KR101774197B1 - Method for producing nontoxic bee venom peptide - Google Patents

Abstract

The present invention relates to a method for producing refined bee venom peptide in which allergic components are removed and refined bee venom in which allergic components are removed by being manufactured by the method. The method comprises the following steps: (a) adding a buffer solution to bee venom and diluting the same; (b) adding protease to a diluted solution diluted in the step (a) and performing enzyme reaction; (c) adding cooled ethanol to a reactant after enzyme reaction in the step (b) and coagulating protein; (d) separating the reactant coagulated in the step (c) by centrifugation and depositing coagulation; and (e) obtaining supernatant from the reactant in which the coagulation in the step (d) is deposited, and drying the same.

Description

TECHNICAL FIELD The present invention relates to a method for producing a nontoxic bee venom peptide,

(A) diluting a bee venom with a buffer solution; (b) adding a proteolytic enzyme to the diluted diluted solution of step (a), followed by enzymatic reaction; (c) coagulating the protein of the reactant by adding cooled ethanol to the reactant reacted in the step (b); (d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And (e) taking a supernatant from the reaction product in which the solidified product of step (d) has been precipitated, followed by drying. The method for producing allergen-free purified beeswax and the method for producing allergen ≪ / 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 detoxified tablet bee venom, in which the allergen component is removed using a proteolytic enzyme 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- And to provide a method for producing a detoxified tablet bee venom using a proteolytic enzyme that can be removed.

In order to solve the above-described problems, the present invention provides a method for preparing a bee venom, comprising: (a) diluting a bee venom with a buffer solution; (b) adding a proteolytic enzyme to the diluted diluted solution of step (a), followed by enzymatic reaction; (c) coagulating the protein of the reactant by adding cooled ethanol to the reactant reacted in the step (b); (d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And (e) removing the supernatant from the reaction product in which the solidified product of step (d) has been precipitated and drying the supernatant, wherein the purified bee venom is prepared by removing the allergen.

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) diluting the bee venom with a buffer solution;

(b) adding a proteolytic enzyme to the diluted diluted solution of step (a), followed by enzymatic reaction;

(c) coagulating the protein of the reactant by adding cooled ethanol to the reactant reacted in the step (b);

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

(e) removing the supernatant from the reaction product in which the solidification product precipitated in step (d) has been precipitated, and drying the supernatant, wherein the purified bee venom is produced by removing the allergen.

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.

In the method of manufacturing the tablet bee venom of the present invention, the buffer solution of step (a) is preferably EDTA, Tris, phosphate or citrate buffer solution at a pH of 5 to 9 and a concentration of 1 to 150 mM. , More preferably a phosphate buffer at a pH of 6 to 9 and a concentration of 50 to 150 mM, most preferably a phosphate buffer at a pH of 7.5 and 100 mM. The buffer solution can remove Ca ions acting as a cofactor of phospholipase A2, an allergen component of bee venom. In addition, if oxygen is present in the buffer solution, an oxidation reaction occurs during the reaction, and the potency of the obtained bee venom may be lowered. Therefore, in order to remove oxygen in the buffer solution, the buffer solution may be heated at a temperature of 90 ° C or more for 5 to 30 minutes, or oxygen may be removed by replacing the purified nitrogen gas in the buffer solution for 30 minutes or more.

In addition, in the method of manufacturing the tablet bee venom of the present invention, the dilution of the step (a) may be diluted to 8-12% (w / v) by adding a buffer solution of pH 6-9 to the bee venom, Preferably bee venom can be diluted to 10% (w / v) by adding pH 7.5 buffer solution. Peptide components represented by melitin in bee venom are about 50% of the solid components of bee venom, and the remaining 50% are allergen-inducing protein components such as phospholipase A2. The total solids content of these two ingredients is about 30% of bee venom. Therefore, the content of allergen protein component in 1 mL of bee venom can reach about 150 mg. In the present invention, the bee venom is diluted in the buffer solution to a concentration of about 10%, so that the content of allergen-induced protein in 1 mL of the reactant is about 15 mg.

In addition, in the method for producing bee venom of the present invention, the protease of step (b) may be selected from the group consisting of animal proteases such as trypsin and pancreatase, vegetable proteinases such as bromelamine and papaya, , And more preferably at least one protein hydrolyzing enzyme selected from the group consisting of Alcalase. The alkalase can effectively remove the allergen component in bee venom as compared with other proteolytic enzymes. The use concentration can be preferably 0.8 to 1.2 unit / mL, more preferably 1 unit / mL. If the concentration is above the above range, melanin and the like, which are effective components of bee venom, are decomposed and the content of the active ingredient is lowered.

Further, in the method of the present invention, in the step (c), the protein of the reaction product may be coagulated by adding ethanol cooled to -15 to -25 ° C to the enzyme-reacted reaction product, Can coagulate the protein of the reaction product by adding ethanol cooled to -20 ° C to the enzyme reaction-reacted product. For cooling the reactants, ethanol cooled at -20 ° C, dry ice addition, liquid nitrogen addition, etc. may be used. However, since bee venom peptides are soluble in ethanol, it is preferable to use ethanol cooled at -20 ° C. The ethanol used may be ethanol of 95% purity. The ethanol may be added to the reactant so that the ethanol content is 30-65%, more preferably 50%. When the amount of ethanol added is less than the above range, the effect of protein coagulation is small and the degree of protein coagulation does not change even if the added amount exceeds the above range.

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

(a) diluting the bee venom with 8 to 12% (w / v) by adding a pH 6-9 buffer solution;

(b) adding an alkalase to the diluted diluted solution of step (a) in an amount of 0.8 to 1.2 unit / ml, and then performing enzyme reaction at 34 to 40 ° C for 5 to 20 minutes;

(c) coagulating the protein of the reaction product by adding ethanol cooled at -15 to -25 ° C to the reaction product of the step (b);

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

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

More specifically,

(a) diluting to 10% (w / v) by adding pH 7.5 buffer solution to bee venom;

(b) adding 1 unit / mL of an alcalase to the diluted diluted solution of step (a), followed by enzymatic reaction at 37 ° C for 10 minutes;

(c) coagulating the protein of the reaction product by adding ethanol cooled at -20 ° C to the reaction product obtained in the step (b);

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

(e) removing the supernatant from the reaction product in which the solidification product of step (d) has been precipitated, followed by drying.

The present invention also provides a tablets bee venom with allergy components removed by the above method.

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) Bee venom was diluted to 10% (w / v) by adding pH 7.5 and 1 mL of 100 mM phosphate buffer to 0.1 g of solid 30% (w / w) bee venom purchased from Korean Beekeeping Association.

(b) An alkalase (Alcalase, Novozyme) was added to the diluted diluted solution of step (a) at 1 unit / mL, followed by enzymatic reaction at 37 ° C for 10 minutes.

(c) 1 ml of 95% (v / v) ethanol cooled to -20 캜 was added to the reaction product of the step (b) to thereby coagulate the protein of the reaction product.

(d) The coagulated reaction product of step (c) 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, followed by low-vacuum drying (Speed Vac) to remove ethanol in the solution to obtain 1 mL of purified bee venom phosphate buffer solution.

Example  One: allergy  Confirming removal of trigger substances

(Preparation Example 1-3) prepared by differently manufacturing the purified beacon (Preparation Example 1-2) prepared by manufacturing the purified beacon according to the method of Production Example 1 and manufactured by differently purchasing the beacon, In the bee venom of tablets, the changes of the components of allergenic substances phospholipase A and hyaluronidase were investigated. The presence or absence of the allergen component was analyzed by liquid chromatography and the column was analyzed using Sephadex 75, Source 15RPC ST, C18, RP-amide and Peptide ES-C18, and the content of melittin, apamin, phospholipase A2, The content of tin is determined by the following formula, and the results are shown in Table 2 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 Tablet bee venom (Preparation Example 1) 61 2.0 0 0 Tablet bee venom (Preparation Example 1-2) 60 2.1 0 0 Tablet bee venom (Preparation Example 1-3) 61 2.0 0 0

Preparation Example 1 Tablet pre-bean purchased from Korea Beek Bong Kang and purified bee venom using alcholase purchased from Novo Co.

Production Example 1-2: Purification of bee venom from bee venom purchased from Ivy Farming Association

Production Example 1-3: Purified beacon using alcalase purchased from Sigma Chemical Co.

As a result of confirming the removal of allergen-inducing phospholipase A and hyaluronidase in the tablet bee venom, as shown in the above Table 2, it was confirmed that the purified bee venom of Preparation Example 1-2, A2 and the hyaluronidase component were not detected, and it was confirmed that the allergen component was not detected even in the preparation bee venous preparation of Production Example 1-3, which was produced with different kinds of alcalase. The purified bee venom produced by the method of Production Example 1 of the present invention was not detected in allergen-inducing substances, phospholipase A2 and hyaluronidase, and melilthin, an active ingredient of bee venom, , And 2% or more of apamin.

Example  2: PCA (Passive cutaneous anaphylaxis) response

In order to confirm the allergen inhibitory effect of the tablet bee venom prepared by the method of Production Example 1 of the present invention, PCA (passive cutaneous anaphylaxis) test was performed as described below.

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 PCA dose should be the same.

8) Administration: Sensitization was administered to the adenocarcinoma of the guinea pig, and the serum of the blood collected after sensitization was used as a PCA response. The PCA reaction was carried out using 2 animal-specific animals per each individual in the test serum on PCA Yagi day. The serum to be tested is serially diluted 2 times from 10 times to 5120 times with injection of physiological saline, and then 100 μl of each is injected into guinea pig embryos. After 4 hours of intradermal administration, the Yagi's stock solution containing 2% of Evan's blue was intravenously injected into the Fuji vein, causing a PCA reaction.

9) Observation and examination: General symptoms and weight test. 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) Determined as the potency (antibody).

As shown in the following Table 4, no specific symptoms and deaths of the bee venom according to the present invention were observed, and the results of the PCA reaction showed that the negative control group G1, G2 in bee venom, and G3 in high dose group were not observed. However, in the positive control group G4, the antibody titer was 5120 times, indicating a clear PCA response. From the above results, it was confirmed that the tablets bee venom prepared according to the present invention had no antigenicity.

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

Claims (5)

(a) diluting the bee venom with a buffer solution;
(b) adding a proteolytic enzyme to the diluted diluted solution of step (a), followed by enzymatic reaction;
(c) coagulating the protein of the reactant by adding cooled ethanol to the reactant reacted in the step (b);
(d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And
(e) removing the supernatant from the reaction product in which the solidified product of step (d) has been precipitated and drying the supernatant. The method according to claim 1, wherein the allergen component is phospholipase A2 and hyaluronidase. The method according to claim 1, wherein the protease of step (b) is Alcalase. The method of claim 3,
(a) diluting the bee venom with 8 to 12% (w / v) by adding a pH 6-9 buffer solution;
(b) adding an alkalase to the diluted diluted solution of step (a) in an amount of 0.8 to 1.2 unit / ml, and then performing enzyme reaction at 34 to 40 ° C for 5 to 20 minutes;
(c) coagulating the protein of the reaction product by adding ethanol cooled at -15 to -25 ° C to the reaction product obtained in the step (b);
(d) centrifuging the solidified reaction product of step (c) to precipitate a solidified product; And
(e) removing the supernatant from the reaction product in which the solidified product of step (d) has been precipitated and drying the supernatant. delete