Histamine removal process for reducing loss of phospholipase A2 and melittin in bee venom

Abstract

The invention relates to a process for removing histamine and reducing loss of phospholipase A2 (PLA2) and melittin in bee venom at the same time.

Description

Technical Field

[0001] The present invention relates to a method for removing histamine inducing allergic reactions from bee venom and a bee venom composition that can be prepared by the method. Background Art

[0002] Bee venom collected from bees has long been used to treat or alleviate various diseases and symptoms. The effects of bee venom include pain suppression by inhibiting the biosynthesis of prostaglandins, improvement of neurological disorders through nerve excitation, improvement of blood circulation due to vasoconstriction and dilation, strong anti-inflammatory effects, promotion of hormone secretion through stimulation of the pituitary gland and adrenal cortex, antibacterial effects, skin improvement effects, etc.

[0003] The raw material of bee venom is a clear, transparent, viscous liquid with a bitter aromatic substance. Its specific gravity is 1.13, which is heavier than water. Its pH value is about 5.2 to 5.5. At room temperature, the acidic substances in bee venom may evaporate and leave about 30% of the dry matter. About 75% of this dry matter is protein.

[0004] Important components of bee venom include peptides such as melittin and apamin, proteases such as phospholipase A2 (PLA2) and hyaluronidase, amines such as histamine and dopamine, etc. It is known that the various pharmacological effects and actions of bee venom are manifested through the activity of all the components or a single main component contained in the bee venom.

[0005] Melittin is a hemolytic and cardiotoxic peptide that accounts for about 40% to 60% of dry bee venom. It attaches to the cell membrane and exhibits pharmacological effects such as hemolysis, antibacterial and anticancer effects. It is especially a core component with anti-inflammatory and anti-pain effects.

[0006] Melittin is a globular peptide that is one of the neurotoxins and is known to account for about 2% to 3% of dry bee venom. According to recent research reports, Melittin significantly increases axonal regeneration by promoting the production of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), which induce nerve regeneration.

[0007] Phospholipase A2 (PLA2) is a phospholipid hydrolase that accounts for about 10% to 12% of dry bee venom, also known as lecithinase. Phospholipase A2 is known to promote inflammation and induce neurotoxicity by inducing the production of prostaglandin E2 (PGE2) through cyclooxygenase-2 (COX2), but recent studies have reported that phospholipase A2 plays a role in protecting the brain in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In addition, phospholipase A2 exhibits neuroprotective and anti-inflammatory effects by inducing the activation of regulatory T cells (Treg).

[0008] Histamine is a substance secreted by basophils, mast cells, etc. in the blood, and is known as a substance that induces allergies. Increase blood flow by dilating capillaries and promote leukocyte binding by the activation of vascular endothelial cells. The histamine contained in bee venom can induce pruritus immediately after administration of bee venom or several hours later. In addition, histamine can induce severe allergic reactions (such as shock), which may be fatal.

[0009] As described above, since bee venom contains a variety of substances that exhibit a variety of activities, technologies for regulating the components in bee venom for a specific purpose or for removing components that may exhibit side effects have been studied. International Publication No. WO2014 / 196674 A1 discloses a method for removing phospholipase A2 and hyaluronidase from bee venom by filtering bee venom mixed with distilled water by ultrafiltration in order to remove allergic components from bee venom. Korean Patent Publication No. 10-2016-0122094 relates to a composition for preventing or treating inflammatory diseases containing purified bee venom that reduces allergy-inducing substances, and discloses a composition that removes phospholipase A2 and histamine by mixing bee venom with distilled water and purifying it using a method such as column chromatography.

[0010] However, there is still a lack of research on methods that can remove most of the histamine while minimizing the loss of phospholipase A2 and melittin, which are currently known to be useful components in bee venom. In particular, in terms of purifying the components of bee venom, attempts to develop specific removal procedures and conditions that can further preserve the effective components are still blank.

[0011] Therefore, the present inventors have invented a novel method for significantly improving the preservation rate of effective ingredients such as melittin and phospholipase A2 while removing most of the histamine by physical means without denaturing or changing the components in bee venom, thereby improving the production efficiency of bee venom compositions that exhibit effective activities.

[0012] Prior art literature

[0013] Patent Literature

[0014] Patent Document 1: International Publication No. WO 2014 / 196674 A1

[0015] Patent Document 2: Korean Patent Publication No. 10-2016-0122094 Summary of the invention

[0016] The object of the present invention is to provide a method for eliminating intracellular histamine which can eliminate most of the histamine that can induce allergies while reducing the loss of effective ingredients such as apamin, melittin and phospholipase A2 in bee venom.

[0017] Another object of the present invention is to provide a bee venom composition in which most of histamine is removed from a bee venom raw material and apamin, melittin and phospholipase A2 are preserved in large amounts.

[0018] In order to achieve the above object, one aspect of the present invention provides a method for removing histamine from bee venom, comprising a permeation step of immersing a permeable membrane containing bee venom raw material in an alkaline solvent.

[0019] Furthermore, another aspect of the present invention provides a bee venom composition prepared by the above method.

[0020] Although certain effects and activities of various components of bee venom are known, the mechanism of action of bee venom is not well understood, and thus bee venom compositions containing various components may be useful as a whole.

[0021] On the other hand, some components of bee venom, such as histamine, which may induce fatal consequences when bee venom is administered, need to be fully removed. The histamine removal process of the present invention has an excellent effect of being able to remove most of the histamine in bee venom. In addition, the method of the present invention has the effect of removing most of the histamine in bee venom while minimizing the loss of effective active ingredients, especially showing an effect of significantly inhibiting the loss of components such as melittin.

[0022] Furthermore, the method of the present invention can show high production efficiency of the bee venom composition at a relatively low cost, and utilizes physical methods to reduce the denaturation of the components in the bee venom, thereby having the advantage of ensuring stability.

[0023] However, the effects of the present invention are not limited to the effects described above, and those skilled in the art to which the present invention pertains can clearly understand other effects not mentioned through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The results of high performance liquid chromatography (HPLC) analysis showed a histamine peak.

[0025] Figure 2 The results of high performance liquid chromatography analysis showed peaks of apamin, phospholipase A2, and melittin.

[0026] Figure 3 The bar graph is a comparison of the histamine clearance rate analyzed by high performance liquid chromatography after bee venom raw materials were placed in a permeable membrane tube at temperatures of 25°C and 4°C and permeated using solvents of different pH values ​​for 60 minutes, 90 minutes, 120 minutes and 180 minutes.

[0027] Figures 4a to 4c This is a bar graph comparing the clearance rates of melittin, apamin and phospholipase A2 by HPLC, after placing bee venom raw material into a permeable membrane tube at a temperature of 25°C and permeating with solvents of different pH values ​​for 60 minutes, 90 minutes, 120 minutes and 180 minutes, with the values ​​in the solvent of pH 7 being taken as the reference (100%).

[0028] Figure 5 For Figure 3 This is a photograph of the permeable membrane tube when the solvents at pH 11 and pH 12 were permeated in the experiment under the same conditions.

[0029] Figure 6 This is a bar graph comparing the clearance rates of histamine, melittin, apamin and phospholipase A2 analyzed by high performance liquid chromatography after placing bee venom raw material into a permeable membrane tube at a temperature of 25°C and permeating using solvents of pH 9 and pH 10 for 120 minutes, 135 minutes, 150 minutes, 165 minutes and 180 minutes.

[0030] Figure 7 This is a bar graph comparing the clearance rates of histamine, melittin, apamin and phospholipase A2 analyzed by high performance liquid chromatography after placing bee venom raw material into a permeable membrane tube at a temperature of 15°C and permeating using solvents of pH 9 and pH 10 for 120 minutes, 135 minutes, 150 minutes, 165 minutes and 180 minutes. DETAILED DESCRIPTION

[0031] Hereinafter, the present invention will be described in detail.

[0032] The present invention provides a method for removing histamine from bee venom.

[0033] The method for removing histamine from bee venom comprises a permeation step of immersing a permeable membrane containing bee venom raw material in an alkaline solvent.

[0034] The term "bee venom (bee venom or apitoxin)" in the present invention is the venom discharged from the ovipositor of bees, and can refer to the bee venom in the state before the histamine elimination process of the present invention or the bee venom in the state after the histamine elimination process, depending on the situation, and can refer to the bee venom in both situations.

[0035] The bee venom and / or bee venom raw material is a substance or composition that is the target of histamine removal in the method of the present invention, and can be directly collected from bees or commercially available, and can also include purified bee venom that has undergone more than one purification or processing process.

[0036] The bee venom and / or bee venom raw material may be derived from bees commonly known as honey bees, and the bees may be Apis dorsta, Apis florea, Apis mellifera and / or Apis cerana, but the bees are not limited as long as they can produce bee venom.

[0037] The bee venom and / or bee venom raw material may contain histamine, apamin, melittin and / or phospholipase A2.

[0038] The above-mentioned permeable membrane (membrane tubing) is a porous membrane having small holes, and substances inside and outside the permeable membrane can move through the holes. The above-mentioned permeable membrane can be prepared in the form of a microtube for use, but it is not limited to the tube form, and any form can be used as long as the liquid bee venom raw material can be placed inside. In the case where the above-mentioned permeable membrane is used in the form of a permeable membrane tube, both ends of the opening of the above-mentioned permeable membrane tube can be sealed. In this case, a tube closure can be used for sealing, but the sealing method is irrelevant as long as it can prevent the substances inside the tube from entering or exiting from the two side ends of the tube.

[0039] The permeable molecular weight of the above-mentioned permeable membrane may be 0.5 kD to 1.5 kD, 0.6 kD to 1.4 kD, 0.7 kD to 1.3 kD, 0.8 kD to 1.2 kD, 0.9 kD to 1.1 kD or 1 kD. The permeable molecular weight may be used in the same or similar sense as the molecular weight cut off (MWCO). If the permeable molecular weight of the above-mentioned permeable membrane is smaller than the above-mentioned range, histamine in bee venom may not be sufficiently removed, and if it is larger than the above-mentioned range, the loss of active ingredients such as apamin, melittin and / or phospholipase A2 may be increased.

[0040] The permeable membrane may be prepared from cellulose ester and / or regenerated cellulose components, but is not limited thereto.

[0041] The bee venom material may be placed in a liquid or solution state in the permeable membrane, specifically, the bee venom material may be dissolved in an appropriate solvent (buffer) and placed in the permeable membrane. The solvent may be the same as the alkaline solvent used to immerse the permeable membrane, or may have a partially different pH condition.

[0042] Before placing the bee venom raw material into the permeable membrane, the method for removing histamine from bee venom of the present invention may further include the step of mixing the bee venom raw material with a solvent. The step of mixing the bee venom raw material with the solvent may be performed together with ultrasonic treatment, stirring, etc. The ultrasonic treatment may be performed for 20 minutes to 50 minutes, 25 minutes to 40 minutes, 25 minutes to 35 minutes, or 35 minutes.

[0043] Before placing the bee venom raw material into the permeable membrane, the method for removing histamine from bee venom of the present invention may further include the step of filtering the bee venom raw material mixed with the solvent. The above filtration may be performed using a polyvinylidene fluoride (PVDF) filter, but the filtering method is not limited as long as it is a means of removing impurities from the bee venom raw material without affecting the main components of the bee venom (phospholipase A2, bee venom peptide, apamin, etc.). The above polyvinylidene fluoride filter may be a polyvinylidene fluoride filter of 0.4 μm to 0.5 μm or 0.45 μm.

[0044] The permeable membrane containing the bee venom raw material may be immersed in the alkaline solvent by placing the permeable membrane in a container containing the alkaline solvent, or by immersing a part or the whole of the permeable membrane. More specifically, the permeable membrane may be immersed in a manner such that an area corresponding to the entire volume of the bee venom raw material placed in the permeable membrane is in contact with the alkaline solvent through the permeable membrane.

[0045] The pH of the alkaline solvent can be above pH8, above pH8.5, above pH9, pH8 to pH12, pH8.5 to pH11 or pH9 to pH10. The alkaline solvent can be prepared based on distilled water, phosphate buffer solution (PBS) and / or physiological saline, but its type is not limited. As long as it does not significantly denature or affect the activity, structure, etc. of the components in the bee venom, it can be used regardless of its type. For example, the alkaline solvent can be prepared by adding sodium hydroxide and / or phosphoric acid to the distilled water, phosphate buffer solution and / or physiological saline to adjust the pH. More specifically, in order to prepare the alkaline solvent of the present invention, sodium hydroxide (NaOH), sodium bicarbonate (NaHCO3), ammonia (NH3), sodium carbonate (Na2CO3), potassium carbonate (K2CO3) and / or calcium oxide (CaO) can be used, but it is not limited thereto.

[0046] In one embodiment of the present invention, it was confirmed that the use of an alkaline solvent has the effect of further reducing the loss of effective ingredients (melittin, melittin, phospholipase A2, etc.) in bee venom compared to the use of an acidic or neutral solvent (distilled water) ( Figure 3). In particular, when alkaline solvents at pH 9 and pH 10 were used, the effect of significantly preventing the loss of the above-mentioned active ingredients was shown. For example, when the permeation step was performed at a temperature of 25°C for 180 minutes, when alkaline solvents at pH 9 or pH 10 were used, while most of the histamine in the bee venom was removed, the loss of active ingredients such as melittin and phospholipase A2 was very small, and it was confirmed that the loss prevention effect was about 18% to 25% better than the melittin removal rate when solvents at pH 7 and pH 8.5 were used.

[0047] The method for removing histamine from bee venom of the present invention may further include the step of stirring the alkaline solvent in which the permeable membrane is soaked.

[0048] The method for removing histamine from bee venom of the present invention may further include a solvent replacement step of removing the alkaline solvent and immersing the permeable membrane in a new alkaline solvent. In this case, the new alkaline solvent may have the same pH as the previous alkaline solvent.

[0049] The method for removing histamine from bee venom of the present invention can more specifically set the time and / or temperature conditions of the permeation step.

[0050] The permeation step may be performed for 120 to 240 minutes, 160 to 240 minutes, 165 to 240 minutes, 180 to 240 minutes, 135 to 230 minutes, 150 to 210 minutes, 160 to 200 minutes, 165 to 200 minutes, or 165 to 180 minutes. In order to achieve a sufficient histamine scavenging effect and an effect of inhibiting the loss of active ingredients (apamin, melittin, phospholipase A2, etc.), the permeation step may be performed for 165 to 240 minutes using an alkaline solvent at pH 9 to pH 10.

[0051] In the permeation step, the temperature may be 20° C. to 35° C., 20° C. to 30° C., 23° C. to 28° C., 20° C. to 25° C., 25° C. to 30° C., specifically, 23° C. to 28° C. or 25° C. In order to achieve a sufficient histamine scavenging effect and an effect of inhibiting the loss of active ingredients (melittin, melittin, phospholipase A2, etc.), the permeation step may be performed using an alkaline solvent of pH 9 to pH 10 at a temperature of 23° C. to 28° C. or 25° C.

[0052] Based on the amount of histamine in the bee venom raw material before the permeation step, the histamine removal rate can be 85% or more, 90% or more, or 95% or more, and specifically, 95% or more. According to the present invention, the histamine removal process in bee venom can remove most of the histamine contained in the bee venom raw material. If the histamine is removed within the above-mentioned removal rate range, considering that the histamine content in the initial bee venom raw material is small, the effect of removing most of the histamine in the bee venom can be achieved.

[0053] In the case of the histamine removal process in bee venom according to the present invention, the preservation rate of melittin can be increased by 15% to 30%, specifically, by 15% to 28% or 15% to 25%, compared to the preservation rate of melittin when using neutral pH. According to a specific embodiment of the present invention, after a 120-minute permeation step, melittin was removed less by 17.3% and 15.1% when using alkaline solvents of pH 9 and pH 10, respectively, compared to the case of using neutral pH 7, and the melittin preservation rate was more than 15%. After a 180-minute permeation step, melittin was removed less by 18.9% and 24.3% when using alkaline solvents of pH 9 and pH 10, respectively, compared to the case of using neutral pH 7, and the melittin preservation rate was about 18% to 25%.

[0054] In the case of the histamine removal process in bee venom according to the present invention, the preservation rate of phospholipase A2 can be increased by 7% to 35%, specifically, by 7% to 34% or 7.5% to 34%, compared to the preservation rate of phospholipase A2 when using neutral pH. The above preservation rate refers to how much the amount of the component in the bee venom before / after the permeation step is reduced and removed. The above preservation rate can be calculated using the clearance rate calculated based on the amount of the component in the bee venom raw material before the permeation step. Therefore, the above preservation rate can be compared by calculating whether the clearance rate of components such as melittin, phospholipase A2 and / or apamin is reduced or increased. According to a specific embodiment of the present invention, after a permeation step of 120 minutes, the phospholipase A2 was removed less by 31.8% and 33.7% when using alkaline solvents of pH 9 and pH 10, respectively, compared with the case of using a neutral pH 7, indicating that the phospholipase A2 preservation rate was approximately 30% to 35%. After a permeation step of 180 minutes, the phospholipase A2 was removed less by 12.4% and 7.8% when using alkaline solvents of pH 9 and pH 10, respectively, compared with the case of using a neutral pH 7, indicating that the phospholipase A2 preservation rate was approximately 7.5% to 15%.

[0055] The preservation rate of melittin and phospholipase A2 using the histamine removal method of the present invention can be calculated by dividing the amount of melittin and / or phospholipase A2 in bee venom after the permeation step by the amount of melittin and / or phospholipase A2 in bee venom before the permeation step, and expressed as a percentage, and the preservation rate of melittin and phospholipase A2 can be increased compared with the preservation rate of melittin and phospholipase A2 using the removal method using a neutral solvent.

[0056] Based on the amount of phospholipase A2 in the bee venom raw material before the permeation step, the clearance rate of phospholipase A2 may be 10% or less, 8% or less, or 5% or less. Furthermore, based on the amount of melittin in the bee venom raw material before the permeation step, the clearance rate of melittin may be 25% or less, 24% or less, 23% or less, or 20% or less. When permeation is performed according to the method of the present invention, the clearance rates of effective ingredients such as phospholipase A2 and melittin are within the above ranges, thereby having the advantage of reducing the loss of these ingredients.

[0057] The present invention provides a bee venom composition prepared by the above-mentioned method for removing histamine from bee venom.

[0058] The bee venom composition may contain apamin, melittin and phospholipase A2. The bee venom composition is characterized in that most of histamine is removed and the loss of effective ingredients (apamin, melittin, phospholipase A2, etc.) is small compared to the components of the bee venom raw material.

[0059] The bee venom composition can be used for pain suppression, improvement of neurological disorders, improvement of blood circulation, promotion of hormone secretion through stimulation of the pituitary gland and adrenal cortex, antibacterial, skin improvement, and / or relief, prevention or treatment of diseases or symptoms related thereto, but is not limited thereto, and can be used for any purpose related to the known efficacy and / or activity of bee venom. In particular, the bee venom composition can be effectively used in people who are sensitive to histamine or have severe allergic reactions, and can reduce the possibility of allergic reactions.

[0060] Hereinafter, the present invention will be described in detail by way of examples and experimental examples.

[0061] However, the following embodiments and experimental examples are only used to specifically illustrate the present invention, and the contents of the present invention are not limited to the following embodiments and experimental examples.

[0062] Example 1

[0063] Analytical method for confirming the histamine-clearing effect of the method of the present invention

[0064] 1-1. High performance liquid chromatography analysis for identification of components in bee venom

[0065] Standard solutions for measuring the amounts of histamine, phospholipase A2, melittin, and apamin in the components contained in bee venom were prepared, and high performance liquid chromatography analysis was performed under the following conditions.

[0066] In order to prepare a quantitative histamine standard solution for histamine, first, 166 mg of a histamine standard (SIGMA-ALDRICH, H7250) was precisely measured and placed in a 50 ml flask, 40 ml of distilled water was added and ultrasonicated for 30 minutes, and then the standard line was calibrated with a solvent. Then, 5 ml was accurately taken and placed in a 50 ml flask, and the standard line was recalibrated with a solvent, and then filtered using a 0.45 μm polyvinylidene fluoride filter to prepare a histamine standard solution.

[0067] The HPLC analysis of histamine was performed with the settings shown in Table 1 below. The HPLC analysis data of histamine are shown in Table 1. Figure 1 shown.

[0068] Table 1

[0069]

[0070] In addition, in order to prepare a standard solution of bee venom for the quantitative determination of phospholipase A2, melittin and apamin in bee venom, 50 mg of bee venom was precisely measured and placed in a 10 ml flask, dissolved in distilled water and ultrasonicated for 30 minutes, and then the standard line was calibrated using the solvent. Then, the solution was filtered using a 0.45 μm polyvinylidene fluoride filter to prepare a standard solution of bee venom. Bee venom was purchased from Bee VenomLab.

[0071] The HPLC analysis of phospholipase A2, melittin and apamin was performed with the settings shown in Tables 2 to 4 below. The HPLC analysis data of phospholipase A2, melittin and apamin were as follows: Figure 2 shown.

[0072] Table 2

[0073] project content Analytical methods Liquid chromatography Testing Instruments UV absorption photometer (measurement wavelength 210nm) column C18 (4.6mm×150mm, 5μm) or equivalent Column temperature 25±2℃ Sample temperature 8±2℃ flow 2ml / min Injection volume 50μl

[0074] Table 3

[0075]

[0076]

[0077] (Mobile phase A: 0.1% trifluoroacetic acid, mobile phase B: acetonitrile: 0.1% trifluoroacetic acid = 80:20)

[0078] Table 4

[0079] Element Relative retention time (RRT) Melittin 0.40 PLA20 0.75 Melittin 1.00

[0080] 1-2. Preparation of Bee Venom Raw Material Sample Solution and Permeable Membrane Tube

[0081] 300 mg of bee venom was precisely measured and placed in a 10 ml flask, dissolved in a solvent and ultrasonicated for 30 minutes, and then the standard line was calibrated with distilled water. Then, 5 ml was precisely taken and placed in a 50 ml flask, and 45 ml of distilled water was added to make the final concentration 5 mg / ml, and then filtered using a 0.45 μm polyvinylidene fluoride filter to prepare a bee venom raw material sample solution.

[0082] In order to prepare a permeable membrane tube for containing bee venom raw materials, first, a permeable membrane (Spectra / Por 6 Dialysis Tubing, 1 kD) purchased from Repligen was cut to about 5 cm and one side was sealed with a tube closer. 4 ml of the bee venom raw material sample solution prepared as described above was placed from the other side of the tube to remove the air inside the permeable membrane to the greatest extent. Then, the open side of the tube was sealed with a closer (weighted closer) and placed in a container with the side facing downward to prepare a permeable membrane tube containing bee venom raw materials for use in subsequent experiments.

[0083] Example 2

[0084] Confirmation of the histamine-clearing effect in bee venom using permeable membrane and alkaline solvent

[0085] After the permeable membrane tube containing the bee venom raw material prepared in the above Example 1-2 was placed in solvents with different pH values ​​to remove histamine, the components of the bee venom sample remaining in the tube were analyzed by high performance liquid chromatography, and the amounts of histamine, phospholipase A2, melittin and apamin were measured for comparison.

[0086] The alkaline solvent used in the histamine removal method according to the present invention was prepared at pH 9, and the acidic solvent used as a control group was prepared at pH 4. The pH of the solvent was adjusted using 1 mM sodium hydroxide or phosphoric acid solution in distilled water.

[0087] First, before the removal process using the permeable membrane, the bee venom raw material sample liquid was subjected to high performance liquid chromatography to measure the amount of histamine, phospholipase A2, bee venom peptide and bee venom peptide contained in the bee venom raw material, and compared with the values ​​after the removal process. The alkaline solvent of the method of the present invention uses a solvent adjusted to pH 8.5, pH 9, pH 10, pH 11 and pH 12, respectively, and an acidic solvent of pH 5 and a neutral solvent (distilled water) of pH 7 are prepared as a control group. In addition, the temperature conditions are set to a low temperature condition of 4°C and 25°C for permeation experiments. After the bee venom raw material permeable membrane tube of Example 1-2 is placed in each solvent, it is stirred with a stirrer and after a removal process of 60 minutes, 90 minutes, 120 minutes and 180 minutes, the permeable membrane tube is recovered and the components of the bee venom in the tube are analyzed by high performance liquid chromatography according to the method of Example 1-1. Furthermore, the clearance rate (%) was calculated and compared by comparing the reduction in the amount of each component after the permeation step through the permeation membrane with the measured values ​​of the amounts of histamine, phospholipase A2, melittin and apamin in the initial bee venom raw material.

[0088] As a result, when the bee venom placed in the permeable membrane was permeated in a solvent at a temperature of 25°C according to the method of the present invention, the amount of histamine before and after the permeation step decreased, thereby confirming that there was a clearing effect ( Figure 3 ). When the permeation time was 120 minutes, the histamine clearance rate was about 90% or more, and when the permeation time was 180 minutes, the histamine clearance rate was 95% or more, and most of the histamine in the bee venom was cleared. On the other hand, the results of the experiment under low temperature conditions of 4°C showed that even if the permeation time was 180 minutes, the histamine clearance rate under all pH conditions was very low, and no sufficient histamine clearance effect was shown ( Figure 3 ).

[0089] Based on the histamine removal effect, the measurement results of other components at 25°C were observed. In the case of the alkaline solvent (pH 8.5, pH 9, pH 10) of the method of the present invention, not only the histamine removal rate was 95% or more at 180 minutes, but also the removal rates of apamin, melittin, and phospholipase A2, which are active ingredients in bee venom, were significantly lower than those of the solvent under acidic or neutral conditions, showing less loss. In particular, in the case of the solvents at pH 9 and pH 10, at 180 minutes, while more than 95% of histamine was removed, the removal rate of melittin was low, showing the minimization of the loss of active ingredients. In the case of 120 minutes, melittin was retained by 17.3% and 15.1% more at pH 9 and pH 10, respectively, compared with the neutral pH 7, and phospholipase A2 was retained by 31.8% and 33.7% more at pH 9 and pH 10, respectively, compared with the neutral pH 7. Compared with the neutral pH 7, melittin was also preserved by 11.8% and 22.9% more at pH 9 and pH 10, respectively. Moreover, over a period of 180 minutes, melittin was preserved by 18.9% and 24.3% more at pH 9 and pH 10, respectively, compared with the neutral pH 7, and phospholipase A2 was preserved by 12.4% and 7.8% more at pH 9 and pH 10, respectively, compared with the neutral pH 7. Compared with the neutral pH 7, melittin was also preserved by 7.0% and 23.0% more at pH 9 and pH 10, respectively. Figures 4a to 4c ).

[0090] The above results confirm that the method of the present invention can reduce the loss of effective ingredients when using an alkaline solvent, which has the effect of significantly reducing the production cost of raw materials.

[0091] On the other hand, when using a permeable membrane with a solvent of pH 11 or pH 12, the components of bee venom aggregated in the tube, and the removal of histamine could not be achieved normally, so it was confirmed that it was not suitable for use. Figure 5 ).

[0092] The above experimental results confirmed that in terms of removing histamine from bee venom, when a permeable membrane and an alkaline solvent are used according to the method of the present invention, histamine can be fully removed while minimizing the loss of effective ingredients such as phospholipase A2, melittin, and apamin, and it was confirmed that the effect of preventing the loss of effective ingredients is particularly excellent when permeating with alkaline solvents of pH 9 and pH 10 at a temperature of 25°C for more than 180 minutes.

[0093] Example 3

[0094] Confirmation of the effect of pH 9 and pH 10 solvents according to additional permeation time and temperature conditions

[0095] The above Example 2 confirmed that when using the alkaline solvents of pH 9 and pH 10 of the present invention, the effect of fully demonstrating the histamine scavenging effect while minimizing the loss rate of active ingredients such as bee venom peptide and phospholipase A2 is the best. The changes in the scavenging rate and preservation rate under specific time and temperature conditions were measured using the solvents of pH 9 and pH 10 for comparison.

[0096] Specifically, the temperature conditions were set to 25°C and 15°C, respectively, and the time conditions were further divided into 120 minutes, 135 minutes, 150 minutes, 165 minutes and 180 minutes, when the histamine clearance rate in Example 2 did not exceed 95%, and the time conditions were further divided into 120 minutes, 135 minutes, 150 minutes, 165 minutes and 180 minutes, and the amounts of histamine, melittin, apamin and phospholipase A2 at each time point were measured for comparison. The preparation method of the tube and the HPLC analysis method were carried out in the same manner as in Example 1, and the clearance confirmation process was carried out in the same manner as in Example 2.

[0097] The experimental results showed that under the condition of 25°C, as the permeation lasted for more than 120 minutes, the histamine clearance rate was generally excellent, especially at 165 minutes and 180 minutes, the histamine clearance rate was more than 95%, indicating that most of the histamine in the bee venom was cleared. Even so, when the solvents at pH 9 and pH 10 were used, the maximum clearance rate of bee venom peptide was 21.4%, which was confirmed to be less than 25%, and the maximum clearance rate of phospholipase A2 was 5.2%, which was confirmed to be less than 10%, indicating that the loss of active ingredients was not large ( Figure 6 ).

[0098] The results of the experiment under the temperature condition of 15℃ showed that the histamine clearance rate was less than 90% in some cases, and at the time points of 165 minutes and 180 minutes, the histamine clearance rate was less than the target value of 95%. Compared with the condition of 25℃, the histamine clearance effect decreased overall ( Figure 7 ).

[0099] By observing the experimental results of the above examples, it can be seen that when the bee venom raw material is placed in a permeable membrane and an alkaline solvent is used to purify the bee venom, most of the histamine that may induce allergies can be removed from the bee venom, and even so, the loss of components such as melittin, apamin, and phospholipase A2, which are effective components of the bee venom, can be minimized. In particular, the histamine removal effect is excellent at a temperature of 25°C compared to a temperature of 4°C or 15°C, and when a pH of 9 or pH 10 solvent is used among alkaline solvents, the loss of effective components such as melittin and phospholipase A2 is low while most of the histamine in the bee venom is removed. Moreover, under the above conditions, the permeation time range that shows the best clearance rate of histamine, melittin, and phospholipase A2 is 165 minutes to 240 minutes.

[0100] The present invention has been described in detail above through embodiments, but a person skilled in the art should understand that various modifications and variations can be made within the technical scope of the present invention, and these modifications and variations certainly fall within the scope of protection of the accompanying invention claims.

Claims

1. A method for removing histamine from bee venom, characterized in that: The method comprises the steps of immersing a permeable membrane containing bee venom raw materials in an alkaline solvent.

2. The method for removing histamine from bee venom according to claim 1, characterized in that: The molecular weight of the permeable membrane is 0.8 kD to 1.2 kD.

3. The method for removing histamine from bee venom according to claim 1, characterized in that: The permeable membrane is made of a component selected from the group consisting of cellulose ester and regenerated cellulose.

4. The method for removing histamine from bee venom according to claim 1, characterized in that: The pH of the alkaline solvent is pH9 to pH10.

5. The method for removing histamine from bee venom according to claim 1, characterized in that: The step of immersing the permeable membrane in the alkaline solvent is performed for 165 to 240 minutes.

6. The method for removing histamine from bee venom according to claim 1, characterized in that: In the step of immersing the permeable membrane in the alkaline solvent, the temperature is 20°C to 35°C.

7. The method for removing histamine from bee venom according to claim 1, characterized in that: Based on the amount of histamine in the bee venom raw material before the permeation step, the histamine removal rate is more than 95%.

8. The method for removing histamine from bee venom according to claim 1, characterized in that: Compared with the preservation rate when a neutral solvent is used in the above-mentioned permeation step, the preservation rate of phospholipase A2 is increased by 15% to 30%.

9. The method for removing histamine from bee venom according to claim 1, characterized in that: Compared with the preservation rate when a neutral solvent was used in the above permeation step, the preservation rate of melittin increased by 7% to 35%.

10. The method for removing histamine from bee venom according to claim 1, characterized in that: The method further comprises the step of stirring the alkaline solvent soaked in the permeable membrane.

11. The method for removing histamine from bee venom according to claim 1, characterized in that: The method further comprises a solvent replacement step of immersing the permeable membrane in a new alkaline solvent after removing the alkaline solvent.

12. A bee venom composition, characterized in that: The method is prepared by the method for removing histamine from bee venom according to any one of claims 1 to 11.

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