CN109890835A - The purification bee venom of the method for the purification bee venom of preparation removal allergic component and the removal allergic component prepared by the method - Google Patents
The purification bee venom of the method for the purification bee venom of preparation removal allergic component and the removal allergic component prepared by the method Download PDFInfo
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- CN109890835A CN109890835A CN201780038902.6A CN201780038902A CN109890835A CN 109890835 A CN109890835 A CN 109890835A CN 201780038902 A CN201780038902 A CN 201780038902A CN 109890835 A CN109890835 A CN 109890835A
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- bee venom
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- reactant
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- allergic component
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/63—Arthropods
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
Abstract
The present invention relates to the method for the purification bee venom of preparation removal allergic component and the purification bee venom of the removal allergic component prepared by the method, which is characterized in that the described method comprises the following steps: (a) adding buffer solution into bee venom and be diluted；(b) Xiang Suoshu step (a) by diluted dilution add protease after carry out enzyme reaction；(c) it is added in the reactant of the step (b) through overcooled ethyl alcohol, thus the protein of Hirschfeld-Klinger reaction object；(d) reactant through supersolidification of the step (c) is centrifuged, so that coagulum be made to precipitate；And it is dried after the supernatant in the reactant for (e) taking the coagulum of the step (d) to precipitate.
Description
Technical field
The present invention relates to the method for the purification bee venom of preparation removal allergic component and the removals prepared by the method
The purification bee venom of quick ingredient, which is characterized in that the described method comprises the following steps: buffer solution (a) is added into bee venom and is carried out
Dilution；(b) Xiang Suoshu step (a) by diluted dilution add protease after carry out enzyme reaction；(c) in the step
(b) it is added in reactant through overcooled ethyl alcohol, thus the protein of Hirschfeld-Klinger reaction object；(d) by the process of the step (c)
The reactant of solidification is centrifuged, so that coagulum be made to precipitate；And the coagulum of the step (d) (e) is taken to precipitate
It is dried after supernatant in reactant.
Background technique
Bee venom refers to poison possessed by honeybee, and since ancient times, the therapeutic effect medically of bee venom is authenticated.According to most
Close result of study discovery bee venom is in addition to having such as antitumaous effect, alleviation arthritis, alleviation artery sclerosis, waist pain-relieving, controlling
The immune effect of skin wound, enhancing, anti-inflammatory effect are treated, reduces blood pressure and increases lymphocyte in blood and red blood cell
Except regenerated medicine function, also have the function of improving wrinkle, whitening function etc. for the purpose of beauty.
The main constituents and its pharmacological function for observing bee venom are as shown in following table 1.
[table 1]
| Ingredient | Pharmacological function | Ingredient ratio (%) |
| Peptide (melittin, apamin) | Sedation, immunization | 50% or so |
| Protein (phospholipase A2) | Destroy cell, allergy | 15% |
| Amine (histamine) | It reduces blood pressure | 1~2% |
Bee venom includes peptide, protein and low molecule reactive amines etc., by 40 kinds or more of material composition, main effective component
As shown in above-mentioned table.
In the composition of bee venom, protein-based is by the phospholipase A2 of the molecular weight with 13kDa or more
(Phospholipase A2, PLA2), hyaluronidase (Hyaluronidase), phosphatase (phosphatase), α-grape
Glycosidase (α-Glucosidase) etc. primarily serves at being grouped as and destroys blood cell film, blood clotting, blood vessel dilatation and infiltration
Thoroughly, promote blood circulation, promote the physiological activities such as hydrolysis of protein.
In particular, phosphatidase and hyaluronidase are to induce strong anaphylactoid bee venom component, have to bee venom
For hypersensitive user, problem for security very serious can be caused.Therefore, in order to the purpose for the treatment of use bee
Poison, it may be said that the inactivation of mentioned component or to completely remove be required.
Accordingly, it is currently known the technology of some substances that above-mentioned induction allergy is removed from bee venom.For example, Korean granted is special
It describes and dissolves bee venom to prepare bee venom solution in benefit the 1382404th, and the bee venom solution is added to ethylenediamine-
Mixture is prepared in N- propyl silane (primary secondary amine, PSA) adsorbent, is carried out accurate mistake later
Filter, thus the method that large scale preparation goes deimpurity pure bee venom.In addition, being disclosed in Korean granted patent the 1364506th
Size using retention (cut-off) molecular weight is that the ultrafiltration membrane of 10kDa or more removes the phosphatidase in bee venom, and makes to refine bee
The method of the melittin of apamin, 50 weight % or more containing 4 weight % or more in poison.In addition, Korean granted patent
It is disclosed in No. 1608045 using the phosphatidase in cation exchange resin removal bee venom, and only separates the technology of melittin.But
It is that there is presently no the methods such as the Non-toxic purification bee venom of the invention for preparing removal allergic component using protease.
Summary of the invention
Technical problems to be solved
The present invention is proposed according to demand as described above, and the purpose of the present invention is to provide prepared using protease
The method that Non-toxic refines bee venom, the method can fully retain the effective component of the bee venom such as melittin and apamin
Meanwhile, it is capable to effectively completely remove the allergic component phospholipase A2 and hyaluronidase of bee venom.
Technical solution
It is special in order to solve the above technical problem, the present invention provides the method for the purification bee venom of preparation removal allergic component
Sign is, the described method comprises the following steps: (a) adding buffer solution into bee venom and is diluted；(b) Xiang Suoshu step (a)
By in diluted dilution add protease after carry out enzyme reaction；(c) it is added and passes through in the reactant of the step (b)
Cooling ethyl alcohol, thus the protein of Hirschfeld-Klinger reaction object；(d) reactant through supersolidification of the step (c) is centrifuged
Separation, so that coagulum be made to precipitate；And the supernatant in the reactant for (e) taking the coagulum of the step (d) to precipitate is laggard
Row drying.
In addition, the present invention provides through the purification bee venom of the removal allergic component of the method preparation.
Beneficial effect
Purification bee venom prepared by the method for the present invention completely remove the strong anaphylactoid phospholipase A2 of induction and
Hyaluronidase so as to prepare fundamentally to the purification bee venom of allergic reaction safety, and fully retains in bee venom
The effective component for including, thus have the advantages that can more to feel at ease for various pharmacological functions using.
Detailed description of the invention
Fig. 1, which is shown, with phosphate buffer solution to be diluted the dilution of non-refined bee venom and is prepared by the method for preparation example 2
The electrophoretic image for refining bee venom phosphate buffer solution analyzes result.
PLA2: phospholipase A2
Specific embodiment
In order to achieve the object of the present invention, the present invention provides the method for the purification bee venom of preparation removal allergic component, special
Sign is, the described method comprises the following steps:
(a) buffer solution is added into bee venom to be diluted；
(b) Xiang Suoshu step (a) by diluted dilution add protease after carry out enzyme reaction；
(c) it is added in the reactant of the step (b) through overcooled ethyl alcohol, thus the protein of Hirschfeld-Klinger reaction object；
(d) reactant through supersolidification of the step (c) is centrifuged, so that coagulum be made to precipitate；And
(e) it is dried after the supernatant in reactant for taking the coagulum of the step (d) to precipitate.
In the method for preparation purification bee venom of the invention, the allergic component can be phospholipase A2 (Phospholipase
A2) and hyaluronidase (Hyaluronidase), but it is not limited to this.
In addition, the buffer solution of the step (a) preferably can be pH and be in the method for preparation purification bee venom of the invention
EDTA, Tris, phosphate (phosphate) or citrate (citrate) buffer solution of 5~9 and 1~150mM concentration, more
It is preferred that can be the phosphate buffer that pH is 6~9 and 50~150mM concentration, most preferably can be pH is 7.5 and 100mM concentration
Phosphate buffer.The buffer solution can remove the co-factor (cofactor) for playing the allergic component phospholipase A2 of bee venom
The Ca ion of effect.In addition, if in buffer solution oxidation reaction can occur in reaction process, cause obtained containing aerobic
The potency of bee venom reduces.Therefore, in order to remove the oxygen in buffer solution, can 90 DEG C or more at a temperature of buffer solution added
Heat 5~30 minutes, or deoxygenation of making a return journey in displacement 30 minutes or more is carried out to buffer with the nitrogen of purification.
In addition, the dilution of the step (a) can preferably add in bee venom in the method for preparation purification bee venom of the invention
Adding pH is 6~9 buffer solution, is diluted to 8~12% (w/v), and it is molten that the buffering that pH is 7.5 can be more preferably added in bee venom
Liquid is diluted to 10% (w/v).It is about the 50% of the solid component of bee venom using melittin as the peptide components of representative in bee venom, remaining
50% protein component for inducing allergy for phospholipase A2 etc..The total solid content that both ingredients add up is reached into bee venom
30%.It is therefore contemplated that the content of the allergy protein ingredient in the bee venom of 1mL reaches about 150mg.With about in the present invention
Bee venom is diluted in buffer solution by 10% concentration, and therefore, the content of the protein of the induction allergy in the reactant of 1mL is about
For 15mg.
In addition, the protease of the step (b) can be for selected from tryptose in the method for preparation purification bee venom of the invention
Enzyme and pancreas enzyme etc. are originated from vegetable proteins enzyme and the eggs from microorganism such as protease, bromelain and the papain of animal
The protease of one or more of white enzyme, more preferably alkali protease (Alcalase).Alkali protease can be serine
Endopeptidase (Serine endopeptidase).Compared with other protease, alkali protease can be effectively removed in bee venom
Allergic component, using concentration preferably can be 0.8~1.2 unit (unit)/mL, 1 unit/mL more preferably can be used.With
Above-mentioned concentration can be effectively removed the ingredient of the induction allergy in bee venom when being handled, but alkali protease using dense
When degree is more than above range, effective component melittin of bee venom etc. can be decomposed, to have asking for the content for reducing effective component
Topic.
In addition, the step (c) can preferably be added cold in reactant in the method for preparation purification bee venom of the invention
But extremely -15~-25 DEG C of ethyl alcohol can be more preferably added in reactant with the protein of Hirschfeld-Klinger reaction object and be cooled to -20 DEG C
Ethyl alcohol with the protein of Hirschfeld-Klinger reaction object.In order to cool down to reactant, the second that addition is cooled to -20 DEG C can use
The methods of alcohol, addition dry ice, addition liquid nitrogen, but the peptide in bee venom has dissolubility to ethyl alcohol, it is therefore preferable that using cooling
To -20 DEG C of ethyl alcohol.Used ethyl alcohol is the ethyl alcohol that purity is 95% at this time, and the content of ethyl alcohol in reactant can be made to reach
30~65% mode adds ethyl alcohol, so that the content of ethyl alcohol is reached 50% mode and add.When the addition of ethyl alcohol
When amount is less than above range, protein coagulating effect is low, even if the additive amount of ethyl alcohol is more than above range, the solidification journey of protein
Degree does not also change, therefore is preferably added with above range.
In the method for preparation purification bee venom of the invention, more specifically, may comprise steps of:
(a) buffer solution that pH is 6~9 is added into bee venom, is diluted to 8~12% (w/v)；
(b) Xiang Suoshu step (a) by diluted dilution add 0.8~1.2 unit/mL alkali protease
(Alcalase), it is then carried out enzyme reaction 5~20 minutes at 34~40 DEG C；
(c) it is added in the reactant of the step (b) and is cooled to -15~-25 DEG C of ethyl alcohol, thus Hirschfeld-Klinger reaction object
Protein；
(d) reactant through supersolidification of the step (c) is centrifuged, so that coagulum be made to precipitate；And
(e) it is dried after the supernatant in reactant for taking the coagulum of the step (d) to precipitate.
It may further include the step (b) in the method for preparation purification bee venom of the invention by enzyme reaction
The step of reactant is heated, more specifically, the method may include following steps:
(a) buffer solution that pH is 6~9 is added into bee venom, is diluted to 8~12% (w/v)；
(b) Xiang Suoshu step (a) by diluted dilution add 0.8~1.2 unit/mL alkali protease
(Alcalase), it is then carried out enzyme reaction 5~20 minutes at 34~40 DEG C；
(c) at 75~85 DEG C, the reactant by enzyme reaction of the step (b) is subjected to heating 10~20 minutes；
(d) addition is cooled to -15~-25 DEG C of ethyl alcohol in the reactant by heating of the step (c), to coagulate
Gu the protein of reactant；
(e) reactant through supersolidification of the step (d) is centrifuged, so that coagulum be made to precipitate；And
(f) it is dried after the supernatant in reactant for taking the coagulum of the step (e) to precipitate.
It will be heat-treated with the reactant that condition as described above carries out enzyme reaction, to interrupt in reactant and include
The enzymatic activity of protease, and make may be present in the bee venom allergy protein phospholipase A2 in reactant and hyaluronidase generation
Thermal denaturation.
In addition, the present invention also provides the purification bee venom of the removal allergic component prepared by the method.Essence of the invention
Bee venom processed is characterized in that, does not detect the phospholipids enzyme A2 (Phospholipase A2) and hyaluronidase that induce allergy
It (Hyaluronidase), include and as the effective component of bee venom 50% or more melittin, 1% or more apamin.
In addition, compared with the LD50 value of the bee venom before purification, purification bee venom of the invention is aobvious in the toxicity assessment using mouse as object
Significantly high LD50 value is shown, almost without toxicity.In addition, with staphylococcus aureus (Staphylococcus
Aureus) bacterial strain and propionibacterium acnes (Propionibacterium acne) bacterial strain are in the antibacterial experiment of object, with essence
Bee venom before system is compared, and purification bee venom of the invention shows similar antibacterial effect, confirms purification bee venom by object of cavy
Inhibition allergy effect as a result, anomalous variation is not observed in cavy, and can be confirmed without antigenicity.
In the following, embodiment through the invention is described in detail.But following embodiments are only used for illustrating this hair
Bright, the contents of the present invention are not limited by following embodiments.
Preparation example 1: removal induces the preparation of the purification bee venom of the substance of allergy
(a) it is to the pH that the solid content purchased from great Han bee-keeping association of 0.1g is addition 1mL in the bee venom of 30% (w/w)
Bee venom is diluted to 10% (w/v) by the phosphate buffer solution of 7.5 and 100mM.
(b) Xiang Suoshu step (a) by adding 1 unit/mL alkali protease in diluted dilution
(Alcalase, Novi believe (Novozyme) company), then carries out enzyme reaction 10 minutes at 37 DEG C.
(c) 95% (v/v) for being cooled to -20 DEG C of 1mL is added in the reactant by enzyme reaction of the step (b)
Ethyl alcohol, thus the protein of Hirschfeld-Klinger reaction object.
(d) reactant through supersolidification of the step (c) is centrifuged, so that coagulum be made to precipitate.
(e) supernatant in reactant for taking the coagulum of the step (d) to precipitate, then carries out low-temperature vacuum drying
(Speed Vac) is to remove the ethyl alcohol in solution, to obtain the purification bee venom phosphate buffer solution of 1mL.
Preparation example 2: removal induces the preparation of the purification bee venom of the substance of allergy
(a) it is to the pH that the solid content purchased from great Han bee-keeping association of 0.1g is addition 1mL in the bee venom of 30% (w/w)
Bee venom is diluted to 10% (w/v) by the phosphate buffer solution of 7.5 and 100mM.
(b) Xiang Suoshu step (a) by adding 1 unit/mL alkali protease in diluted dilution
(Alcalase, Novi believe (Novozyme) company), then carries out enzyme reaction 10 minutes at 37 DEG C.
(c) at 80 DEG C, the reactant by enzyme reaction of the step (b) is subjected to heating 15 minutes.
(d) 95% (v/v) second for being cooled to -20 DEG C of 1mL is added in the reactant by heating of the step (c)
Alcohol, thus the protein of Hirschfeld-Klinger reaction object.
(e) reactant through supersolidification of the step (d) is centrifuged, so that coagulum be made to precipitate.
(f) supernatant in reactant for taking the coagulum of the step (e) to precipitate, then carries out low-temperature vacuum drying
(Speed Vac) is to remove the ethyl alcohol in solution, to obtain the purification bee venom phosphate buffer solution of 1mL.
Embodiment 1: the antibacterial ability of bee venom is refined
In order to confirm whether purification bee venom obtained in preparation example 2 keeps physiological activity, pass through minimum inhibitory concentration
(minimum inhibition concentration, MIC) and minimum bactericidal concentration (minimum bactericidal
Concentration, MBC) it analyzes to staphylococcus aureus (Staphylococcus aureus) bacterial strain and acne propionic acid bar
The antibacterial ability of bacterium (Propionibacterium acne) bacterial strain.
[table 2]
The comparison of bee venom and the antibacterial ability of bee venom after purification before refining
As a result, the MIC and MBC of the purification bee venom of preparation example 2 increased compared with refining preceding bee venom, but it increases
The amplitude added is little, and the fully holding original antibacterial effect of bee venom can be confirmed.
Embodiment 2: change the composition transfer of the purification bee venom of bee venom type
Use the dilution and following purification bee venom non-refined bee venom being diluted in phosphate buffer solution, that is, pass through
The method preparation purification bee venom of the preparation example 1 but the purification bee venom (preparation example 1-2) prepared with buying for changing bee venom
The purification bee venom (preparation example 1-3) prepared with the purchase of change alkali protease, to induction allergy in purification bee venom
The composition transfer of phospholipids enzyme A and hyaluronidase is analyzed.
For whether removing allergic component, analyzed using liquid chromatograph, used column is Sephadex TM
75, Source 15RPC ST, C18, RP-amide, Peptide ES-C18, and the bee venom in bee venom is calculated by following formula
The content of peptide, apamin, phospholipase A2, hyaluronidase, the results are shown in following Table 3.
The amount (mg) of the standard items taken × (purity/100 of standard items) × (peak area of required ingredient in bee venom)/mark
The peak area of quasi- product) × (amount of 100/ bee venom)
[table 3]
Refine the composition transfer (%) of bee venom
| Component content (%) | Melittin | Apamin | Phospholipase A2 | Hyaluronidase |
| Bee venom before refining | 65 | 2.5 | 11 | 1.8 |
| It refines bee venom (preparation example 1) | 61 | 2.0 | 0 | 0 |
| It refines bee venom (preparation example 1-2) | 60 | 2.1 | 0 | 0 |
| It refines bee venom (preparation example 1-3) | 61 | 2.0 | 0 | 0 |
Preparation example 1: using bee venom before the purification purchased from great Han bee-keeping association and purchased from Novozymes Company (Novo Co.)
The purification bee venom of alkali protease
Preparation example 1-2: using purchased from bee venom and purchase before the purification of IBEE Agricultural Union Corporation
From the purification bee venom of the alkali protease of Novozymes Company (Novo Co.)
Preparation example 1-3: bee venom and the basic protein purchased from Sigma Corporation before the purification purchased from great Han bee-keeping association are utilized
The purification bee venom of enzyme
Be confirmed whether removal purification bee venom in induction allergy phospholipids enzyme A and hyaluronidase as a result, such as institute
It states in the purification bee venom for the preparation example 1-2 that can be confirmed prepared by the type for changing bee venom shown in table 3 and does not detect phosphatidase
A2 and hyaluronic acid enzyme component change and also do not examine in the purification bee venom of preparation example 1-3 prepared by the type of alkali protease
Measure allergic component.Furthermore, it is possible to confirm not detect to induce in the purification bee venom of the method preparation of preparation example 1 of the invention
The phospholipids enzyme A2 (Phospholipase A2) and hyaluronidase (Hyaluronidase) of allergy, and as bee venom
Effective component include 60% or more melittin, 2% or more apamin.
Embodiment 3: change the composition transfer of the purification bee venom of protease
The dilution of non-refined bee venom and the purification bee venom of the method preparation by preparation example 2 are diluted with phosphate buffer solution
The electrophoretic image analysis result of phosphate buffer solution is as shown in Figure 1.In electrophoretic image, compared with non-refined bee venom, the present invention
Purification bee venom in phospholipase A2 be removed completely, and compared with non-refined bee venom, there is no show for the concentration of melittin
Very big variation out.
In addition, the purification bee venom phosphate buffer solution prepared using the method by preparation example 2 and the side by preparation example 2
Purification bee venom phosphate buffer solution difference prepared by method preparation purification bee venom but the type for changing the step the protease in (b)
The powder being freeze-dried analyzes melittin, apamin, phospholipase A2, hyaluronic acid enzyme component.
[table 4]
Refine the composition transfer (%) of bee venom
As a result, the phospholipids enzyme A2 for not detecting to induce allergy in the purification bee venom of preparation example 2 can be confirmed
(Phospholipase A2) and hyaluronidase (Hyaluronidase), and include 50% as the effective component of bee venom
Above melittin, 1% or more apamin.On the other hand, can be confirmed using neutral proteinase or compound protease and
In the case where purification bee venom prepared by unused alkali protease, compared with preparation example 2, the content of the effective component of bee venom
It substantially reduces, and the substance of the induction allergy of bee venom fails to be removed completely.
Embodiment 4: models of passive skin irritability (Passive cutaneous anaphylaxis, PCA) reaction is logical in order to confirm
The effect for crossing the inhibition allergy of the purification bee venom of the method preparation of preparation example 1 and 2 of the invention, carries out following models of passive skin irritability
(Passive cutaneous anaphylaxis, PCA) test is reacted, experimental method is as follows.
1) substances: the purification bee venom of preparation example 1 or preparation example 2
2) excipient: normal saline solution
3) positive control: ovalbumin (Ovalbumin: is originated from egg)
4) positive control substance excipient: normal saline solution
5) experimental animal: the cavy (guinea pig) of the no-special pathogen (SPF) of 300~380g
6) composition of test group, the decision of amount of application, the separation and application of group
[table 5]
The decision of the composition and amount of application of test group
| Group | Size of animal | It applies liquid measure (ml/kg) | Amount of application (mg/kg) |
| G1 (excipient control group) | 5 | 1 | 0 |
| G2 (low dosage group) | 5 | 1 | 0.025 |
| G3 (high dosage group) | 5 | 1 | 0.05 |
| G4 (OVA+ adjuvant (adjuvant) group) | 5 | 1 | 2.5 |
7) decision of amount of application: keep sensitization identical with the amount of application of PCA.
8) apply: sensitization is to be applied to the nape of the neck of cavy, and the initiation of PCA reaction is using the blood acquired after sensitization
Serum.For causing the tested serum of PCA that day, each serum of Different Individual, which is served only for the animal caused using 2, to be come in fact
Apply PCA reaction.Tested serum is diluted to 5120 times from 10 times with normal saline solution, every time with 2 times of progress serial dilutions,
Then it is intradermal with 100 μ l to be applied to guinea pig back respectively.After intradermal administration 4 hours, 2% Evans blue (Evan's will be contained
Blue initiation antigen liquid) is applied in hind leg vein, to cause PCA reaction.
9) it observes and checks: general symptom and weight are checked.The judgement of PCA reaction is as follows, that is, is being caused
After PCA reacts 30 minutes, experimental animal is sacrificed with etherization method, the skin of back of cavy is then subjected to peeling, and observe
Whether bluish white color spot is had.When there is bluish white color spot, major diameter and minor axis are measured, the average value that will be calculated by (major diameter+minor axis)/2
Judgement for 5mm or more is positive, and would indicate that positive final serum diluting multiple (maximum dilution multiple) is set to the blood
Clear final potency (antibody titer).
[table 6]
The PCA result (positive reaction size of animal/experimental animal total quantity) of the purification bee venom of preparation example 1 and 2
To the effect of the inhibition allergy of the purification bee venom prepared by the method for preparation example 1 and 2 confirmed as a result, such as
Shown in the table 6, it can be confirmed and do not observe specific symptoms and dead animal as caused by purification bee venom of the invention, and
PCA reaction result is not observed in the low dosage administration group G2 of bee venom, high dosage administration group G3 including negative control group G1
To bluish white color spot, but in positive controls G4, antibody titer is 5120 times, apparent PCA reaction occurs.By above-mentioned knot
Fruit may finally confirm that purification bee venom prepared in accordance with the present invention is without antigenicity.
Claims (7)
1. the method for the purification bee venom of preparation removal allergic component, which is characterized in that the described method comprises the following steps:
(a) buffer solution is added into bee venom to be diluted；
(b) Xiang Suoshu step (a) by diluted dilution add protease after carry out enzyme reaction；
(c) it is added in the reactant of the step (b) through overcooled ethyl alcohol, thus the protein of Hirschfeld-Klinger reaction object；
(d) reactant through supersolidification of the step (c) is centrifuged, so that coagulum be made to precipitate；And
(e) it is dried after the supernatant in reactant for taking the coagulum of the step (d) to precipitate.
2. the method for the purification bee venom of preparation removal allergic component according to claim 1, which is characterized in that the allergy
Ingredient is phospholipase A2 and hyaluronidase.
3. the method for the purification bee venom of preparation removal allergic component according to claim 1, which is characterized in that the step
(b) protease is alkali protease.
4. the method for the purification bee venom of preparation removal allergic component according to claim 1, which is characterized in that the method
It further comprise by the step (b) the step of heating by the reactant of enzyme reaction.
5. the method for the purification bee venom of preparation removal allergic component according to claim 3, which is characterized in that the method
The following steps are included:
(a) buffer solution that pH is 6~9 is added into bee venom, is diluted to 8~12% (w/v)；
(b) Xiang Suoshu step (a) by diluted dilution add 0.8~1.2 unit/mL alkali protease, then
It is carried out enzyme reaction 5~20 minutes at 34~40 DEG C；
(c) it is added in the reactant of the step (b) and is cooled to -15~-25 DEG C of ethyl alcohol, thus the albumen of Hirschfeld-Klinger reaction object
Matter；
(d) reactant through supersolidification of the step (c) is centrifuged, so that coagulum be made to precipitate；And
(e) it is dried after the supernatant in reactant for taking the coagulum of the step (d) to precipitate.
6. the method for the purification bee venom of preparation removal allergic component according to claim 4, which is characterized in that the method
The following steps are included:
(a) buffer solution that pH is 6~9 is added into bee venom, is diluted to 8~12% (w/v)；
(b) Xiang Suoshu step (a) by diluted dilution add 0.8~1.2 unit/mL alkali protease, then
It is carried out enzyme reaction 5~20 minutes at 34~40 DEG C；
(c) at 75~85 DEG C, the reactant by enzyme reaction of the step (b) is subjected to heating 10~20 minutes；
(d) addition is cooled to -15~-25 DEG C of ethyl alcohol in the reactant by heating of the step (c), to solidify anti-
Answer the protein of object；
(e) reactant through supersolidification of the step (d) is centrifuged, so that coagulum be made to precipitate；And
(f) it is dried after the supernatant in reactant for taking the coagulum of the step (e) to precipitate.
7. removing the purification bee venom of allergic component, method preparation according to any one of claim 1 to 6.
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020160079374A KR101659894B1 (en) | 2016-06-24 | 2016-06-24 | Method for producing nontoxic bee venom peptide having excellent thermal stability and removing allergy |
| KR10-2016-0079375 | 2016-06-24 | ||
| KR10-2016-0079374 | 2016-06-24 | ||
| KR20160079375 | 2016-06-24 | ||
| KR10-2017-0037709 | 2017-03-24 | ||
| KR1020170037709A KR101774197B1 (en) | 2016-06-24 | 2017-03-24 | Method for producing nontoxic bee venom peptide |
| PCT/KR2017/006527 WO2017222300A1 (en) | 2016-06-24 | 2017-06-21 | Method for producing allergenic component-free purified bee venom, and allergenic component-free purified bee venom produced thereby |
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| KR101364506B1 (en) * | 2013-06-07 | 2014-02-21 | 주식회사 청진바이오텍 | Preparation of bee venom removed allergic ingredients |
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| KR20150036856A (en) * | 2013-09-30 | 2015-04-08 | 주식회사 청진바이오텍 | Method for manufacturing functional cosmetic composite using no allergic bee venom |
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| KR101659894B1 (en) * | 2016-06-24 | 2016-09-26 | 김용수 | Method for producing nontoxic bee venom peptide having excellent thermal stability and removing allergy |
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| CN103006530A (en) * | 2012-12-20 | 2013-04-03 | 中国科学院南海海洋研究所 | Bee venom compound with sun-proof effect |
| KR101418067B1 (en) * | 2012-12-20 | 2014-07-14 | 주식회사 삼양제넥스 | Preparation method of hypoallergenic royal jelly by enzyme treatment |
| KR101364506B1 (en) * | 2013-06-07 | 2014-02-21 | 주식회사 청진바이오텍 | Preparation of bee venom removed allergic ingredients |
| KR20150036856A (en) * | 2013-09-30 | 2015-04-08 | 주식회사 청진바이오텍 | Method for manufacturing functional cosmetic composite using no allergic bee venom |
| KR20160057662A (en) * | 2014-11-14 | 2016-05-24 | 대한민국(농촌진흥청장) | Method for seperating non-allergenic bee venom |
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| CN109890835B (en) | 2022-05-17 |
| WO2017222300A1 (en) | 2017-12-28 |
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