction method comprises the following steps: taking a living animal medicinal material, freezing it at a temperature of -20°C to -40°C for 4 to 8 hours, adding 2 to 5 times water to homogenize it, and then adding trypsin and proteinase K for enzymolysis to obtain an enzymolysis solution; the ratio of the enzyme activity of the trypsin and proteinase K is 4:1 to 10:1; the dosage of the trypsin and proteinase K is 500 to 1000 U of trypsin and proteinase K per 1 gram of fresh medicine; the temperature of the enzymolysis is 45-65°C; the living animal medicinal material is at least one of leeches, cockroaches, earthworms, earthworms, scorpions, centipedes, geckos, seahorses, geckos, white flower snakes, black-necked snakes and horseflies; The extraction method further comprises an ultrafiltration step: after the enzymatic hydrolysate is microfiltered, ultrafiltration is performed using an ultrafiltration membrane to collect an ultrafiltrate having a relative molecular mass of 3KDa to 30KDa; The oligonucleotide extraction method also includes a chromatography purification step: the ultrafiltrate is chromatographed through anion exchange column, washed with a balance solution, and then eluted with an eluent to obtain the oligonucleotide in the animal fresh medicine.

2. The method for extracting oligonucleotides from fresh animal medicines according to claim 1, wherein: The extraction method comprises the following steps: taking a living animal medicinal material, freezing it at a temperature of -30°C to -40°C for 6 hours, adding 2 to 5 times water to homogenize it, then adding trypsin and proteinase K, adjusting the pH to 7.5 to 8.5, and performing enzymolysis at a temperature of 50 to 65°C; after the enzymolysis is completed, heating it to 80 to 90°C and keeping it warm for 30 to 60 minutes to obtain an enzymolysis solution; the ratio of the enzyme activity of the trypsin and proteinase K is 5:1 to 8:1; and the amount of the protease used is 750U of protease per 1 gram of fresh medicine.

3. The method for extracting oligonucleotides from fresh animal medicines according to claim 1, wherein: The pH of the enzymatic hydrolysate is adjusted to 6.5-7.5, and then the enzymatic hydrolysate is filtered through a 0.5-2 μm microporous filter membrane to obtain a microfiltrate.

4. The method for extracting oligonucleotides from fresh animal medicines according to claim 1, wherein: The pH of the microfiltrate was adjusted to 6.5-7.5, and then passed through 30KDa and 3KDa ultrafiltration membranes respectively, and the ultrafiltrate with a relative molecular mass of 3KDa-30KDa was collected, and the pH was adjusted to 8.5-9.

5.

5. The method for extracting oligonucleotides from fresh animal medicines according to claim 1, characterized in that: The equilibrium solution is a Tris-hydrochloric acid buffer or a diethanolamine-hydrochloric acid buffer with a pH of 8.5-9.

5.

6. The method for extracting oligonucleotides from fresh animal medicines according to claim 1, characterized in that: The eluent is a 0.25-0.5 mol / L NaCl solution.

7. The method for extracting oligonucleotides from fresh animal medicines according to claim 1, characterized in that: The chromatographic medium in the anion exchange column chromatography is at least one of anion exchange resins with styrene-divinylbenzene copolymer, polymethacrylic acid, ethyl cellulose, cross-linked dextran and agarose as a matrix.

8. An oligonucleotide, characterized in that The oligonucleotide is obtained by the method for extracting oligonucleotide from fresh animal medicine as claimed in claim 1.