KR20220071937A - Composition for maintaining bee venom stability - Google Patents

Abstract

The present invention relates to a composition for maintaining stability of bee venom, a method for maintaining stability of bee venom, and a bee venom composition with improved stability. The composition for maintaining stability of bee venom according to the present invention is formed by adding a preservative to saline solution or sterilized distilled water. When adding chlorocresol or cresol to the saline solution as the preservative, or adding benzalkonium chloride or benzetonium chloride to the sterilized distilled water as the preservative, the stability of the bee venom can be maintained. Especially, when adding the chlorocresol to the saline solution or adding mixed chlorocresol and cresol to the saline solution, the stability of the bee venom is highly maintained. Accordingly, the composition for maintaining stability of bee venom of the present invention is usefully used to manufacture and store a bee venom herbal acupuncture solution formed by the bee venom as a raw material, biologics, and cosmetics.

Description

Composition for maintaining bee venom stability}

The present invention relates to a composition for maintaining stability of bee venom, a method for maintaining stability of bee venom using the same, and a composition for improving stability of bee venom.

Bongchim therapy is a field of traditional medicine and is used to treat various kinds of inflammation and pain. In the past, live bees were caught with tweezers and stinged on the affected area, or only the sting was extracted and injected into the affected area. are harvesting The dried bee venom product is manufactured through a relatively simple purification process of centrifugation, filtration, and freeze-drying.

Bee venom is a venom secreted by bees ( Apis mellifera L.) from the ovipositor to defend against external stimuli or attacks. Bee venom has been recognized for its medicinal therapeutic effect since ancient times. About 75% of live bee venom consists of protein, and only ingredients effective for the human body are extracted from bee venom and used to treat diseases such as neuralgia, rheumatism, arthritis, and facial paralysis. As a result of recent research, bee venom has anticancer action, arthritis relief, arteriosclerosis relief, back pain relief, skin wound treatment, immune enhancement action, anti-inflammatory action, lowering blood pressure, and medical functions such as increased regeneration of lymphocytes and red blood cells in the blood. For the purpose of wrinkle improvement and whitening, etc. have been found.

Among the components of bee venom, melittin is a protein composed of 26 amino acids and constitutes 40-50% of dried bee venom. , stimulation of enzyme secretion, etc. have been reported. However, melittin is very unstable in temperature and pH as well as in other organic solvent formulations such as acids and bases, and it is difficult to keep melittin stable because its components decrease rapidly. There is a need for research on a method for maintaining the stability of tin.

On the other hand, as a storage method for clinical use of bee venom, it is recommended to store it frozen in a powder state. When a substance derived from living organisms is used as a raw material or material, stricter storage conditions are required to maintain the effectiveness and quality of the product and to prevent denaturation. Accordingly, the present inventors applied the general matters of the 'Guidelines for Biologics Stability Test' of the Ministry of Food and Drug Safety, added an aqueous solution and a preservative, and then conducted a stability test to find a way to keep bee venom for longer while maintaining its effectiveness. wanted to

Korean Patent Registration No. 10-1659894

The present inventors have studied a method of maintaining the stability of bee venom in aqueous solution and storing it for a longer period of time. As a result, a combination of an aqueous solution and a preservative that maintains high stability of bee venom when each of 9 preservatives is added to sterile physiological saline or sterile distilled water was confirmed. Based on this, the present invention was completed.

Accordingly, it is an object of the present invention to provide a composition for maintaining the stability of bee venom.

Another object of the present invention is to provide a method for maintaining stability of bee venom, comprising adding the composition for maintaining stability of bee venom to bee venom.

Another object of the present invention is to provide a bee venom composition with improved stability comprising bee venom and the composition for maintaining stability of bee venom as an active ingredient.

However, the technical task to be achieved by the present invention is not limited to the tasks mentioned above, and other tasks not mentioned may be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. There will be.

In order to achieve the above object, the present invention provides a composition for maintaining bee venom stability comprising sterile water and a preservative as active ingredients,

The sterile water is at least one selected from the group consisting of sterile physiological saline and sterile distilled water,

The preservative is chlorocresol, cresol (Cresol), methyl paraoxybenzoate (Methyl ρ-Hydroxybenzoate), propyl paraoxybenzoate (Propyl ρ-Hydroxybenzoate), butyl paraoxybenzoate (Butyl ρ-Hydroxybenzoate), chlorobutanol ( It provides a composition for maintaining bee venom stability, characterized in that at least one selected from the group consisting of Chlorobutanol), benzalkonium chloride, and benzethonium chloride.

The present invention also provides a method for maintaining stability of bee venom, comprising adding the composition for maintaining stability of bee venom to bee venom.

In addition, the present invention provides a bee venom composition with improved stability comprising bee venom and the composition for maintaining stability of bee venom as an active ingredient,

There is provided a bee venom composition with improved stability, characterized in that the stability of bee venom is improved by the composition for maintaining stability of bee venom.

In one embodiment of the present invention, the composition for maintaining stability of bee venom includes sterile physiological saline; And it may include one or more selected from the group consisting of chlorocresol and cresol as an active ingredient, but is not limited thereto.

In another embodiment of the present invention, the composition for maintaining stability of bee venom includes sterile distilled water; And it may include one or more selected from the group consisting of benzalkonium chloride and benzethonium chloride as an active ingredient, but is not limited thereto.

As another embodiment of the present invention, the composition for maintaining the stability of bee venom may maintain the storage stability of bee venom for 1 to 3 months, but is not limited thereto.

In another embodiment of the present invention, the chlorocresol may be included in a concentration of 0.01% (w/v) to 0.2% (w/v) with respect to the composition for maintaining bee venom stability, but is not limited thereto.

As another embodiment of the present invention, the cresol may be included in a concentration of 0.01% (w/v) to 0.4% (w/v) with respect to the composition for maintaining bee venom stability, but is not limited thereto.

As another embodiment of the present invention, the composition for maintaining bee venom stability comprises sterile physiological saline, chlorocresol, and cresol as active ingredients,

Sterile physiological saline and cresol: Sterile physiological saline and chlorocresol may be included in a volume ratio of 1:0.5 to 2 based on the total composition, but is not limited thereto.

As another embodiment of the present invention, the benzalkonium chloride or benzethonium chloride may be included in a concentration of 0.001% (w/v) to 0.02% (w/v) with respect to the composition for maintaining bee venom stability, but is not limited thereto. does not

As another embodiment of the present invention, the composition may maintain the stability of melittin in bee venom, but is not limited thereto.

As another embodiment of the present invention, the composition for maintaining bee venom stability may be added to bee venom so that the concentration of bee venom is 0.5 mg/mL to 3 mg/mL, but is not limited thereto.

In addition, the present invention is sterile physiological saline; and at least one selected from the group consisting of chlorocresol and cresol for maintaining stability of bee venom.

In addition, the present invention is sterile physiological saline; and at least one selected from the group consisting of chlorocresol and cresol for the preparation of a formulation for maintaining stability of bee venom.

The composition for maintaining bee venom stability according to the present invention is prepared by adding a preservative to sterile physiological saline or sterile distilled water. It was confirmed that the addition of tonium contributed to the maintenance of the stability of bee venom. In particular, it was confirmed that the stability of bee venom was maintained high when chlorocresol was added to sterile physiological saline or when chlorocresol and cresol were mixed. Accordingly, the composition for maintaining the stability of bee venom of the present invention is expected to be usefully used in the manufacture and storage of bee venom immersion liquid, biopharmaceuticals, cosmetics, etc. using bee venom as a raw material.

1 is a view showing the HPLC chromatogram according to the addition of sterile distilled water (DW) and each preservative for bee venom according to an embodiment of the present invention.
2 is a view showing HPLC chromatograms according to the addition of sterile physiological saline (NaCl) and each preservative for bee venom according to an embodiment of the present invention.
3 is a result confirming the change in melittin content (%) for 31 days after adding sterile distilled water or sterile physiological saline, and a preservative to bee venom according to an embodiment of the present invention.
4 is a result of confirming the melittin content (%) on days 0 and 31 after adding sterile distilled water or sterile physiological saline, and a preservative to bee venom according to an embodiment of the present invention.
5 is a result confirming the change in melittin content (%) for 8 weeks after adding sterile distilled water or sterile physiological saline, and a preservative to bee venom according to an embodiment of the present invention.
6 is a result of confirming the change in melittin content (%) on days 0 and 8 after adding sterile distilled water or sterile physiological saline, and a preservative to bee venom according to an embodiment of the present invention.
7 is a result confirming the change in melittin content (%) for 3 months after adding sterile distilled water or sterile physiological saline, and a preservative to bee venom according to an embodiment of the present invention.
8 is a result confirming the change in melittin content (%) at 0 days and 3 months after adding sterile distilled water or sterile physiological saline, and a preservative to bee venom according to an embodiment of the present invention.
9 to 11 are results of confirming the change in melittin content (%) according to the addition ratio of sterile physiological saline and preservative for bee venom according to an embodiment of the present invention, respectively, for 4 weeks (FIG. 9), 8 weeks ( 10) and 3 months (FIG. 11) after confirming the change in melittin content.

In an experimental example of the present invention, in order to confirm the composition for increasing the storage stability of bee venom in aqueous solution, methyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzalkonium chloride in sterile physiological saline or sterile distilled water , benzethonium chloride, cresol, chlorocresol, or benzyl alcohol were added to each of nine preservatives, and after dissolving bee venom, stability was tested through HPLC. As a result, in the case of sterile physiological saline, when chlorocresol or cresol was added, the stability of bee venom was maintained high (82%, 60%, respectively) until 31 days compared to the case where no preservative was added. When added, it was confirmed that melittin in bee venom was maintained at about 64% until 3 months. In addition, in the case of sterile distilled water, it was confirmed that more than 50% of melittin in bee venom was maintained until 31 days when benzalkonium chloride, benzethonium chloride, methyl paraoxybenzoate, or cresol was added compared to the case where no preservative was added ( See Experimental Example 1).

In another experimental example of the present invention, sterile physiological saline containing 0.4% (w/v) cresol and sterile physiological saline containing 0.2% (w/v) chlorocresol were mixed with 1:1, 1:2, or 2: When mixed in a ratio of 1 and added in a ratio of 1:2, melittin in bee venom was maintained at about 83% after 4 weeks and at 60% or more after 3 months, so bee venom stability was improved. It was confirmed that the optimal ratio of the preservative for maintenance (see Experimental Example 2).

Accordingly, the present invention provides a composition for maintaining bee venom stability comprising sterile water and a preservative as active ingredients,

The sterile water is at least one selected from the group consisting of sterile physiological saline and sterile distilled water,

The preservative is chlorocresol, cresol (Cresol), methyl paraoxybenzoate (Methyl ρ-Hydroxybenzoate), propyl paraoxybenzoate (Propyl ρ-Hydroxybenzoate), butyl paraoxybenzoate (Butyl ρ-Hydroxybenzoate), chlorobutanol ( It provides a composition for maintaining bee venom stability, characterized in that at least one selected from the group consisting of Chlorobutanol), benzalkonium chloride, and benzethonium chloride.

The present invention also provides a method for maintaining stability of bee venom, comprising adding the composition for maintaining stability of bee venom to bee venom.

In the present invention, “bee venom” is an animal-derived natural physiologically active material that is stored in the venom sac at the tip of the bee’s abdomen and is connected to the bee needle and secreted upon stimulation. It is composed of bioactive amines. Bee venom is involved in antibacterial action, antioxidant action and anti-inflammatory action, and is used for the purpose of treating diseases such as arthritis, back pain, and arteriosclerosis.

In one embodiment of the present invention, dried purified bee venom from Bplus was purchased and used (see Example 2), but there is no particular limitation on the type of bee venom, the method of obtaining it, and the place of obtaining it.

In the present invention, “Melittin” is a small bipolar membrane active peptide naturally occurring in bee venom, and has antibacterial and antifungal action as well as hemolytic action. Recently, anticancer action, anti-inflammatory action, anti-pain action, anti- Many effects have been reported as a composition for a virus preparation.

In the present invention, “bee venom stability” refers to the degree to which the structure, function, and/or biological activity of bee venom is retained when stored for a predetermined period, and according to an embodiment of the present invention, the stability of bee venom is As meaning the stability of melittin, the main component of bee venom, it can be confirmed through the content of melittin after storage of bee venom dissolved in sterile physiological saline or sterile distilled water, but is not limited thereto.

In the present invention, "maintenance" is meant to include both the state in which the stability of bee venom is preserved or continues to exist, and the delay in the decrease in the stability of bee venom.

In the present invention, "sterile water" means water from which microorganisms such as bacteria are removed by medicine or heat, and according to an embodiment or an experimental example of the present invention, the sterile water is selected from the group consisting of sterile physiological saline and sterile distilled water. It may be one or more, but is not limited thereto.

In the present invention, “preservative” means any compound or substance that helps to prevent changes in the structure or activity of proteins or peptides in bee venom. Chlorocresol, cresol, methyl paraoxybenzoate (Methyl ρ-Hydroxybenzoate), propyl paraoxybenzoate (Propyl ρ-Hydroxybenzoate), butyl paraoxybenzoate (Butyl ρ-Hydroxybenzoate), chlorobutanol (Chlorobutanol), It may be at least one selected from the group consisting of benzalkonium chloride, benzethonium chloride, and benzylalcohol, and “preservative solution” may mean an aqueous solution containing the preservative, but is limited thereto. doesn't happen

In the present invention, the composition for maintaining bee venom stability is sterile physiological saline; And it may include one or more selected from the group consisting of chlorocresol and cresol as an active ingredient, but is not limited thereto.

In the present invention, the composition for maintaining bee venom stability may include sterile physiological saline and chlorocresol as active ingredients.

In the present invention, the composition for maintaining stability of bee venom may include sterile physiological saline and cresol as active ingredients.

In the present invention, the composition for maintaining bee venom stability may include sterile physiological saline, chlorocresol, and cresol as active ingredients, in this case, sterile physiological saline and cresol: sterile physiological saline and chlorocresol are 1: 0.1 to 3, 1:0.1 to 2.5, 1:0.5 to 3, 1:0.5 to 2.5, 1:0.5 to 2, 1:1 to 3, 1:1 to 2.5, 1:1 to 2, 1:1.5 to 3, 1:1.5 to 2.5, 1:1.5 to 2 may be included in a volume ratio of, but is not limited thereto.

In the present invention, “chlorocresol” may have a chemical formula of C ₇ H ₇ ClO, and “cresol” may have a chemical formula of C ₇ H ₈ O.

At this time, the chlorocresol is 0.01 % (w/v) to 0.2 % (w/v), 0.05 % (w/v) to 0.2 % (w/v), 0.07 % (w/v) with respect to the composition for maintaining bee venom stability v) to 0.2 % (w/v), 0.1 % (w/v) to 0.2 % (w/v), 0.12 % (w/v) to 0.2 % (w/v), 0.15 % (w/v) to 0.2% (w/v), 0.17% (w/v) to 0.2% (w/v), or 0.2% (w/v), but is not limited thereto.

In addition, the cresol is 0.01% (w/v) to 0.4% (w/v), 0.05% (w/v) to 0.4% (w/v), 0.1% (w/v) with respect to the composition for maintaining bee venom stability ) to 0.4 % (w/v), 0.15 % (w/v) to 0.4 % (w/v), 0.2 % (w/v) to 0.4 % (w/v), 0.25 % (w/v) to a concentration of 0.4 % (w/v), 0.3 % (w/v) to 0.4 % (w/v), 0.35 % (w/v) to 0.4 % (w/v), or 0.4 % (w/v) may be included, but is not limited thereto.

In the present invention, the composition for maintaining bee venom stability is sterile physiological saline; And when at least one selected from the group consisting of chlorocresol and cresol is included as an active ingredient, bee venom stability may be maintained for 1 to 3 months after storage, but is not limited thereto. According to an experimental example of the present invention, sterile physiological saline; And when bee venom is treated with a composition for maintaining stability of bee venom comprising at least one selected from the group consisting of chlorocresol and cresol as an active ingredient, the melittin content in bee venom for 1 to 3 months after storage is 50% or more, such as 50 % to 100%, 55% to 100%, 60% to 100%, 65% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65% , 50% to 60%, 50% to 55%, 60% to 90%, or 60% to 85%, bee venom stability can be improved when compared to the control group treated with sterile physiological saline alone, but is not limited thereto does not

In the present invention, the composition for maintaining bee venom stability is sterile distilled water; And it may include one or more selected from the group consisting of benzalkonium chloride and benzethonium chloride as an active ingredient, but is not limited thereto.

In the present invention, “Benzalkonium chloride” has the chemical formula of C ₆ H ₅ CH ₂ N(CH ₃ ) ₂ RCl (R=C ₈ H ₁₇ ~C ₁₈ H ₃₇ ), “benzethonium chloride” (Benzethonium chloride)” may have a chemical formula of C ₂₇ H ₄₂ ClNO ₂ .

At this time, the benzalkonium chloride or benzethonium chloride is 0.001 % (w/v) to 0.02 % (w/v), 0.005 % (w/v) to 0.02 % (w/v), with respect to the composition for maintaining bee venom stability, 0.01 % (w/v) to 0.02 % (w/v), 0.015 % (w/v) to 0.02 % (w/v), or 0.02 % (w/v) may be included, but is not limited thereto does not

In the present invention, the composition for maintaining bee venom stability is sterile distilled water; And when at least one selected from the group consisting of benzalkonium chloride and benzethonium chloride is included as an active ingredient, bee venom stability may be maintained up to one month after storage, but is not limited thereto. According to an experimental example of the present invention, sterile distilled water; And when bee venom is treated with a composition for maintaining stability of bee venom comprising at least one selected from the group consisting of benzalkonium chloride and benzethonium chloride as an active ingredient, the melittin content in bee venom is 50% or more, such as 50 % to 100%, 52% to 100%, 54% to 100%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, or 50% to 55% may be maintained to improve bee venom stability compared to the control group treated with sterile distilled water alone, but is not limited thereto.

As another aspect of the present invention, the present invention may provide a composition for maintaining the stability of bee venom in an aqueous solution. In this case, the aqueous solution may be sterile water, the sterile water may be sterile physiological saline or sterile distilled water, and the composition is chlorocresol, cresol, methyl paraoxybenzoate (Methyl ρ-Hydroxybenzoate), para At least one selected from the group consisting of propyl oxybenzoate (Propyl ρ-Hydroxybenzoate), butyl paraoxybenzoate (Butyl ρ-Hydroxybenzoate), chlorobutanol (Chlorobutanol), benzalkonium chloride, and benzethonium chloride The stability of bee venom dissolved in sterile physiological saline or sterile distilled water may be maintained by including a preservative as an active ingredient, but is not limited thereto.

In the present invention, the composition for maintaining stability of bee venom may be added as a preservative to a pharmaceutical composition or cosmetic composition containing bee venom to maintain storage stability of bee venom in pharmaceuticals or cosmetics.

Accordingly, as another aspect of the present invention, the present invention provides a bee venom composition with improved stability comprising bee venom and the composition for maintaining stability of bee venom as an active ingredient,

There is provided a bee venom composition with improved stability, characterized in that the stability of bee venom is improved by the composition for maintaining stability of bee venom.

In the present invention, "bee venom composition with improved stability" refers to a bee venom composition in which the stability of bee venom is maintained in an aqueous solution by adding a composition for maintaining stability of bee venom to bee venom, and thus the duration of bee venom stability is increased or extended.

In the present invention, the bee venom composition with improved stability may be in the form of a pharmaceutical composition or a cosmetic composition, but is not limited thereto.

The composition for maintaining bee venom stability according to the present invention has a bee venom concentration of 0.5 mg/mL to 3 mg/mL, 0.5 mg/mL to 2.5 mg/mL, 0.5 mg/mL to 2 mg/mL, 0.5 mg/mL to 1.5 mg/mL, 0.5 mg/mL to 1 mg/mL, 1 mg/mL to 3 mg/mL, 1 mg/mL to 2.5 mg/mL, 1 mg/mL to 2 mg/mL, 1 mg/mL to 1.5 mg/mL or 1 mg/mL may be added to bee venom, and with respect to the bee venom composition, bee venom may be included at the above concentration to maintain stability, but is not limited thereto.

In the present invention, when the composition for maintaining bee venom stability is added to the pharmaceutical composition, it may further include an appropriate carrier, excipient and diluent commonly used in the preparation of the pharmaceutical composition. The excipient may be, for example, at least one selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a humectant, a film-coating material, and a controlled-release additive.

The pharmaceutical composition may be prepared in a powder, granule, sustained-release granule, enteric granule, liquid, eye drop, elsilic, emulsion, suspension, spirit, troche, fragrance, limonaade, tablet, sustained-release, respectively, according to a conventional method. Form tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, liquid extracts, injections, capsules, irrigation liquids, warnings, lotions It can be formulated and used in the form of external preparations such as preparations, pasta preparations, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external preparations are creams, gels, patches, sprays, ointments, warning agents, lotions, It may have a formulation such as liniment agent, pasta agent or cataplasmase agent.

Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium hydrogen carbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methyl cellulose, sodium carboxymethyl cellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methyl excipients such as cellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin , hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch powder, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. Hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, silicic anhydride, 1-hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, Disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodite, kaolin, petrolatum, sodium stearate, cacao fat, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogen Added soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, A lubricant such as starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, light anhydrous silicic acid; may be used.

As additives for the liquid formulation according to the present invention, water, diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

In the syrup according to the present invention, a sucrose solution, other sugars or sweeteners may be used, and if necessary, a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifying agent, thickening agent, etc. may be used.

Purified water may be used in the emulsion according to the present invention, and if necessary, an emulsifier, preservative, stabilizer, fragrance, etc. may be used.

Suspension agents according to the present invention include acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, HPMC 2910, etc. An agent may be used, and a surfactant, a preservative, a stabilizer, a colorant, and a fragrance may be used as needed.

The injection according to the present invention includes distilled water for injection, 0.9% sodium chloride injection, Ringel injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), lactated Ringel injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; Solubilizing aids such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethyl acetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethyl acetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, buffers such as albumin, peptone and gum; isotonic agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO ₃ ) carbon dioxide gas, sodium metabisulfite (Na ₂ S ₂ O ₅ ), sodium sulfite (Na ₂ SO ₃ ), nitrogen gas (N ₂ ), ethylenediaminetetraacetic acid; sulphating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, acetone sodium bisulfite; analgesic agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; suspending agents such as SiMC sodium, sodium alginate, Tween 80, or aluminum monostearate.

The suppository according to the present invention includes cacao fat, lanolin, witepsol, polyethylene glycol, glycerogelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lanet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolene (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Butyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium, A, AS, B, C, D, E, I, T, Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository IV type (AB, B, A, BC, BBG, E, BGF, C, D, 299), supostal (N, Es), Wecobi (W, R, S, M, Fs), tester triglyceride base (TG-95, MA, 57) and The same mechanism may be used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The pharmaceutical composition is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.

The pharmaceutical composition may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention pertains.

The pharmaceutical composition may be administered to an individual by various routes. All modes of administration can be envisaged, for example, transdermal administration via microneedle, oral administration, subcutaneous injection, intradermal injection, topical administration, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection. , sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, and the like.

The pharmaceutical composition is determined according to the type of drug as an active ingredient along with several related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.

In the present invention, when the composition for maintaining bee venom stability is added to the cosmetic composition, the formulation of the cosmetic composition is skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutritional lotion, massage cream, It may be in the form of nourishing cream, mist, moisture cream, hand cream, hand lotion, foundation, essence, nourishing essence, pack, soap, cleansing foam, cleansing lotion, cleansing cream, cleansing oil, cleansing balm, body lotion or body cleanser. .

The cosmetic composition may further include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, and sphingolipids.

As water-soluble vitamins, any type of vitamin can be used as long as it can be formulated in cosmetics. Examples include vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, and vitamin H. Also, their salts (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-phosphate sodium salt, ascorbic acid-2-phosphate magnesium salt, etc.) are also included in the water-soluble vitamins that can be used in the present invention. Included. Water-soluble vitamins can be obtained by conventional methods, such as a microbial transformation method, a purification method from a microbial culture, an enzymatic method, or a chemical synthesis method.

The oil-soluble vitamin may be any type of vitamin that can be formulated in cosmetics, and for example, vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol), etc. , their derivatives (ascorbine palmitate, ascorbine stearate, ascorbine dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherol nicotinic acid, vitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl ethers, etc.) are also included in the oil-soluble vitamins used in the present invention. Oil-soluble vitamins can be obtained by conventional methods such as a microbial transformation method, a purification method from a microbial culture, and an enzyme or chemical synthesis method.

The macromolecular peptide may be any compound as long as it can be formulated in cosmetics, and examples thereof include collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, and keratin. Polymeric peptides can be purified and obtained by conventional methods such as purification from a microbial culture solution, enzyme method or chemical synthesis method, or can be purified and used from natural products such as dermis of pigs or cattle, silk fiber of silkworms, etc.

The high molecular weight polysaccharide may be any compound as long as it can be formulated in cosmetics, and examples thereof include hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate, or a salt thereof (sodium salt, etc.). For example, chondroitin sulfate or a salt thereof can be used after purification from mammals or fish.

The sphingolipid may be any as long as it can be formulated in cosmetics, and examples thereof include ceramide, phytosphingosine, and sphingoglycolipid. Sphingo lipids can be purified by conventional methods from mammals, fish, shellfish, yeast or plants, or can be obtained by chemical synthesis.

In addition to the above essential components, the cosmetic composition may contain other components commonly formulated in cosmetics as needed.

Other ingredients that may be added include oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, UV absorbers, preservatives, bactericides, antioxidants, plant extracts, pH adjusters, alcohols, colorants, fragrances, A blood circulation promoter, a cooling agent, a restrictive agent, purified water, etc. are mentioned.

When the formulation of the present invention is a lotion, paste, cream or gel, animal fiber, vegetable fiber, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. can be used

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, linolin derivative or ethoxylated glycerol fatty acid ester and the like may be used.

In addition, the present invention is sterile physiological saline; and at least one selected from the group consisting of chlorocresol and cresol for maintaining stability of bee venom.

In addition, the present invention is sterile physiological saline; and at least one selected from the group consisting of chlorocresol and cresol for the preparation of a formulation for maintaining stability of bee venom.

Hereinafter, preferred examples and experimental examples are presented to help the understanding of the present invention. However, the following Examples and Experimental Examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

[Example]

Example 1. Preparation of bee venom preservation solution

As a preservative that can preserve protein and peptide, the main component of bee venom, an acceptable preservative was selected according to the guidelines for drug additives presented by the Ministry of Food and Drug Safety (MFDS). The selected preservatives are methyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzalkonium chloride, benzethonium chloride, cresol, chlorocresol, or benzyl alcohol. (Louis, MO, USA).

Each preservative was dissolved in sterile distilled water or sterile physiological saline according to the maximum concentration range allowed by the Ministry of Food and Drug Safety, and stored in a refrigerator after blocking the light.

The conditions for using the added aqueous solution and preservative for maintaining the stability of bee venom are shown in Table 1 below.

Example 2. Preparation of bee venom stability test samples

Dried bee venom manufactured in June 2020 was purchased from Bplus and used. Powdered dry bee venom was precisely weighed 1.0 mg each and completely dissolved in sterile distilled water or sterile physiological saline and each preservative added to a concentration of 1.0 mg/mL, 3 lots were prepared. Each sample was filtered once with a 0.2 μm polyvinylidene fluoride (PVDF) syringe filter immediately after sample preparation, and stability data on day 0 was obtained by HPLC. Thereafter, the stability test sample was stored refrigerated (5 ℃ ± 3 ℃), and the stability was evaluated by comparing the content and composition of bee venom in the HPLC chromatogram until the 31st day at intervals of 3 to 4 days.

Example 3. HPLC analysis

Analysis was performed with a Waters ALLIANCE e2695 HPLC and 2998 Photodiode Array (PDA) detector. A YMC triart C ₁₈ column (4.6 × 150 mm, 5 μm) was selected as the stationary phase, and the column temperature was maintained at 30 °C. The sample temperature was maintained at 4 °C in order to minimize the effect of the temperature of the analysis sample as the analysis time increases due to the large number of samples. As the mobile phase, water (Solvent A) and acetonitrile (Solvent B) to which 0.1% trifluoroacetic acid (TFA) was added were used to increase resolution. 10 μL of the sample was injected, and the flow rate was maintained at 0.4 mL/min. Immediately after sample injection, 10% B was held for 3 minutes, and B was increased to 70% by 15 minutes. B (16 minutes) maintained at 70% for 1 minute was developed as a gradient system in which it returned to the initial solvent condition (10% B) at 17 minutes and maintained for 3 minutes (10% B, 20 minutes). The optimal detection wavelength was set to 220 nm so that the detection wavelength could be detected with the highest sensitivity by comparing the chromatograms according to the 190-400 nm UV wavelength.

HPLC analysis conditions for the bee venom stability test are shown in Table 2 below.

[Experimental example]

Experimental Example 1. Stability test result

1-1. 0-31 days

Day 0 and Day 31 HPLC chromatograms of bee venom are shown in FIGS. 1 and 2 . 1 is an HPLC chromatogram for bee venom according to the addition of sterile distilled water and each preservative, and FIG. 2 shows an HPLC chromatogram according to the addition of sterile physiological saline and each preservative for bee venom. Stability for 31 days was evaluated based on the change in the content of melittin, a peak showing an area value of 85% on the 0th day HPLC chromatogram of bee venom.

As a result of confirming the stability of bee venom in aqueous solution according to the addition of aqueous solution and preservative for bee venom, as shown in FIGS. 3 and 4, when 0.2% (w/v) chlorocresol is added to sterile physiological saline (NaCl), It was confirmed that the main component (Melittin) was maintained at an average of 82.4% even after 31 days, showing the highest stability. In addition, the melittin peak of bee venom was maintained at an average of 60% after 31 days in a preservative solution containing 0.4% (w/v) cresol in sterile physiological saline, showing the second highest stability.

On the other hand, in the case of sterile distilled water, it was confirmed that the highest level of melittin was maintained in the preservation solution to which 0.02% (w/v) benzalkonium chloride was added. It did not completely dissolve but turned cloudy, and the lowest melittin content and rapid peak reduction were also confirmed in the HPLC chromatogram.

When no preservative was used, the melittin peak of bee venom dissolved in sterile physiological saline decreased by an average of 71.5%, and the melittin peak of bee venom dissolved in sterile distilled water decreased by an average of 62.1% after 31 days of manufacture. An increasing trend was confirmed.

From the above results, as a composition of a preservation solution for maintaining the stability of bee venom in aqueous solution, sterile physiological saline and 0.2% (w/v) chlorocresol, or sterile physiological saline and 0.4% (w/v) It was confirmed that cresol can be used, and 0.02 % (w/v) benzalkonium chloride, 0.02 % (w/v) benzethonium chloride, 0.18 % ( When w/v) methyl paraoxybenzoate or 0.4% (w/v) cresol was added, it was found that the stability of bee venom was maintained at an average of 50% or more after 31 days.

1-2. 0-8 weeks

On the 0th day HPLC chromatogram of bee venom, HPLC analysis was performed once a week based on the change in the content of melittin, a peak showing an area value of 85%, to evaluate stability for 8 weeks.

As a result of confirming the stability of bee venom in aqueous solution according to the addition of aqueous solution and preservative to bee venom, as shown in FIGS. 5 and 6, when 0.2% (w/v) chlorocresol is added to sterile physiological saline, the main component (melitin) in bee venom ) was maintained at an average of 73.5% even after 8 weeks, showing the highest stability. In addition, the melittin peak of bee venom was maintained at an average of 42.0% in a preservative solution containing 0.4% (w/v) cresol in sterile physiological saline, showing the second highest stability. At this time, in the case of the sterile physiological saline alone treatment group used as a control group, it was confirmed that the melittin content was as low as about 5%.

In addition, the melittin peak of bee venom dissolved in sterile distilled water was lower than about 15% when 8 weeks had elapsed after preparation, and bee venom was added when benzalkonium chloride, benzethonium chloride, methyl paraoxybenzoate, or cresol was added to sterile distilled water. of melittin was maintained at about 30-38%, it was confirmed that even with the addition of the preservative, the melittin peak was reduced by an average of 63.0% or more.

1-3. 0-3 months

On the 0th day HPLC chromatogram of bee venom, HPLC analysis was performed once a month based on the change in the content of melittin, a peak showing an area value of 85%, to evaluate stability for 3 months.

As a result of confirming the stability of bee venom in aqueous solution according to the addition of aqueous solution and preservative to bee venom, as shown in FIGS. 7 and 8, when 0.2% (w/v) chlorocresol is added to sterile physiological saline, the main component (melitin) in bee venom ) was maintained at an average of 64.1% even after 3 months, showing the highest stability. In addition, the melittin peak of bee venom was maintained at an average of 27.9% in the preservative solution in which 0.4% (w/v) cresol was added to sterile physiological saline, showing the second highest stability, but when other preservatives were added to sterile physiological saline After 3 months, it was confirmed that the melittin peak decreased by an average of 98.3% or more.

In addition, the melittin peak of bee venom dissolved in sterile distilled water was low at less than about 5% after 3 months of manufacture, and it was confirmed that the melittin peak, the main component, decreased by an average of 77.7% or more even if a preservative was added.

Experimental Example 2. Stability test results according to the combination of preservatives

Sterile physiological saline containing 0.2% (w/v) chlorocresol and 0.4% (w/v) cresol were added, which showed the highest stability in the stability test for 31 days performed in Experimental Example 1-1. The effect on the stability of bee venom by combining the saline solution in a 1:2, 1:1, or 2:1 ratio (total amount to volume ratio) was evaluated through HPLC analysis. After dissolving bee venom at a concentration of 1 mg/mL in each combination preservation solution, it was filtered once with a 0.2 μm syringe filter and analyzed. The deployment time was partially changed.

The results of the bee venom stability test according to the preservative combination were as a control, sterile physiological saline, sterile physiological saline containing 0.2% (w/v) chlorocresol, and sterile physiological saline containing 0.4% (w/v) cresol were added. It was compared with the chromatogram of a 1 mg/mL sample of bee venom. All samples were prepared three by one, refrigerated (5 ℃ ± 3 ℃) after securing the HPLC chromatogram of week 0, and analyzed by HPLC once a week for 4 weeks to confirm the change in the content of the melittin peak.

As a result, as shown in FIG. 9, in the HPLC analysis 4 weeks after sample preparation, sterile physiological saline containing 0.4% (w/v) cresol and 0.2% (w/v) chlorocresol were added to 1: 2, the bee venom melittin in the preservative solution showed the highest stability, maintaining an average of 83.3%. On the other hand, in sterile physiological saline containing 0.4% (w/v) cresol and sterile physiological saline containing 0.2% (w/v) chlorocresol in a 2:1 ratio, the average concentration of bee venom melittin was 53.1 % decreased, and showed lower stability than in sterile physiological saline in which 0.4 % (w/v) cresol and 0.2 % (w/v) chlorocresol were added alone.

In addition, as a result of confirming bee venom melittin after 8 weeks and 3 months, as shown in FIGS. 10 (after 8 weeks) and 11 (after 3 months), sterile physiological saline with cresol: sterile physiological with chlorocresol When the saline solution was mixed in 2:1, bee venom melittin decreased almost after 3 months, whereas when mixed in 1:2, bee venom melittin was maintained at an average of about 70% after 8 weeks and about 60% or more after 3 months. It was confirmed that the highest stability was confirmed, and it was found that even when added in a 1:1 ratio, it was maintained up to almost 60% after 3 months.

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (12)

In the composition for maintaining bee venom stability comprising sterile water and a preservative as active ingredients,
The sterile water is at least one selected from the group consisting of sterile physiological saline and sterile distilled water,
The preservative is chlorocresol (Chlorocresol), cresol (Cresol), methyl paraoxybenzoate (Methyl ρ-Hydroxybenzoate), propyl paraoxybenzoate (Propyl ρ-Hydroxybenzoate), butyl paraoxybenzoate (Butyl ρ-Hydroxybenzoate), chlorobutanol ( Chlorobutanol), benzalkonium chloride (Benzalkonium chloride), and bee venom stability maintenance composition, characterized in that at least one selected from the group consisting of benzethonium chloride.
According to claim 1,
The composition is sterile physiological saline; and at least one selected from the group consisting of chlorocresol and cresol as an active ingredient, the composition for maintaining bee venom stability.
According to claim 1,
The composition is sterile distilled water; and at least one selected from the group consisting of benzalkonium chloride and benzethonium chloride as an active ingredient, the composition for maintaining bee venom stability.
According to claim 1,
The composition for maintaining bee venom stability, characterized in that the composition maintains the storage stability of bee venom for 1 to 3 months.
3. The method of claim 2,
The composition for maintaining bee venom stability, characterized in that the chlorocresol is contained in a concentration of 0.01% (w/v) to 0.2% (w/v) with respect to the composition for maintaining bee venom stability.
3. The method of claim 2,
The composition for maintaining bee venom stability, characterized in that the cresol is contained in a concentration of 0.01% (w/v) to 0.4% (w/v) with respect to the composition for maintaining bee venom stability.
3. The method of claim 2,
The composition comprises sterile physiological saline, chlorocresol, and cresol as active ingredients,
Sterile physiological saline and cresol: A composition for maintaining bee venom stability comprising sterile physiological saline and chlorocresol in a volume ratio of 1:0.5 to 2 with respect to the total composition.
4. The method of claim 3,
The composition for maintaining bee venom stability, characterized in that the benzalkonium chloride or benzethonium chloride is contained in a concentration of 0.001% (w/v) to 0.02% (w/v) with respect to the composition for maintaining bee venom stability.
According to claim 1,
The composition for maintaining stability of bee venom, characterized in that the composition maintains the stability of melittin in bee venom.
According to claim 1,
The composition for maintaining bee venom stability, characterized in that the composition is added to bee venom so that the concentration of bee venom is 0.5 mg/mL to 3 mg/mL.
A method for maintaining stability of bee venom, comprising adding the composition for maintaining stability of bee venom according to any one of claims 1 to 10 to bee venom.
A bee venom composition with improved stability, comprising as an active ingredient the bee venom and the composition for maintaining the stability of bee venom according to any one of claims 1 to 10,
A bee venom composition with improved stability, characterized in that the stability of bee venom is improved by the composition for maintaining stability of bee venom according to any one of claims 1 to 10.

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