WO2017222300A1 - Method for producing allergenic component-free purified bee venom, 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) diluting a bee venom by adding a buffer solution thereto; (b) adding a protease to the diluted solution diluted in step (a), and then having same undergo an enzymatic reaction; (c) coagulating a protein of the reactant of step (b) by adding cold ethanol to the reactant; (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, then drying same.

Description

Method for producing tablet bee venom with allergens removed and tablet bee venom with allergens

The present invention (a) adding a buffer solution to bee venom dilution; (b) adding proteolytic enzyme to the diluted solution of step (a) and then performing enzymatic reaction; (c) adding cooled ethanol to the reaction of step (b) to solidify the protein of the reaction; (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 drying the mixture, wherein the allergy component is prepared and the allergy prepared by the method. A tablet bee venom with components 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 a detoxified purified bee venom from which allergen components have been removed using the protease 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. The present invention provides a method for preparing detoxified purified bee venom using a protease that can be removed.

In order to solve the above problems, the present invention (a) adding a buffer solution to bee venom dilution; (b) adding proteolytic enzyme to the diluted solution of step (a) and then performing enzymatic reaction; (c) adding cooled ethanol to the reaction of step (b) to solidify the protein of the reaction; (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 then drying the prepared supernatant.

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.

Figure 1 shows the results of analyzing the electrophoresis pattern of the dilution diluted the crude bee venom in phosphate buffer and the purified bee venom phosphate buffer prepared by the method of Preparation Example 2.

PLA2: phospholipase A2

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

(a) diluting by adding a buffer solution to bee venom;

(b) adding proteolytic enzyme to the diluted solution of step (a) and then performing enzymatic reaction;

(c) adding cooled ethanol to the reaction of step (b) to solidify the protein of the reaction;

(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 then 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 preparation method of the purified bee venom of the present invention, the buffer solution of step (a) is preferably pH 5-9 and 1 ~ 150 mM concentration of EDTA, Tris, phosphate or citrate buffer solution And, more preferably, phosphate buffers at pH 6-9 and 50-150 mM concentrations, most preferably pH 7.5 and 100 mM phosphate buffers. The buffer solution can remove Ca ions acting as a cofactor of phospholipase A2, which is an allergic component of bee venom. In addition, if oxygen is present in the buffer solution, an oxidation reaction may occur during the reaction, thereby lowering the titer of bee venom obtained. 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 the purified nitrogen gas may be replaced with the buffer for 30 minutes or more to remove oxygen.

In addition, in the preparation method of the purified bee venom of the present invention, the dilution of step (a) is preferably diluted to 8 to 12% (w / v) by adding a pH 6-9 buffer solution to the bee venom, more Preferably, bee venom may be diluted to 10% (w / v) by adding pH 7.5 buffer. The peptide component represented by melittin in bee venom is about 50% of the solid component of bee venom, and the remaining 50% is an allergen protein component such as phospholipase A2. The combined solids add up to 30% of the bee venom. Therefore, the content of the allergic protein component in 1 mL of bee venom may amount to about 150 mg. In the present invention, bee venom is diluted in a buffer solution at a concentration of about 10% so that the content of allergen protein in 1 mL of the reaction is about 15 mg.

In addition, in the method for producing purified bee venom of the present invention, the proteolytic enzyme of step (b) is an animal-derived protease such as trypsin and pancrease, vegetable protease such as bromelain and papaya and microbial-derived protease It may be one or more proteolytic enzymes selected from the group consisting of, more preferably Alcalase (Alcalase). Alcalase may be Serine endopeptidase. Alcalase was able to effectively remove allergen components in bee venom compared to other proteolytic enzymes, the use concentration is preferably 0.8 ~ 1.2 unit / mL, more preferably 1 unit / mL can be used. Treatment with the concentration could effectively remove allergens of bee venom, but if the use concentration exceeds the above range, there is a problem in that the active ingredient content is degraded by decomposing melatine, which is an active ingredient of bee venom.

In addition, in the preparation method of the purified bee venom of the present invention, step (c) is preferably added to the reactant cooled to -15 ~ -25 ℃ ethanol to coagulate the protein of the reactant, more preferably to the reactant Ethanol cooled to −20 ° C. may be added to coagulate the protein of the reaction. There may be methods such as ethanol cooled to −20 ° C., dry ice addition, liquid nitrogen addition, etc. to cool the reactants. However, since bee venom peptides are soluble in ethanol, ethanol cooled to −20 ° C. is preferably used. In this case, the ethanol used is 95% pure ethanol, the ethanol may be added to the reactant so that the ethanol content of 30 ~ 65%, more preferably 50%. If the amount of ethanol is less than the above range, the protein coagulation effect is small, and even if the amount exceeds the above range, the degree of protein coagulation does not change.

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

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

(b) adding Alcalase (0.8-1.2 unit / mL) to the diluted dilution of step (a) and enzymatically reacting at 34-40 ° C. for 5-20 minutes;

(c) adding ethanol cooled to -15 to -25 ° C to the reactant of step (b) to solidify the reactant protein;

(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 drying.

In the method for preparing purified bee venom of the present invention, the step (b) may further comprise the step of heating the reaction reacted in the enzyme, more specifically

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

(b) adding Alcalase (0.8-1.2 unit / mL) to the diluted dilution of step (a) and enzymatically reacting at 34-40 ° C. for 5-20 minutes;

(c) heating the reactant reacted in step (b) at 75 to 85 ° C. for 10 to 20 minutes;

(d) coagulating the reactant protein by adding ethanol cooled to −15˜-25 ° C. to the heated reactant of step (c);

(e) centrifuging the solidified reactant of step (d) to precipitate the coagulum; And

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

By heat-treating the reactant reacted under the conditions described above, the enzyme activity of the protease contained in the reactant is stopped, and heat denatured phospholipase A2 and hyaluronidase, which may be present in the reaction product. .

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 substance), 50% of melittin, an active ingredient of bee venom, more than 1% of apamin Characterized in that. In addition, in the toxicity evaluation in mice, the LD50 value was significantly higher than that of the bee venom before purification, indicating little toxicity. 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 Tablet Bee Venom with Allergens Removed

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

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

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

(d) The coagulated reactant of step (c) was centrifuged to precipitate a coagulated product.

(e) The supernatant was taken from the reactant in which the coagulum of step (d) was precipitated, followed by low temperature vacuum drying (Speed Vac) to remove ethanol from the solution to obtain 1 mL of purified bee venom phosphate buffer.

Preparation Example 2 Preparation of Tablet Bee Venom with Allergens Removed

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

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

(c) The reaction product of step (b) was heated at 80 ° C. for 15 minutes.

(d) 1 mL of 95% (v / v) ethanol cooled to −20 ° C. was added to the heated reaction product of step (c) to coagulate the protein of the reaction product.

(e) The coagulated reactant of step (d) was centrifuged to precipitate a coagulum.

(f) The supernatant was taken from the reactant in which the coagulum of step (e) was precipitated, followed by low temperature vacuum drying (Speed Vac) to remove ethanol from the solution to obtain 1 mL of purified bee venom phosphate buffer.

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 2 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	0.18	0.21	0.71	0.83
Propionibacterium acne	0.08	0.10	0.31	0.40

As a result, the purified bee venom of Preparation Example 2 compared with the bee venom before purification, although the MIC and MBC slightly increased, the increase was not large, it was confirmed that the original antibacterial effect is maintained as it is.

Example 2: Changes in Components of Tablet Bee Venom with Different Bee Venom Types

Prepared bee venom prepared by dilution of crude bee venom in phosphate buffer solution and the method of Preparation Example 1, but manufactured by different bee venom purchase destination (Preparation Example 1-2) and alcalase purchase destination The purified bee venom (Preparation Example 1-3) was used to investigate the component changes of allergens phospholipase A and hyaluronidase in the purified bee venom.

Allergen removal was analyzed using liquid chromatography, column was analyzed using Sephadex TM 75, Source 15RPC ST, C18, RP-amide, Peptide ES-C18, melittin, apamin, phospholipase A2, hyaluro The content of the nidase is obtained by the following formula, 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)

% Change in Component of Purified Bee Venom

Component Content (%)	Melittin	Apamin	Phospholipase A2	Hyaluronidase
Bee Venom Before Purification	65	2.5	11	1.8
Refined Bee Venom (Manufacturing Example 1)	61	2.0	0	0
Purified Bee Venom (Manufacturing Example 1-2)	60	2.1	0	0
Purified Bee Venom (Manufacturing Example 1-3)	61	2.0	0	0

Preparation Example 1: Purified bee venom purchased from Korea Beekeeping Association and purified bee venom using alcalase purchased from Novo Co.

Preparation Example 1-2: Bee Venom before Purification from IBF Co., Ltd. and Bee Venom using Alcalase from Novo Co.

Preparation Example 1-3: Bee Venom before Purification from Korea Beekeeping Association and Tablet Bee Venom using Alcalase from Sigma

As a result of confirming the removal of phospholipase A and hyaluronidase, which are allergens from the purified bee venom, as shown in Table 3 above, phospholipase from the purified bee venom of Preparation Example 1-2 prepared by different bee venom types The A2 and hyaluronidase components were not detected, and it was confirmed that the allergen component was not detected even in the purified bee venom of Preparation Example 1-3 prepared by different kinds of alcalase. Purified bee venom prepared by the method of Preparation Example 1 of the present invention did not detect allergen-producing phospholipase A2 and hyaluronidase, and at least 60% of melittin, an active ingredient of bee venom, was detected. It was confirmed that 2% or more of apamin was included.

Example 3: Component Changes of Purified Bee Venom with Different Proteolytic Enzymes

As a result of analyzing the electrophoretic pattern of the dilution diluted the crude bee venom in the phosphate buffer solution and the purified bee venom phosphate buffer solution prepared by the method of Preparation Example 2. The purified bee venom of the present invention showed complete elimination of phospholipase A2 in the electrophoresis pattern, and the concentration of melittin was not significantly changed compared to the untreated bee venom.

In addition, purified bee venom phosphate buffer solution prepared by the method of Preparation Example 2 and purified bee venom prepared by the method of Preparation Example 2, but the purified bee venom phosphate buffer solution prepared by varying the type of protease in step (b) The dry powder was analyzed for melatin, apamin, phospholipase A2 and hyaluronidase components.

% Change in Component of Purified Bee Venom

Ingredient Content (%)	Melittin	Apamin	Phospholipase A2	Hyaluronidase
Bee Venom Before Purification	65	2.5	11	1.8
Preparation Example 2	52	1.8	0	0
Comparative Example 1 (Neutrase)	44	1.2	2.5	0.2
Comparative Example 2 (Protamex)	45	1.4	3.4	0.5

As a result, the purified bee venom of Preparation Example 2 did not detect phospholipase A2 and hyaluronidase, which are allergens, and 50% or more of melittin, an active ingredient of bee venom, and It could be confirmed that it contains 1% or more. On the other hand, in the case of the purified bee venom prepared without using alcalase and using Neutrase or Protamex, the active ingredient content of bee venom is remarkably reduced compared to Preparation Example 2, and the allergen of bee venom is not completely removed. Could be confirmed.

Example 4: Passive cutaneous anaphylaxis reaction

In order to confirm the allergic inhibitory effect of the tablet bee venom prepared by the methods of Preparation Examples 1 and 2 of the present invention, the following PCA (Passive cutaneous anaphylaxis) test was carried out.

1) Test substance: Purified bee venom of Preparation Example 1 or Preparation Example 2

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).

PCA results of purified bee venom of Preparation Examples 1 and 2 (number of positive reaction animals / total number of experimental animals)

division	Dilution factor	G1	G2	G3	G4
Preparation Example 1	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
Preparation Example 2	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 confirming the allergic inhibitory effect of the tablet bee venom prepared by the methods of Preparation Examples 1 and 2, as shown in Table 6, the specific symptoms and death animals due to the tablet bee venom according to the present invention was not observed, PCA response results negative Blue and white spots were not observed in the bee venom low-dose group G2 and the high-dose group G3 as well as the control group G1. However, in the positive control group G4, the antibody titer was 5120-fold, indicating a clear PCA response. From the above results, it was finally confirmed that the purified bee venom prepared according to the present invention was not antigenic.

Claims (7)

1. (a) diluting by adding a buffer solution to bee venom;

   (b) adding proteolytic enzyme to the diluted solution of step (a) and then performing enzymatic reaction;

   (c) adding cooled ethanol to the reaction of step (b) to solidify the protein of the reaction;

   (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 then drying the prepared allergy component.
2. The method of claim 1, wherein the allergic component is phospholipase A2 and hyaluronidase.
3. The method of claim 1, wherein the proteolytic enzyme of step (b) is an alcalase.
4. The method of claim 1, further comprising the step of heating the reactant reacted in the step (b) of the method for producing a purified bee venom with allergen components removed.
5. The method of claim 3,

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

   (b) adding Alcalase (0.8-1.2 unit / mL) to the diluted dilution of step (a) and enzymatically reacting at 34-40 ° C. for 5-20 minutes;

   (c) adding ethanol cooled to -15 to -25 ° C to the reactant of step (b) to solidify the reactant protein;

   (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 then drying the prepared allergy component.
6. The method of claim 4, wherein

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

   (b) adding Alcalase (0.8-1.2 unit / mL) to the diluted dilution of step (a) and enzymatically reacting at 34-40 ° C. for 5-20 minutes;

   (c) heating the reactant reacted in step (b) at 75 to 85 ° C. for 10 to 20 minutes;

   (d) coagulating the reactant protein by adding ethanol cooled to −15˜-25 ° C. to the heated reactant of step (c);

   (e) centrifuging the solidified reactant of step (d) to precipitate the coagulum; And

   (f) 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 (e) is precipitated and drying.
7. A purified bee venom from which allergen components prepared by the method of any one of claims 1 to 6 have been removed.

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