KR20160057662A - Method for seperating non-allergenic bee venom - Google Patents

Abstract

The present invention relates to a separation method of purified bee venom, with inactivated phospholipase A2 and hyaluronidase. The separation method of purified bee venom comprises the steps of: i) diluting 1g of purified bee venom in 50-150 ml of sterilized and distilled water; ii) heating and boiling the diluted and purified bee venom solution for one to eight hours at the temperature of 65-85°C in a constant-temperature water tank; iii) obtaining a cleaned solution by filtering the boiled and purified bee venom with 0.45 μm of a membrane filter; and iv) freeze-drying the cleaned solution.

Description

METHOD FOR SEPERATING NON-ALLERGENIC BEE VENOM BACKGROUND OF THE INVENTION 1. Field of the Invention < RTI ID = 0.0 >

The present invention relates to a method of separating purified bee venom in which an allergenic substance including phospholipase A2 and hyaluronidase is inactivated.

Bee venom is composed of more than 40 substances including peptides and proteins (eg enzymes) and active amines. These components are used for anti-inflammatory, anti-inflammatory and antimicrobial actions, as well as promoting the secretion of pituitary hormones and promoting blood circulation. It is known to be effective.

Among the bee venom components, the protein is a phospholipase A2 (PLA2), hyaluronidase, phosphatase, and alpha-glucosidase having a molecular weight of about 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 A2 and hyaluronidase are known to be a bee venom composition that induces a potent allergic response and can cause very serious safety problems for users with hypersensitivity to bee venom (Stefan Bogdanov; Bee Venom: Composition, Health, Medicine: A Review, Bee Product Science (2011))

Due to these side effects, the effective amount of bee venom is limited, and as the amount of bee venom is limited, the effect of physiological activity of bee venom is also limited, resulting in a problem that it is not fully realized.

Open Patent Publication No. 10-2012-0003178 relates to a composition for whitening and moisturizing skin containing bee venom extract, which comprises pre-treating bee venom or extracting bee venom extract from bee venom, and skin whitening and skin moisturizing effect Lt; RTI ID = 0.0 > cosmetic < / RTI >

However, the above conventional techniques do not include the process of removing phospholipase A2 and hyaluronidase, which induce a strong allergic reaction. Thus, a composition containing the bee venom extract prepared by the conventional technique as an active ingredient and a cosmetic composition Can cause very serious safety problems when used by a user who is sensitive to bee venom.

In addition, in the past, a method of adding an allergic reaction inhibitor to inhibit the allergic reaction has been devised. However, this method does not fundamentally solve the allergies and causes a side effect due to the allergic reaction inhibitor.

In addition, there are HPLC (high performance liquid chromatography) or gel filtration methods for separating and removing phospholipase A2 and hyaluronidase from conventional bee venom, but as the separation or purification process proceeds, There is a problem that mass production is difficult.

Therefore, the present inventors have developed a method for easily inactivating allergen-inducing substances, phospholipase A2 and hyaluronidase, from purified bee venom.

Korean Patent Publication No. KR 2012-0003178A

It is an object of the present invention to provide a method for separating tablets bee venom characterized in that phospholipase A2 and hyaluronidase are inactivated.

Another object of the present invention is to provide a taboo beeper which is characterized in that the allergen causing substance including phospholipase A2 and hyaluronidase isolated by the above method is inactivated.

In order to achieve the above object,

i) diluting 1 g of tablets bee venom to 50 to 150 ml of sterile distilled water;

ii) soaking the diluted purified beeotene solution in a constant temperature water bath at a temperature of 65 to 85 DEG C for 1 to 8 hours;

iii) filtering the beaked purified beeotene solution with a 0.45-μm membrane filter to obtain a supernatant; And

iv) lyophilizing said subculture solution;

Wherein the phospholipase A2 and the hyaluronidase are inactivated. The present invention also provides a method for separating purified bee venom.

As used herein, the term "bee venom (BV)" is a mixture of acidic and basic secretions produced in the abdomen of a bee (Apis mellifera), which is in the form of a colorless bitter liquid, the main ingredient of which is melittin such as melittin, apamin, and mast cell degranulating (MCD) peptides and enzymes, phospholipase A2 (PLA2), histamine and dopamine, and others And contains trace amounts of components. In embodiments of the present invention, it refers to the major components isolated from domestic beekeeping products.

As used herein, the term " purified bee venom " can be obtained by dissolving bee venom collected from bees or the like with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, and then filtering and deactivating impurities and lyophilization . In the embodiment of the present invention, the bee venom was collected by using a bee venom collecting device of a bee of a Western bee ( Apis mellifera L. ), and purified bee venom was recovered using the simple purification method of bee venom.

In an embodiment of the invention, 1 g of the recovered tablet bee venom can be diluted preferably in 50 to 150 ml of sterile distilled water, and most preferably in 90 to 110 ml of sterile distilled water.

The diluted tablet bee venom can be boiled using a constant temperature water bath. In the embodiment of the present invention, the allophilous substances phospholipase A2 and hyaluronidase may be inactivated by the warm water condition using the constant temperature water bath. In an embodiment of the present invention, the bathing conditions can be carried out at a temperature of 65 to 85 캜 in a constant temperature water bath for 1 to 8 hours, most preferably at 65 캜 for about 4 hours. If it is outside the above range, the allergenic substances phospholipase A2 and hyaluronidase may not be completely inactivated. With the above method of the present invention, the allergen-inducing phospholipase A2 and hyaluronidase can be inactivated.

In the prior art, a method of separating and removing phospholipase A2 and hyaluronidase from bee venom using high performance liquid chromatography (HPLC) or gel filtration has been used. However, when this method is used, the desired allergen The components of bee venom other than the bee venom are separated and removed, so that the separation or purification process progresses, and the yield is rapidly lowered, and mass production is difficult. However, the present invention uses a method of inactivation, rather than separating and removing the allergen-inducing substance, and is characterized in that the yield of tablets beeing is very high by accurately inactivating phospholipase A2 and hyaluronidase.

The supernatant is filtered through a 0.45-μm membrane filter to obtain a supernatant, whereby the allergen-inducing phospholipase A2 and hyaluronidase can be completely inactivated to obtain a purified bee venom.

In an embodiment of the present invention, after the step iii), the obtained supernatant is subjected to high performance liquid chromatography (HPLC) to confirm the presence of an allergen causing substance including phospholipase A2 and hyaluronidase; . ≪ / RTI > In an embodiment of the present invention, UPLC (Ultra Performance Liquid chromatography) may be additionally used in addition to the above-mentioned high performance liquid chromatography in order to confirm the presence of allergen in the bee venom.

After confirming that the phospholipase A2 and hyaluronidase, which are the allergen substances of the tablet bee venom, are inactivated, the supernatant is lyophilized to obtain the tablets bee poisoning in which the allergen material is inactivated.

The present invention also provides a tablet bee venom characterized in that the phospholipase A2 and hyaluronidase isolated by the above method are inactivated. In an embodiment of the present invention, the tablet bee venom may be characterized by containing at least 60% by weight of melittin and at least 2% by weight of an acanthin as an active ingredient. The allergenic substances phospholipase A2 and hyaluronidase Is 0% by weight.

In an embodiment of the present invention, purified bee venom, in which phospholipase A2 and hyaluronidase are inactivated, was isolated by the above method and purified allergen-inactivated purified bee venom was used to examine the allergic inhibitory effect As a result, no abnormal changes were observed in guinea pigs and no antigenicity was confirmed.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

With the separation method of the present invention, it is possible to easily and easily separate the tablet bee venom in which phospholipase A2 and hyaluronidase are inactivated. In addition, bee venom, in which the allergen is inactivated, can alleviate pain and itching when injecting blood vessels and muscles with natural antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a process of separating purified bee venom in which an allergenic substance including phospholipase A2 and hyaluronidase is inactivated according to an embodiment of the present invention. FIG.
FIG. 2 is a photograph of a guinea pig subjected to a passive cutaneous anaphylaxis (PCA) staining reaction in which purified bee venom is inactivated in accordance with an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example  One. From the tablets 포스폴화  A2 and hyaluronidase Component separation

In order to inactivate allergenic substances phospholipase A2 and hyaluronidase in tablet bee venom, purified bee venom was isolated as follows.

Apis mellifera L. ) were collected by bee venom collecting device. The collected bee venom was recovered from the bee venom using a simple purification method of bee venom disclosed in Korean Patent No. 10-0758814. The obtained tablet bee venom inhibited the activity of phospholipase A2 and hyaluronidase which cause pain and itching accompanied by blood vessel and muscle injection by the processor as shown in FIG.

Specifically, 1 g of tablets bee venom was diluted in 100 ml of sterilized distilled water.

Then, the diluted bee venom solution was treated at intervals of 10 ° C from 50 to 80 ° C in a constant temperature water bath at intervals of 1 hour and 8 hours. The temperature and time interval were then reduced to find the optimal temperature and time. The optimum temperature was about 65 ° C and the time was about 4 hours.

The beeotene solution obtained after the soaking treatment was filtered with a 0.45 μm membrane filter to recover the supernatant. The recovered bee venom solution was subjected to high performance liquid chromatography to confirm the presence of allergenic substances. After confirmation, the bee venom solution with inactivated allergen was lyophilized to obtain 0.8 g of allergen inactive bee venom.

Experimental Example  One. 포스폴화  A2 and hyaluronidase Inactivity check

Melittin (From bee venom.), Apamin (From bee venom), phospholipase A2 (From bee venom), and hyaluronidase, which are the main components of bee venom according to the temperature and time treatment described in Example 1 above (Hyaluronidase) were analyzed.

Liquid chromatography (AKTA explorer) and UPLC (Ultra performance liquid chromatography) were used for the analysis.

The columns used were Sephadex TM75 and Source 15RPC ST columns, C18, RP-amide, and peptide ES-C18. Melitin, apamin, phospholipase A2 and hyaluronidase content in bee venom components were calculated as follows.

X (Purity of the standard product / 100) X (Peak area of the desired component in bee venom / Peak area of the standard product) X (100 /

Inert enzyme identification - Purified beet poison melittin, Afamin, phospholipase A2 and hyaluronidase content Component content (%)
Condition Melitin Apamin phospholipase A2 hyaluronidase Before processing 65 2.5 11 1.8 50 ℃ 8 hours 65 ± 3.2 2.3 ± 0.1 12 ± 0.5 1.7 ± 0.1 60 ℃ 8 hours 64 ± 8.9 2.7 ± 0.6 8 ± 0.5 0.8 ± 0.5 65 ℃ 1 hour 67 ± 2.1 2.3 ± 0.5 8 ± 0.5 0.9 ± 0.8 65 ℃ 4 hours 63 ± 5.6 2.4 ± 0.5 0 0 65 ℃ 8 hours 63 ± 5.9 2.2 ± 0.8 0 0 70 ℃ 1 hour 61 ± 9.3 1.9 ± 0.7 0 0 80 ℃ 1 hour 64 ± 9.5 1.9 ± 0.5 0 0

Experimental Example  2. PCA ( Passive cutaneous anaphylaxis ) reaction

A PCA test was conducted to confirm the allergen inhibitory effect using the bee venom solution of Example 1 in which the allergy substance was inactivated. The conditions are as follows.

① Test substance: Confirmation of the allergic inhibitory effect of the enzyme inactive bee venom obtained after reaction at 65 ℃ for 4 hours

② Excipient: saline solution

③ Positive control substance: Ovalbumin (OVA, Albumin Chicken egg)

④ Positive control substance excipient: saline injection

⑤ Experimental animals Species and systems Specific pathogen member (SPF) Guinea pig, Hartley Reason for selection Guinea pigs are one of the most frequently used test animals for susceptibility testing and are used for antigenicity testing. gender cock Number of animals Upon receipt 53 Upon administration 50 Weight range Upon receipt 282.05 + - 305.53 g Upon administration 316.73 + - 382.15 g Treatment of residual animals Euthanasia treatment

⑥ Test group composition, dose setting, group separation and administration

Sensitization 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 (high dose + adjuvant group) 5 One 0.05 G5 (OVA (ovalbumin) + adjuvant group)
Positive control group 5 One 2.5

PCA Reaction Test 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 (high dose + adjuvant group) 5 One 0.05 G5 (OVA + adjuvant group)
Positive control group 5 One 2.5

⑦ Dose setting: The dose of sensitization and induction was made the same. Generally, the amount of antigen which causes anaphylactic shock was used in the present invention since a high dose at the time of sensitization was used. The positive control material was set at a commonly used amount of 2.5 mg / kg.

⑧ Administration: Sensitization was administered to avoid guinea pig warts, and PCA response was induced by using blood serum after cryopreservation. In the study, two sera of each animal were used for the PCA reaction. The serum to be tested was serially diluted (× 10, × 20, × 40, × 80, × 160, × 320, × 640, × 1,280, × 2,560, × 5,120) 100 μl each was administered intraperitoneally to the guinea pig. After 4 hours of intradermal administration, Yagi's stock solution containing Evan's blue (about 2%) was administered into the Fuji vein, causing PCA reaction.

⑨ Observation and examination: General symptoms and body weight were measured. PCA response was determined by ether anesthesia 30 min after the PCA reaction, and the presence or absence of blue patches was observed by avoiding the guinea pig's diffuse skin. When the blue half was observed, the long diameter and the short diameter were measured, and the average value of 5 mm or more calculated as (long diameter + short diameter) / 2 was judged to be positive. The most positive dilution ratio (Antibody value).

The experimental results are as follows:

 GENERAL SYMPTOMS: No specific symptoms and deaths from enzyme inactivated (phospholipase A2 and hyaluronidase inactivated) bee venom were observed.

 Weight change: No change in body weight was observed by inactivation of the enzyme.

PCA Reaction Determination: As shown in Table 5 and FIG. 2, the antigenicity of the enzyme inactive bee venom was evaluated by using the guinea pig-guinephik system as the indicator of passive cutaneous anaphylaxis reaction induction. (G3, 0.05 mg / kg), high dose + adjuvant (G4, 0.05 mg / kg) and positive control (G5, , 2.5 mg / kg, and OVA + adjuvant). The dose was set at 0.05 mg / kg for G1-G4 and 2.0 mg / kg for G5. PCA response was not observed in all of the negative control G1, low dose G2, and high dose G3 groups, which are bee venom-administered groups. In the positive control group G5, the antibody titer was 5120 times, indicating a clear PCA response. In the high dose + adjuvant group G4, the antibody titer was 5120 times. As a result, the inactivated enzyme inactivated phospholipase A2 and hyaluronidase of the present invention were not found to be antigenic.

PCA results (number of positive animals / total number of test animals) meeting place G1 G2 G3 G4 G5 × 10 0/10 0/10 0/10 10/10 10/10 × 20 0/10 0/10 0/10 10/10 10/10 × 40 0/10 0/10 0/10 10/10 10/10 × 80 0/10 0/10 0/10 10/10 10/10 × 160 0/10 0/10 0/10 10/10 10/10 × 320 0/10 0/10 0/10 10/10 10/10 × 640 0/10 0/10 0/10 6/10 10/10 × 1280 0/10 0/10 0/10 5/10 10/10 × 2560 0/10 0/10 0/10 2/10 10/10 × 5120 0/10 0/10 0/10 2/10 10/10

Claims (4)

i) diluting 1 g of tablets bee venom to 50 to 150 ml of sterile distilled water;
ii) soaking the diluted purified beeotene solution in a constant temperature water bath at a temperature of 65 to 85 DEG C for 1 to 8 hours;
iii) filtering the purified beeotene solution with a 0.45-μm membrane filter to obtain a supernatant; And
iv) lyophilizing said subculture solution;
Wherein the phospholipase A2 and the hyaluronidase are inactivated. The method according to claim 1,
Confirming the presence or absence of an allergen causing substance including phospholipase A2 and hyaluronidase using the high performance liquid chromatography after the step iii) by using the high performance liquid chromatography. How to separate bee venom. A tablet bee venom characterized in that the phospholipase A2 and hyaluronidase isolated by the method of claim 1 or 2 are inactivated. The tablet bee venom of claim 3, wherein the tablet bee venom contains at least 60% by weight of melitin and at least 2% by weight of an apamin as an effective ingredient.

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