WO2017222305A1 - Method for producing allergenic component-free purified bee venom using yolk, and allergenic component-free purified bee venom produced thereby - Google Patents

Abstract

The present invention relates to a method for producing an allergenic component-free purified bee venom, and an allergenic component-free purified bee venom produced thereby, the method enabling the production of an allergenic component-free purified bee venom by comprising the steps of: (a) preparing a buffer solution containing yolk; (b) adding a bee venom to the buffer solution prepared in step (a), and then stirring same; (c) coagulating a protein of the stirred reactant of step (b) by cooling the reactant while adding cold ethanol thereto; (d) precipitating the coagulated material by centrifuging the coagulated reactant of step (c); and (e) obtaining a supernatant from the reactant of step (d) in which the coagulated material is precipitated, and concentrating and drying same.

Description

Preparation method of purified bee venom from which allergic component is removed using egg yolk and purified bee venom from which allergic component prepared by the above method is removed

The present invention (a) preparing a buffer containing egg yolk; (b) adding bee venom to the buffer solution prepared in step (a) and then stirring; (c) solidifying the reactant protein by adding cooled ethanol while cooling the stirred reactant of step (b); (d) centrifuging the coagulated reactant of step (c) to precipitate the coagulum; And (e) taking the supernatant from the reactant in which the coagulum of step (d) is precipitated, and concentrating and drying the preparation. It relates to a tablet bee venom from which allergens have been removed.

Bee venom is a poison that bees have, and bee venom has long been recognized for its therapeutic effects. Recent studies have shown that bee venom has been shown to be effective in anti-cancer, arthritis relief, atherosclerosis relief, back pain relief, treatment of skin wounds, immunopotentiation, anti-inflammatory action, lowering blood pressure and increasing the regeneration of lymphocytes and red blood cells in the blood. Wrinkle improvement, whitening and the like have also been found for the purpose.

The main components of bee venom and their pharmacological functions are shown in Table 1 below.

ingredient	Pharmacological function	Component ratio (%)
Peptides (meltin, apamin)	Sedative, immune	Around 50%
Protein (phospholipase A2)	Cell destruction, allergy	15%
Amine (histamine)	Lowering blood pressure	1 to 2%

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

Among the bee venom components, proteins include phospholipase A2 (Phospholipase A2, PLA2), hyaluronidase, phosphatase, and α-glucosidase having a molecular weight of 13 kDa or more. Consists of physiological activities such as blood cell membrane destruction, blood coagulation, vasodilation and permeation, blood circulation, and protein hydrolysis.

In particular, phospholipase and hyaluronidase are bee venom constituents that cause a strong allergic reaction and 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 are known to remove the allergen from bee venom. For example, Korean Patent No. 1382404 prepares a bee venom solution by dissolving bee venom, adding the bee venom solution to a primary secondary amine (PSA) adsorbent to make a mixture, and then fine filtration to remove impurities. A method for mass production of bee venom is described. In addition, Korean Patent No. 1364506 removes phospholipase from bee venom by using an ultrafiltration membrane having a molecular weight cut-off size of 10 kDa or more, and at least 4% by weight of apamin and 50 melitines in purified bee venom. A method is disclosed for containing at least% by weight. In addition, Korean Patent No. 1608045 discloses a technique for removing phospholipase in bee venom and separating only melittin using a cation exchange resin. However, there is no method for producing tablet bee venom from which allergic components are removed using egg yolk as in the present invention.

The present invention has been made by the above-described demands, and an object of the present invention is to effectively maintain the active ingredients of bee venom such as melittin and apamin, while allergy components of phospholipase A2 and hyaluronidase are effectively and completely. It is to provide a method for producing purified bee venom that can be removed.

In order to solve the above problems, the present invention (a) preparing a buffer containing egg yolk; (b) adding bee venom to the buffer solution prepared in step (a) and then stirring; (c) solidifying the reactant protein by adding cooled ethanol while cooling the stirred reactant of step (b); (d) centrifuging the coagulated reactant of step (c) to precipitate the coagulum; And (e) taking the supernatant from the reactant in which the coagulum of step (d) is precipitated, and concentrating and drying.

The present invention also provides a purified bee venom from which allergen components prepared by the above method are removed.

Purified bee venom prepared by the method of the present invention can completely remove the phospholipase A2 and hyaluronidase which induce a strong allergic reaction, thereby preparing a purified bee venom which is essentially safe from the allergic reaction, By keeping it intact, there is an advantage that it can be used more safely for various pharmacological functions.

In order to achieve the object of the present invention, the present invention

(a) preparing a buffer solution containing egg yolk;

(b) adding bee venom to the buffer solution prepared in step (a) and then stirring;

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

(d) centrifuging the coagulated reactant of step (c) to precipitate the coagulum; And

(e) providing a method for preparing purified bee venom from which allergen components are removed, comprising the step of taking the supernatant from the reactant in which the coagulum of step (d) is precipitated and concentrating and drying.

In the preparation method of the purified bee venom of the present invention, the allergic component may be, but is not limited to, phospholipase A2 and hyaluronidase.

In addition, in the manufacturing method of the purified bee venom of the present invention, the egg yolk of the step (a) is a raw egg yolk derived from algae eggs, such as eggs, duck eggs, quail eggs, etc. can be used that obtained by separating the egg white and egg yolk by activating the eggs have.

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

In addition, in the preparation method of the purified bee venom of the present invention, step (b) is preferably added to the buffer solution bee venom 0.5 ~ 1.5% (w / v) and then stirred for 5 to 20 minutes at 30 ~ 50 ℃. More preferably, 1% (w / v) bee venom is added to the buffer solution, and then stirred at 35 ° C. for 10 minutes. The addition of bee venom to the buffer solution under the above conditions and stirring allowed allergens contained in bee venom to bind to the yolk lipoprotein effectively.

In addition, in the preparation method of the purified bee venom of the present invention, the step (c) is preferably added to the reactant protein by adding ethanol cooled to -10 ~ -20 ℃ while cooling the stirred reaction to -5 ~ -15 ℃ It is possible to coagulate, and more preferably, the reactant can be coagulated by adding ethanol cooled to −15 ° C. while cooling the stirred reaction to −10 ° C. When the coagulation of the yolk lipoprotein combined with the allergen, as described above, it is possible to easily remove the allergen by centrifugation to precipitate the coagulum.

The manufacturing method of the refined bee venom of this invention is more concretely.

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

(b) adding bee venom 0.5-1.5% (w / v) to the buffer solution prepared in step (a) and stirring for 5-20 minutes at 30-50 ° C .;

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

(d) centrifuging the coagulated reactant of step (c) to precipitate the coagulum; And

(e) taking the supernatant from the reactant in which the coagulum of step (d) is precipitated, and may include the step of concentrating and drying,

More specifically

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

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

(c) solidifying the reactant protein by adding ethanol cooled to −15 ° C. while cooling the stirred reactant of step (b) to −10 ° C .;

(d) centrifuging the coagulated reactant of step (c) to precipitate the coagulum; And

(e) taking the supernatant from the reactant in which the coagulum of step (d) is precipitated and may include the step of concentrating and drying.

The present invention also provides a purified bee venom from which allergen components prepared by the above method are removed. Purified bee venom of the present invention was not detected phospholipase A2 and hyaluronidase (allergen-inducing substances), 60% or more of melittin, active ingredient of bee venom, 2% or more of apamin Characterized in that. In addition, the purified bee venom of the present invention showed a similar antimicrobial effect when compared to bee venom before purification in antibacterial experiments against Staphylococcus aureus strain and Propionibacterium acne strain. As a result of confirming the allergic inhibitory effect of the purified bee venom in the guinea pigs, no abnormal change was observed in the guinea pigs, and it was confirmed that there was no antigenicity.

Hereinafter, the embodiment of the present invention will be described in detail. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Preparation Example 1 Preparation of Allergen-Removed Tablet Bee Venom

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

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

(c) The stirred reaction product of step (b) was transferred to an ice pocket of -10 ° C, and then cooled, and slowly added 95% (v / v) ethanol cooled to -15 ° C to coagulate the protein of the reaction product.

(d) The coagulated reactant at -10 ° C of step (c) was centrifuged to precipitate a coagulated product.

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

Example 1 Antimicrobial Activity of Tablet Bee Venom

In order to confirm that the physiological activity of the purified bee venom obtained in Preparation Example 1 was maintained, the antimicrobial activity against Staphylococcus aureus strain and Propionibacterium acne strain was determined by MIC (minimum inhibition concentration). And MBC (minimum bacteriocidal concentration) were analyzed.

Comparison of antimicrobial activity between bee venom before purification and bee venom after purification

Strain	MIC (μg / ml)		MBC (μg / ml)
	Bee Venom Before Purification	Bee venom after purification	Bee Venom Before Purification	Bee venom 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 Preparation Example 1 retained its antibacterial effect as compared with bee venom before purification.

Example 2: Component Changes of Tablet Bee Venom

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

As analytical device, liquid chromatography, Sephadex TM 75, Source 15RPC ST, C18, RP-amide, Peptide ES-C18 were used, and melittin, apamin, phospholipase A2 and hyaluronidase in bee venom were used. The content was determined by the following equation, and the results are shown in Table 3 below.

Standard product intake amount (mg) X (purity / 100 of standard product) X (the desired ingredient peak area in bee venom) / peak area of standard product) X (100 / bee venom amount)

Changes in the Components of Tablet Bee Venom

Ingredient Content (%)	Melittin	Apamin	Phospholipase A2	Hyaluronidase
Bee Venom Before Purification	65	2.5	11	1.8
Preparation Example 1	63	2.7	0	0

As a result, the purified bee venom of the present invention did not detect phospholipase A2 and hyaluronidase, which are allergens, and 60% or more of melittin, which is an active ingredient of bee venom, and 2 aphamines. It was confirmed that it contains more than%.

Example 3: Allergic inhibitory effect test of tablet bee venom

In order to confirm the allergic inhibitory effect of the purified bee venom according to the present invention, the following PCA (Passive cutaneous anaphylaxis: passive skin anaphylaxis) test was performed.

1) Test substance: Purified bee venom of Preparation Example 1

2) excipient: saline injection

3) Positive control: Ovalbumin (from egg)

4) Positive Control Excipient: Physiological Salt Injection

5) Test animals: 300-380 g SPF guinea pig

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

Test Group Composition and Dose Determination

group	The number of animals	Dosage amount (ml / kg)	Dose (mg / kg)
G1 (excipient control group)	5	One	0
G2 (low dose group)	5	One	0.025
G3 (high dose group)	5	One	0.05
G4 (OVA + adjuvant group)	5	One	2.5

7) Dose Determination: The sensitization and PCA doses were equal.

8) Administration: The sensitization was administered to the cervix of guinea pigs, and the serum of the blood collected after sensitization was used to induce PCA response. Test sera on the PCA challenge day performed a PCA response using two causing animals per serum per subject. Test serum was serially diluted 2-fold from 10 to 5120-fold with physiological saline injection solution, and then 100 μl of each was administered into guinea pig-distributed skin. After 4 hours of intradermal administration, a causative antigen solution containing 2% Evan's blue was administered intravenously to cause a PCA response.

9) Observation and examination: General symptoms and body weight were examined. The determination of the PCA response was carried out 30 minutes after the PCA reaction was induced by slaughtering the test animal by ether anesthesia, and then the guinea pig-dorned skin was cut out to observe the presence or absence of alum. In the case of blue and white patches, the long and short diameters were measured, and the mean value calculated as (long diameter + short diameter) / 2 was 5mm or more.The positive serum dilution factor (maximum dilution factor) was found to be positive. Titer (antibody titer).

10) Test result

General symptoms: No specific symptoms and deaths caused by bee venom according to the present invention were observed.

Body weight change: Body weight change by bee venom according to the present invention was not observed.

-PCA determination: The antigenicity of bee venom according to the present invention was carried out with the induction of PCA response using guinea pigs, and the results are shown in Table 5 below.

PCA results (number of positive animals / total experimental 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

As a result of the PCA response, blue spots were not observed in the negative control group G1, 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 fold, indicating a clear PCA response. As a result, it is determined that the purified bee venom prepared according to the present invention is not antigenic.

Claims (5)

1. (a) preparing a buffer solution containing egg yolk;

   (b) adding bee venom to the buffer solution prepared in step (a) and then stirring;

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

   (d) centrifuging the coagulated reactant of step (c) to precipitate the coagulum; And

   (e) a method for producing a purified bee venom from which allergen components are removed, comprising the step of taking the supernatant from the reactant in which the coagulum of step (d) is precipitated and concentrating and drying.
2. The method of claim 1, wherein the allergic component is phospholipase A2 and hyaluronidase.
3. The method according to claim 1, wherein the buffer solution of step (a) is a pH 7-9 buffer containing egg yolk 1-10% (w / v).
4. The method of claim 1,

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

   (b) adding bee venom 0.5-1.5% (w / v) to the buffer solution prepared in step (a) and stirring for 5-20 minutes at 30-50 ° C .;

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

   (d) centrifuging the coagulated reactant of step (c) to precipitate the coagulum; And

   (e) a method for producing a purified bee venom from which allergen components are removed, comprising the step of taking the supernatant from the reactant in which the coagulum of step (d) is precipitated and concentrating and drying.
5. Tablet bee venom from which the allergic component prepared by the method of any one of claims 1 to 4 has been removed.