KR101839061B1 - Method for manufacturing polydeoxyribonucleotide and polydeoxyribonucleotide manufactured thereby - Google Patents

Abstract

A method for producing a polydioxyribonucleotide is disclosed. DNA fragments containing polydioxyribonucleotides in salmon and eggs can be easily and rapidly obtained using a manufacturing method including an ultrasonic disruption step.

Description

METHOD FOR MANUFACTURING POLYDEOXYRIBONUCLEOTIDE AND POLYDEOXYRIBONUCLEOTIDE MANUFACTURED THEREBY BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing a polydioxyribonucleotide,

The present invention relates to a method for producing a polydioxyribonucleotide and a polydioxyribonucleotide prepared thereby, and more particularly, to a method for producing a polydioxyribonucleotide using ultrasonic disruption.

Since the DNA polymer is composed of a biopolymer such as a base or a phosphate deoxyribose, researches for use as a drug or the like are actively under way. Among the components of the DNA polymer, polydeoxyribonucleotide has recently been widely used because it has been found to be effective for skin regeneration, cartilage regeneration, wound healing, and the like.

Polydioxyribonucleotides are commonly extracted from fish testis and / or eggs. In order to extract polydioxyribonucleotides from fish testis and / or eggs, methods such as enzymatic process extraction, filtration process extraction, and extraction using a surfactant are used. However, these methods are industrially unfavorable due to low yield compared to high investment costs, too complicated processes, and environmental problems due to organic solvents and the like.

Therefore, there is a need to develop a process that can simplify the process and increase the extraction yield of the polydeoxyribonucleotide.

Korean Patent Publication No. 10-2013-0044195 (Feb. European Patent Registration No. 0226254 (December, 1993)

The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a method for producing a polydioxyribonucleotide which can be easily extracted at a high yield, and a polydioxyribonucleotide prepared by the method.

As a technical means for achieving the above technical object, a method for producing a polydioxyribonucleotide according to one aspect of the present invention comprises the steps of preparing a fish testis and / or egg, ultrasonic disruption of fish testis and / or eggs, And filtering the resulting crushed material.

Here, the fish testis and / or egg may be the testis and / or the alleys of salmon and fish.

The preparing step may include a step of water-defrosting the frozen fish testis and / or eggs.

The ultrasonic disruption step may be a ultrasonic disintegration of the fish testis and / or eggs that have been thawed in a low temperature container.

The ultrasonic wave breaking step may be performed by repeating ultrasonic wave breaking and stopping.

The ultrasonic disruption step may be performed at 45 캜 or lower.

The ultrasonic disruption step may be performed at a rate of 20 to 55% of the ultrasonic disintegration time relative to the stop time.

The ultrasonic wave crushing may be performed at an ultrasonic frequency of 10 to 50 kHz and an ultrasonic energy amount of 20 to 2,000 W, and the ultrasonic crushing strength may be ultrasonic wave crushing with an output intensity of 10 to 100% of a maximum value.

The filtration step may be such that the density of the polydioxyribonucleotide after the filtration step is 5 times or more.

A polydioxyribonucleotide according to another aspect of the present invention may be one prepared by the above-described production method.

The method for producing a polydioxyribonucleotide of the present invention can extract a polydioxyribonucleotide easily from a testis and / or an egg of a fish while extracting it at a high yield.

FIG. 1 shows the results of confirming the extraction concentration of polydioxyribonucleotides prepared according to an embodiment of the present invention.
FIG. 2 shows the results of measuring the density of polydioxyribonucleotides prepared according to an embodiment of the present invention.
3 is a conceptual diagram of an ultrasonic wave breaking apparatus according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail. However, it should be understood that the present invention is not limited thereto, and the present invention is only defined by the scope of the following claims.

A method for producing a polydioxyribonucleotide according to an aspect of the present invention includes preparing a fish testis and / or egg, ultrasonically disrupting a fish testis and / or egg, and filtering the ultrasonic shredded lysate.

The polydeoxyribonucleotide is a small molecule having an average molecular weight of 350 KD extracted from spermatozoa, testes or eggs of fishes such as salmon and trout, and is a DNA fragment having a composition similar to a human body. Polydioxyribonucleotides are forms in which purines and pyridine nucleotides are joined by phosphodiester bonds and have a length of 50 to 2000 bp. Since the polydioxyribonucleotide is formed of a deoxyribonucleotide polymer, it is possible to supply purines and pyridines to the human body as well as deoxyribonucleotides and deoxyribonucleosides.

Polydioxyribonucleotides activate the body's repair mechanism and have effects on wound healing, skin regeneration, and cartilage regeneration. Damaged or destroyed cells generally promote regeneration through the de-novo pathway. However, polydioxyribonucleotides act on the adenosine A2 receptor to activate several growth factors to stimulate the proliferation of stem cells and promote regeneration through the salvage pathway using purines and pyridines. The salvage pathway is effective for damaged tissue because it shows a faster action with lower energy than the dinobosyl pathway.

Polydioxyribonucleotides also have antiinflammatory action and promote the secretion of growth factors such as VEGF and angiopoietin, and thus can be used for the treatment of musculoskeletal disorders such as cartilage damage.

The fish means a vertebrate with gills that live in water, and may be sea water fish and / or freshwater fish. The reproductive organs of fish are gonads, gonads are composed of testis and ovaries, and eggs are in ovaries. Testis and ovaries are rich in nutrients such as omega 3 and vitamins, as well as nucleic acids and proteins, which are the main constituents, and thus have cell regeneration, immune function, and antioxidant ability. Polydioxyribonucleotides extracted from testis and / or eggs of fish can be extracted in large quantities using a manufacturing method using ultrasonic disruption, which is economical and can extract high quality polydioxyribonucleotides without destroying the components, And the like.

The fish preferably can be fish of at least one species selected from the group consisting of oncorhynchus, salmo, salvelius, brachymystax, and hucho belonging to Salmoninae. have. More preferably, it may be at least one of trout of salmon, salmon of oncorhynchus. Since the DNA of trout and salmon is almost identical to that of humans, the effect of skin regeneration and growth promotion is better than that of polydioxyribonucleotides extracted from other fish.

The preparation step of the fish testis and / or egg may preferably be a step of washing the testis and / or eggs of the fish, a defrosting step, a defrosting step, and more preferably a defrosting step. The testis and / or eggs of the fish may require cleaning and cooling for freshness, and a step of thawing the cooled fish testis and / or eggs to extract the polydioxyribonucleotide is required. The defrosting can be preferably performed by one or more of gentle defrosting and rapid defrosting according to a desired production time, and thawing can be carried out by any one of a defrosting method, an electric defrosting method, and a heating defrosting method using air or water as the defrosting method can do.

The thawing may more preferably be a water-water-thawing step. Flowing thawing is a method of thawing in running water. The water temperature can be kept constant, the water can be thawed in a large amount, and the cost is reduced. For example, it can be thawed by immersion in water having a flow rate of 0.5 m / sec or higher at a water temperature of 20 ° C or lower.

The step of ultrasonic disruption of the fish testis and / or eggs is necessary in order to smoothly extract the polydioxyribonucleotides by splitting and dividing the components of the testis and / or eggs of the fish. Ultrasonic wave crushing is a mechanical extraction method in which a cell or the like is crushed using a sound wave of about 20 kHz which is the maximum audible frequency of a human being. Ultrasonic wave crushing can be performed by using all the instruments capable of crushing and accelerating fish testicles and / or eggs at supersonic speed, and preferably an ultrasonic dispersing machine can be used. A general ultrasonic dispersing device uses a piezoelectric ceramics and an ultrasonic vibrator that convert electric energy into mechanical vibration energy by using an inverse piezoelectric effect and generates ultrasonic waves. In order to increase the amplitude of the generated ultrasonic waves, And an ultrasonic oscillator for attaching and driving a horn. The ultrasonic dispersing device uses high temperature and high pressure shock waves generated when cavitation bubbles having high temperature and pressure generated by irradiating ultrasonic waves to fish testis and / or eggs are grown and ruptured as an energy source, Alternatively, the polydioxyribonucleotides can be isolated by crushing the eggs.

The step of filtering the ultrasound-pulverized fish testis and / or eggs is a step for increasing the purity by removing impurities other than the polydioxyribonucleotide. The filtration step may be performed to remove debris such as debris or protein coagulation after crushing. The filtration step may be performed by filtering through a 1 mm mesh and filter paper. In the case of a large amount of filtration, the filtration can be preferably performed by any one of batch filtration, continuous filtration, cross flow filtration and centrifugation. Batch filtration may be a filtration method of either a plate and frame filter or a pressure leaf filter. Centrifugation is performed using any centrifuge selected from basket centrifugation, tubular bowl centrifugation, decanter centrifugation and multi chamber centrifugation. W You can have fruit.

The ultrasonic disruption step may be performed by a method of ultrasonic disruption of the fish testis and / or egg from the water-defrosted fish in a low-temperature container. Since the testis and / or eggs of fish are sensitive to temperature, even small temperature changes are prone to spoilage. When the testis and / or eggs of a fish are ultrasonically disrupted, heat is generated during the disintegration time, and the testis and / or eggs of the fish may be denatured. It is therefore desirable to carry out ultrasonic disruption in a low temperature container. For example, in the crushing step, for example, ice can be put in the container for maintaining the low temperature, and the container can be cooled through the cooling fan outside the container.

The ultrasonic wave breaking step may be performed by repeating ultrasonic wave breaking and stopping. This is to control the temperature of the stage of ultrasonic disruption to prevent protein denaturation and to extract high quality polydioxyribonucleotides. The testis and / or eggs of a fish are used in their original state extracted from fish, not in a heated state. Therefore, when heated above the appropriate temperature, proteins bound to higher order structures are physically or chemically modified, making it impossible to extract polydioxyribonucleotides. Therefore, it is necessary to regulate the whole temperature by repeatedly ultrasonically breaking and stopping the testis and / or eggs of the fish in the ultrasonic disintegration step.

The ultrasonic disruption step may preferably be manufactured at less than 45 캜, more preferably less than 40 캜. If the testis and / or eggs of fishes, especially salmonids and fishes, are subjected to denaturation at a temperature of 40 ° C or higher, the production yield may deteriorate. If the temperature exceeds 45 ° C, the proportion of denatured portions may increase, .

The ultrasonic disruption step may be performed at a rate of 20% to 55%, more preferably 40% or 50%, with respect to the stopping time. As the time to crush the fish testicles and / or eggs with ultrasonic waves increases, heat is generated and protein denaturation occurs. Therefore, it is necessary to perform repeated ultrasonic disruption and stop. The testis and / or eggs of the fish are not broken so that the polydioxyribonucleotide can be sufficiently extracted from the testis and / or eggs of the fish when the ultrasonic disruption time to the stop time is less than 20%. If the ultrasonic disruption time exceeds the stop time by more than 55%, there may be a problem that the testis and / or eggs of the fish are broken so that the extraction of the polydioxyribonucleotide can not be performed smoothly.

The ultrasonic disruption may be ultrasonic disruption with an energy amount of 20 to 2,000 watts (W). In the case of ultrasonic disruption with an energy amount of less than 20 watts, fish testis and / or eggs may not be broken enough to extract the polydioxyribonucleotide because the energy to be applied to fish testis and / or eggs is low. In the case of ultrasonic disruption with an energy amount exceeding 2,000 watts, fish testis and / or eggs may be excessively disrupted to make it difficult to extract the polydioxyribonucleotide.

The ultrasonic disruption may be ultrasonic disruption at a frequency of 10 to 50 kHz. Ultrasonic pulverization transmits ultrasound vertical vibration to fish testicles and / or eggs through a horn of an ultrasonic crusher. At this time, the testis and / or eggs of the fish are expanded and contracted by the vibration of constant amplitude, and the fine bubbles generated at this time are crushed through the cavitation phenomenon in which the amplified positive pressure is vigorously destroyed . When the ultrasonic wave is less than 10 kHz, the amplitude of the fish transmitted to the testis and / or the ostium is so small that expansion and contraction do not occur inside, so that it is difficult to extract the polydioxyribonucleotide because the cavitation phenomenon is not sufficiently generated. If the ultrasonic wave exceeds 50 kHz, there may be a problem that the polydioxyribonucleotide can not be extracted from the testis and / or eggs of fish due to excessive cavitation phenomenon.

The ultrasonic wave crushing may have an ultrasound crush strength of 10% to 100% as compared with the maximum intensity. It is to prevent destruction and denaturation of testis and / or eggs of fish by controlling ultrasonic crushing strength. If the ultrasonic crushing strength is less than 10%, the testis and / or eggs of the fish can not be sufficiently broken and can not be preserved in testes and / or eggs to extract the polydioxyribonucleotide. If the ultrasonic breaking strength is more than 100%, the testis and / or eggs of the fish may be excessively broken to destroy the polydioxyribonucleotide, so that only the deoxyribonucleotide can be extracted, which may be unsuitable for the intended use.

The filtration step may be filtration of the polydioxyribonucleotide density between 10 and 25 AU after the filtration step. The density is a density to show a high level of therapeutic effect on the dose of the polydioxyribonucleotide. When the density is less than 10 AU, other components other than the polydioxyribonucleotide are contained so that the total amount of the drug to be administered to the human body may be excessively increased. When the density exceeds 25AU, there may be a problem that the polydioxyribonucleotides coagulate with each other. It is preferable that the filtration is performed at a density of 5 times or more as compared with before filtration.

A polydioxyribonucleotide according to another aspect of the present invention is prepared by preparing a fish testis and / or egg, ultrasonically disrupting fish testis and / or eggs, and filtering the ultrasonic disrupted lysate Lt; / RTI > The detailed description of each step is as described above and is omitted.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Example: Preparation of Polydioxyribonucleotide

The eggs of trout were used for the preparation of polydioxyribonucleotides. 30, 35, and 40 were adjusted to 30, 40, and 50%, respectively, according to the respective time ratios, and the polydioxyribonucleotides having a length of 50 to 2000 bp were regulated with ultrasound breaking strengths of 20, 25, 30, .

Experimental Example: Electrophoresis experiment

5.5 g of frozen trout eggs were placed in a 50 ml conical tube and water was thawed. After it was confirmed to be divided into individual eggs, it was moved to an ice bucket. Ultrasonic wave breaking was performed with ultrasound intensity of 20, 25, 30, 35, 40 and 30%, 40%, and 50% of ultrasonic breaking time compared with stopping time. Ultrasonic waves were filtered through a 1 mm mesh and filter paper to remove impurities from trout eggs.

DNA electrophoresis was carried out to examine the degree of separation of the filtered polydioxyribonucleotides and electrophoresis was carried out with a 250bp DNA ladder of Takara Inc. having a range of 250bp to 4.5kbp and an interval of 250bp for identification with polydioxyribonucleotides . The 6x loading dye and sample were loaded and the final volume of 20 microliters was loaded on a 1.6% agarose gel and confirmed in an ultraviolet lamp after 30 minutes. The classification display of each sample is shown in Table 1, and the results of each sample are shown in FIGS. 1 and 2. FIG.

Strength (%)
Duty (%) 20 25 30 35 40 50% L11 L12 L13 L14 L15 40% L6 L7 L8 L9 L10 30% L1 L2 L3 L4 L5

As shown in Table 1, Figs. 1 and 2, when the ratio of the stop time to the ultrasonic disruption time was 50%, it was confirmed that polydioxyribonucleotide was abundant compared with the other groups. At 40%, the density of the polydioxyribonucleotide was found to be high between 20 and 30%.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It will be possible. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description and all changes or modifications derived from the meaning and scope of the claims and their equivalents are to be construed as being included within the scope of the present invention do.

Claims (10)

Preparing a material composed of any one or more of fish testis and eggs;
Ultrasonic disruption of a material composed of any one or more of the fish testimony and eggs; And
And filtering the ultrasonic shredded material,
The fish testimony and eggs are,
The testis and eggs of salmon and fish,
In the preparation step,
Characterized in that a material composed of at least one of frozen fish testimony and eggs is immersed in water having a flow velocity of 0.5 m / sec or higher at a water temperature of 20 DEG C or lower and thawed,
The ultrasonic disruption step may comprise:
An ultrasonic dispersing machine is used,
And ultrasonically crushing a material composed of any one or more of the above water-extracted fish testimony and eggs in a low-temperature container,
The ultrasonic disruption and the stop are repeatedly performed,
Lt; RTI ID = 0.0 > 40 C, < / RTI &
The ultrasonic disintegration time relative to the stop time is performed at a rate of 40 to 50%
An ultrasonic frequency of 10 to 50 kHz and an ultrasonic energy amount of 20 to 2,000 W,
Characterized in that the ultrasonic crushing strength is 10 to 100% of the maximum value,
Wherein the filtration step is characterized in that the filtration is performed so that the density of the polydioxyribonucleotide after the filtration step is 5 times or more and 10 to 25 AU
A method for producing a polydioxyribonucleotide having a length of 50 to 2,000 bp. delete delete delete delete delete delete delete delete delete

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