KR101667995B1 - Method for producing nontoxic purified bee venom not causing allergy using yolk - Google Patents

Abstract

The present invention relates to a method for preparing refined bee venom from which an allergic component is removed and allergic component-removed and refined bee venom prepared by the method, wherein the preparation method includes the following steps of: (a) preparing a buffer including yolk; (b) adding bee venom to the prepared buffer of step (a) and then stirring the resultant; (c) adding cooled ethanol while cooling the stirred reactant of step (b) to coagulate a protein in the reactant; (d) centrifuging the coagulated reactant of step (c) to precipitate the coagulate; and (e) taking a supernatant from the reactant of step (d) in which the coagulate is precipitated, and then concentrating and drying the resultant.

Description

TECHNICAL FIELD The present invention relates to a method for producing a non-toxic bee venom purified bee venom not causing allergy using yolk sac,

(A) preparing a buffer solution containing egg yolk; (b) adding beeswax to the buffer solution prepared in step (a) and stirring the mixture; (c) coagulating the reactant protein by adding cooled ethanol while cooling the stirred reaction product of step (b); (d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And (e) concentrating and drying the supernatant in the reaction product in which the solidification product of step (d) has been precipitated, and drying the purified beverage preparation. ≪ RTI ID = 0.0 > allergic < / RTI >

Bee venom (bee venom) is a poison of bee, and bee venom has been recognized for medical treatment effects since ancient times. Recent studies have shown that bee venom can be used in a variety of medical applications such as anti-cancer treatment, arthritis alleviation, arteriosclerosis relief, back pain relief, treatment of skin wounds, immune enhancement, anti- inflammatory action, lowering of blood pressure and increase of lymphocytes and red blood cells in blood, For the purpose of improving wrinkles, whitening, and has been found.

The major constituents of bee venom and their pharmacological functions are shown in Table 1 below.

ingredient Pharmacological function Component Ratio (%) Peptides (melitin, apamin) Sedative, immune function Around 50% Protein (phospholipase A2) Cell destruction, allergy 15% Amines (histamine) Blood pressure drop 1 to 2%

Bee venom is composed of over 40 substances, including peptides, proteins and low molecular active amines, and the main active ingredients are as shown in the table above.

Among the bee venom components, the proteins include components such as phospholipase A2 (PLA2), hyaluronidase, phosphatase, and? -Glucosidase having a molecular weight of 13 kDa or more And has physiological activities such as destruction of blood cell membranes, blood coagulation, vasodilation and permeation, promotion of blood circulation, and promotion of hydrolysis of proteins.

In particular, phospholipase and hyaluronidase are bee venom components that cause a strong allergic reaction, which can cause very serious safety problems for users with hypersensitivity to bee venom. Therefore, in order to use bee venom for the purpose of treatment, inactivation or complete removal of the above components is inevitable.

Accordingly, techniques for removing allergen-inducing substances from bee venom are known. For example, in Korean Patent No. 1382404, a bee venom lysis solution is prepared by dissolving bee venom, and the bee venom lysis solution is put into a PSA (primary secondary amine) adsorbent to prepare a mixture, which is then subjected to microfiltration to remove pure impurities A method for mass production of bee venom is described. In Korean Patent No. 1364506, phospholipase in bee venom is removed by using an ultrafiltration membrane having a molecular weight cut-off size of 10 kDa or more, and the content of apatine is 4 wt% or more, melitin is 50 By weight or more. Korean Patent No. 1608045 discloses a technique for removing phospholipase from bee venom and separating only melitin using a cation exchange resin. However, there is no method for producing a tablets bee venom having allergy components removed using egg yolk as in the present invention.

The present invention has been made in view of the above-mentioned needs, and it is an object of the present invention to overcome the above-mentioned problems, and it is an object of the present invention to provide a bee venom- The present invention provides a method of manufacturing a tablet bee venom that can be removed.

In order to solve the above-mentioned problems, the present invention provides a method for preparing an egg yolk comprising the steps of: (a) preparing a buffer solution containing egg yolk; (b) adding beeswax to the buffer solution prepared in step (a) and stirring the mixture; (c) coagulating the reactant protein by adding cooled ethanol while cooling the stirred reaction product of step (b); (d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And (e) concentrating and drying the supernatant in the reaction product in which the solidification product of step (d) has been precipitated, and drying the purified beverage preparation.

In addition, the present invention provides a tablets bee venom having allergen components removed by the above method.

Tablet bee venom prepared by the method of the present invention can completely eliminate the phospholipase A2 and hyaluronidase that induce a strong allergic reaction to produce a tablet bee venom that is fundamentally safe from the allergic reaction, Which is advantageous in that it can be used more reliably for various pharmacological functions.

In order to achieve the object of the present invention,

(a) preparing a buffer solution containing egg yolk;

(b) adding beeswax to the buffer solution prepared in step (a) and stirring the mixture;

(c) coagulating the reactant protein by adding cooled ethanol while cooling the stirred reaction product of step (b);

(d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And

(e) concentrating and drying the supernatant in the reaction product in which the solidification product of step (d) has been precipitated, and drying the purified supernatant.

In the method for producing a tablet bee venom of the present invention, the allergen component may be, but is not limited to, phospholipase A2 and hyaluronidase.

Further, in the method for producing a tablet bee venom of the present invention, the yolk of step (a) may be egg yolk derived from algae such as egg, duck, and quail eggs, have.

In addition, in the method for producing a tablet bee venom of the present invention, the buffer solution of step (a) may preferably be a pH 7 to 9 buffer solution containing 1 to 10% (w / v) of egg yolk, May be a pH 8 buffer solution containing 5% (w / v) egg yolk. When egg yolk is dissolved in the buffer solution, egg yolk protein, yolk lecithin, neutral lipid and cholesterol are emulsified by egg yolk lecithin to form an emulsified suspension in colloidal state.

In addition, in the method of manufacturing the tablet bee venom of the present invention, the step (b) may be performed by adding 0.5 to 1.5% (w / v) of bee saponification to a buffer solution and stirring at 30 to 50 ° C for 5 to 20 minutes , And more preferably 1% (w / v) of bee venom is added to the buffer solution, followed by stirring at 35 DEG C for 10 minutes. The addition of bee venom to the buffer solution under the above conditions, followed by stirring, could effectively bind allergen-inducing substances contained in the bee venom to the yolk lipid protein.

In addition, in the method of the present invention, the step (c) is preferably performed by cooling the stirred reaction product to -5 to -15 ° C while adding ethanol cooled to -10 to -20 ° C, And more preferably, the stirred reaction product can be cooled to -10 DEG C and ethanol cooled at -15 DEG C may be added to solidify the protein of the reaction product. When the yolk lipid protein bound to the allergen-inducing substance is coagulated as described above, the allergen-inducing substance can be easily removed by precipitating the coagulum by centrifugation.

The method of producing the tablet bee venom of the present invention, more specifically,

(a) preparing a pH 7 to 9 buffer solution containing 1 to 10% (w / v) egg yolk;

(b) 0.5 to 1.5% (w / v) of bee venom is added to the buffer solution prepared in step (a), followed by stirring at 30 to 50 ° C for 5 to 20 minutes;

(c) coagulating the reaction product protein by adding ethanol cooled to -10 to -20 ° C while cooling the stirred reaction product of step (b) to -5 to -15 ° C;

(d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And

(e) concentrating and drying the supernatant in the reaction product in which the solidification product of step (d) has been precipitated,

More specifically,

(a) preparing a pH 8 buffer solution containing 5% (w / v) egg yolk;

(b) adding 1% (w / v) of bee venom to the buffer solution prepared in step (a) and stirring at 35 DEG C for 10 minutes;

(c) coagulating the reactant protein by adding ethanol cooled to -15 캜 while cooling the stirred reactant in step (b) to -10 캜;

(d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And

(e) concentrating and drying the supernatant in the reaction product in which the solidification product of step (d) has been precipitated.

The present invention also provides a tablets bee venom with allergy components removed by the above method. In the bee venom of the present invention, allergen-inducing substances such as phospholipase A2 and hyaluronidase were not detected, and melitin, an active ingredient of bee venom, was contained in an amount of 60% or more and 2% or more . In addition, the tablet bee venom of the present invention can be obtained by using Staphylococcus aureus strain and Propionibacterium acne ) showed similar antimicrobial effects when compared with pre - bee - bee. In the case of guinea pigs, it was found that there was no abnormal change in guinea pig, It was confirmed that there was no antigenicity.

Hereinafter, embodiments of the present invention will be described in detail. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Manufacturing example  One: allergy  Purified material removed Bee venom  Produce

(a) 100 mL of a 10 mM EDTA buffer solution having a pH of 8 dissolved in 5% (w / v) egg yolk isolated from a live egg was prepared.

(b) 1% (w / v) of bee venom purchased from the Beekeeping Association for the buffer solution prepared in the step (a) was added, followed by stirring at 35 ° C for 10 minutes.

(c) The reaction product of step (b) was transferred to ice pockets at -10 DEG C, and 95% (v / v) ethanol cooled at -15 DEG C was slowly added thereto to coagulate the protein.

(d) The coagulated reaction product of step (c) above was centrifuged to precipitate a solidified product.

(e) The supernatant was taken out from the reaction product in which the solidified product of step (d) was precipitated, concentrated in vacuo to remove ethanol, and lyophilized to obtain 0.75 g of purified bee venom.

Example  1: Tablets Bee venom  Antibacterial activity

To confirm whether the physiological activity of the tablet beacon prepared in Production Example 1 was maintained, Staphylococcus aureus ( Staphylococcus aureus) aureus strain and Propionibacterium < RTI ID = 0.0 > The minimum inhibition concentration (MIC) and minimum bacteriocidal concentration (MBC) of the isolates were analyzed.

Comparison of antibacterial activity of bee venom before and after purification Strain MIC ([mu] g / ml) MBC (占 퐂 / ml) Cleansing before cleansing Bee after purification Cleansing before cleansing Bee after purification Staphylococcus aureus 0.16 0.18 0.61 0.62 Propionibacterium acne 0.06 0.07 0.25 0.24

As a result, it was confirmed that the purified bee venom of Production Example 1 retains the antimicrobial effect when compared with the bee venom before purification.

Example  2: Tablets Bee venom  Component change

Melanin, apamin, phospholipase A2 and hyaluronidase components of bee venom purified by pre-purification bee venom and the method of Preparation Example 1 were analyzed.

Sephadex TM 75, Source 15RPC ST, C18, RP-amide and Peptide ES-C18 were used as the analytical devices. The concentrations of melittin, apamin, phospholipase A2, hyaluronidase The content was determined by the following formula and the results are shown in Table 3 below.

(Purity of standard product / 100) x (peak area of desired component in bee venom) / peak area of standard product) x (100 / bee amount)

Changes in Ingredients of Tablet Beeop Component content (%) Melitin Apamin Phospholipase A2 Hyaluronidase Cleansing before cleansing 65 2.5 11 1.8 Production Example 1 63 2.7 0 0

As a result, the purified bee venom of the present invention did not detect allergen-inducing substances such as phospholipase A2 and hyaluronidase, and found that 60% or more of melitin, an active ingredient of bee venom, % Or more.

Example  3: Tablets Bee venom allergy  Inhibitory effect test

In order to confirm the allergen inhibitory effect of the tablet bee venom according to the present invention, the following PCA (passive cutaneous anaphylaxis) test was performed.

1) Test substance: Purified beacon of Preparation Example 1

2) Excipient: Injection of physiological saline

3) Positive control: Ovalbumin (Egg-derived)

4) Positive control substance excipient: injection of physiological saline

5) Test animals: 300 ~ 380 g Specific pathogen member (SPF) Guinea pig

6) Test group composition, dose determination, group separation and administration

Test group composition and dosage determination group Number of animals Amount of solution (ml / kg) Dose (mg / kg) G1 (vehicle control group) 5 One 0 G2 (low dose group) 5 One 0.025 G3 (high capacity group) 5 One 0.05 G4 (OVA + adjuvant group) 5 One 2.5

7) Dose determination: The sensitization and the PCA dose were made equal.

8) Administration: Sensitization was administered to the adventitious portion of the guinea pig, and serum of the blood obtained after sensitization was used as the cause of PCA reaction. The PCA reaction was carried out on two PCA aged animals using 2 animals per each individual serum. The serum to be tested was continuously diluted with a 2-fold dilution series from 10 times to 5120 times with an injection of physiological saline, and then 100 μl each of the diluted sera was administered into the guinea pig embryos. After 4 hours of intradermal administration, Yagi's stock solution containing 2% of Evan's blue was administered into the Fuji vein to cause PCA reaction.

9) Observation and examination: General symptoms and weight were checked. The PCA response was determined 30 min after the PCA reaction, and the test animals were slaughtered by ether anesthesia. The mean value of long and short diameters (long diameter + short diameter) / 2 was 5 mm or more, and the last serum dilution factor (maximum dilution factor) (Antibody value).

10) Test results

- General symptoms: No specific symptoms and dead animals due to bee venom according to the present invention were observed.

- Weight change: No change in body weight by bee venom according to the present invention was observed.

- PCA judgment: The antigenicity of bee venom according to the present invention was evaluated using the induction of PCA reaction using guinea pig as an index, and the results are shown in Table 5 below.

PCA results (number of positive animals / total number of test animals) Dilution factor G1 G2 G3 G4 10 0/10 0/10 0/10 10/10 20 0/10 0/10 0/10 10/10 40 0/10 0/10 0/10 10/10 80 0/10 0/10 0/10 10/10 160 0/10 0/10 0/10 10/10 320 0/10 0/10 0/10 10/10 640 0/10 0/10 0/10 8/10 1280 0/10 0/10 0/10 6/10 2560 0/10 0/10 0/10 5/10 5120 0/10 0/10 0/10 3/10

PCA response showed no negative control group G1, low bee venom low dose group G2 and high dose group G3. On the other hand, in the positive control group G4, the antibody titer was 5120 times, indicating a clear PCA reaction. As a result, the tablet bee venom prepared according to the present invention does not have antigenicity.

Claims (5)

(a) preparing a buffer solution containing egg yolk;
(b) adding beeswax to the buffer solution prepared in step (a) and stirring the mixture;
(c) coagulating the reactant protein by adding cooled ethanol while cooling the stirred reaction product of step (b);
(d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And
(e) concentrating and drying the supernatant in the reaction product in which the solidification product of step (d) has been precipitated, and drying the purified supernatant. The method according to claim 1, wherein the allergen component is phospholipase A2 and hyaluronidase. The method according to claim 1, wherein the buffer solution of step (a) is a buffer solution of pH 7 to 9 containing 1 to 10% (w / v) egg yolk. The method according to claim 1,
(a) preparing a pH 7 to 9 buffer solution containing 1 to 10% (w / v) egg yolk;
(b) 0.5 to 1.5% (w / v) of bee venom is added to the buffer solution prepared in step (a), followed by stirring at 30 to 50 ° C for 5 to 20 minutes;
(c) coagulating the reaction product protein by adding ethanol cooled to -10 to -20 ° C while cooling the stirred reaction product of step (b) to -5 to -15 ° C;
(d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And
(e) concentrating and drying the supernatant in the reaction product in which the solidification product of step (d) has been precipitated, and drying the purified supernatant. delete