KR20230133595A - Composition for maintaining stability of serotonin contained in Vespa Velutina venom - Google Patents

Abstract

The present invention relates to a stability-maintaining composition capable of suppressing a decrease in the content of serotonin contained in Vespa velutina venom in an aqueous solution and an aqueous Vespa velutina venom formulation comprising the same. The stability-maintaining composition according to the present invention comprises: sterile distilled water or sterile physiological saline; and one or two more stabilizers selected from Methyl ρ-Hydroxybenzoate, cresol, and chlorocresol as an active ingredient, wherein the composition can suppress a decrease in the content of serotonin contained in Vespa velutina venom in an aqueous solution. The stability-maintaining composition and the aqueous formulation of the present invention can be advantageously applied to pharmaceutical compositions, such as pharmacopunctures, biopharmaceuticals, etc., health foods, cosmetic products, etc.

Description

Composition for maintaining stability of serotonin contained in Vespa Velutina venom}

The present invention relates to a composition for maintaining the stability of serotonin contained in black wasp venom. In particular, a composition for maintaining stability that can suppress the decrease in the content of serotonin contained in black wasp venom in an aqueous solution and the same. It relates to an aqueous solution formulation of black wasp venom comprising:

Bee sting therapy, also called bee sting or bee sting therapy, is a method of treating diseases by activating the body's immune function by injecting purified bee venom into acupuncture points. The history of bengchim is very old, and it is currently established as an alternative medicine. Bee needles are known to be particularly effective in treating inflammatory and painful diseases. They have excellent anti-inflammatory effects and have effects such as blood purification, hemolysis, nerve revitalization, sterilization, and tissue creation and destruction. It is known to have

The main ingredient of bee venom extracted and purified from bees is melittin. Melittin is a protein composed of 26 amino acids and makes up 40-50% of dried bee venom, and has been reported to be antibacterial, antiviral, induce hormone secretion, and stimulate enzyme secretion.

Unlike bee venom, which has been actively used and studied in the treatment of diseases from ancient times to the present, wasp venom obtained from wasps is not properly used for therapeutic purposes and research is insufficient. Wasp venom refers to a toxin used by wasps when looking for food or when an enemy invades their territory. It has a significant difference in composition from bee venom. The components of wasp venom vary greatly depending on the type of wasp, and known ingredients include histamine, serotonin, dopamine, adrenaline, and acetylcholine.

The black-backed wasp ( Vespa velutina ) belongs to the Hymenoptera wasp family and is an exotic insect that was introduced into Korea from Southeast Asia in the early 2000s and is spreading nationwide. Black-backed hornets are omnivorous insects that prey on a variety of insects, but they mainly hunt adult bees and prefer Western honey bees ( Apis mellifera ), causing great damage to the beekeeping industry. The main ingredient with the highest content in black wasp venom is serotonin.

Republic of Korea Patent Publication No. 10-2019-0101980 Republic of Korea Patent Publication No. 10-2015-0137314 Republic of Korea Patent Publication No. 10-2018-0052128 Republic of Korea Patent Publication No. 10-2012-0029328

When black wasp venom is left at room temperature as is or in an aqueous solution, the main component, serotonin, decreases rapidly, decreasing by about 50% or more in about 4 weeks. Because of these problems, it has been difficult to develop black wasp venom into an aqueous solution formulation. In addition, even when using black wasp venom after drying it into powder and storing it in the freezer, it was difficult to dissolve it in a solvent to make an aqueous solution before using it immediately each time it was used. When it was made into an aqueous solution, it reacted sensitively to temperature and humidity, so it was difficult to use it immediately. Therefore, significant serotonin decline may begin within a few days in aqueous solution. This problem has become a major obstacle to developing or utilizing black wasp venom for various purposes.

The purpose of the present invention is to provide a composition for maintaining stability that suppresses a decrease in the content of serotonin contained in black wasp venom in an aqueous solution.

In addition, the purpose of the present invention is to provide an aqueous solution formulation of black wasp venom with improved stability that can suppress the decrease in the content of serotonin contained in black wasp venom in an aqueous solution.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art to which the present invention belongs from the description below. will be.

In order to achieve the above object, in the present invention,

A composition for maintaining stability that inhibits the decrease in the content of serotonin contained in black wasp venom in an aqueous solution,

Sterile distilled water or sterile saline solution; and

Provided is a composition comprising one or two or more stabilizers selected from Methyl ρ-Hydroxybenzoate, Cresol, and Chlorocresol as an active ingredient.

The composition, preferably in an aqueous solution, inhibits the content of serotonin contained in black wasp venom from being reduced by more than 15% based on HPLC peak area for at least 4 weeks.

Black wasp venom can be dissolved in the composition at a predetermined concentration and used. In a preferred embodiment, black wasp venom is dissolved and used in the composition at a concentration of 2 to 8 mg/ml.

Additionally, in the present invention,

Black-backed wasp venom;

Sterile distilled water or sterile saline solution; and

An aqueous solution formulation containing one or two or more stabilizers selected from Methyl ρ-Hydroxybenzoate, Cresol, and Chlorocresol,

An aqueous solution formulation of black wasp venom is provided, which is characterized in that it inhibits the content of serotonin contained in the black wasp venom from being reduced by more than 15% for at least 4 weeks based on HPLC peak area.

In a preferred embodiment, the aqueous solution formulation of black wasp venom contains black wasp venom at a concentration of 2 to 8 mg/ml.

The composition for maintaining stability of the present invention can significantly suppress the decrease in the content of serotonin contained in black wasp venom in an aqueous solution. After dissolving black wasp venom in the stability maintaining composition of the present invention, a decrease in serotonin content of more than 15% based on HPLC peak area can be suppressed for at least 4 weeks.

The stability-maintaining composition and the aqueous solution formulation of black wasp venom according to the present invention can be usefully applied to pharmaceutical compositions such as herbal medicine solutions and biological medicines, health foods, cosmetics, etc.

Figure 1 is an HPLC chromatogram showing the results of testing the stability of a composition in which black wasp venom was dissolved in sterilized distilled water and a stabilizer was added.
Figure 2 is an HPLC chromatogram showing the results of testing the stability of a composition in which black wasp venom was dissolved in sterilized saline and a stabilizer was added.
Figure 3 shows a comparison of the HPLC chromatographic changes over time of a composition in which black wasp venom was dissolved in sterilized distilled water without adding a stabilizer.
Figure 4 shows a comparison of the HPLC chromatographic changes over time of a composition in which black wasp venom was dissolved in sterilized saline without adding a stabilizer.
Figure 5 shows the results of confirming the change in serotonin content (%) over 4 weeks after preparing a composition in which black wasp venom was dissolved in sterilized distilled water or sterilized saline and a stabilizer was added.
Figure 6 shows the results of confirming the change in serotonin content (%) at 0 and 4 weeks after preparing a composition in which black wasp venom was dissolved in sterilized distilled water or sterilized saline and a stabilizer was added.

The composition for maintaining stability provided by the present invention,

A composition that inhibits the decrease in the content of serotonin contained in black wasp venom in an aqueous solution, comprising: sterilized distilled water or sterilized physiological saline; and one or two or more stabilizers selected from Methyl ρ-Hydroxybenzoate, Cresol, and Chlorocresol as active ingredients.

In the present invention, "black-backed wasp venom" is defined as a toxin used by the black-backed wasp (Vespa velutina) when looking for food or when an enemy invades its territory, and contains serotonin as its main ingredient. It is obtained by separating and purifying the venom from the venom sac of the black-backed wasp.

In the present invention, “stabilizer” is defined as an additive for suppressing the decrease in serotonin content in black wasp venom.

In the present invention, the black-backed wasp venom is produced by separating the venom from the venom sac of the black-backed wasp. It can be purified and used, and to increase preservability, it can also be dried and powdered using a method such as freeze-drying.

The solvent forming the aqueous solution phase is sterilized distilled water or sterilized physiological saline solution.

As the stabilizer, one or two or more types selected from Methyl ρ-Hydroxybenzoate, Cresol, and Chlorocresol are used. Among the three stabilizers, cresol and chlorocresol can be used more preferably because the increase in by-product peaks is relatively small.

Methyl paraoxybenzoate is preferably included in the composition at a concentration of 0.05-0.18% (w/v), and more preferably at a concentration of 0.1-0.18% (w/v).

Cresol is preferably included in the composition at a concentration of 0.15-0.4% (w/v), and more preferably at a concentration of 0.3-0.4% (w/v).

Chlorocresol is preferably included in the composition at a concentration of 0.1-0.2% (w/v), and more preferably at a concentration of 0.15-0.2% (w/v).

Most preferably, the serotonin content contained in black wasp venom is maintained at the highest level when sterilized saline solution is used as a solvent and chlorocresol at a concentration of 0.2% (w/v) is added.

Black wasp venom is used by dissolving it in the stability-maintaining composition at a desired concentration. In a preferred embodiment, black wasp venom may be included in the composition at a concentration of 2 to 8 mg/ml, and is more preferably included at a concentration of 4 to 6 mg/ml. If black wasp venom is dissolved in a composition for maintaining stability to form an aqueous solution, the serotonin content can be stably maintained at a very high level even after 4 weeks of dissolution. Preferably, the content of serotonin in the black wasp venom contained in the composition for maintaining stability can be maintained at 85% or more for at least 4 weeks based on HPLC peak area, and more preferably at 90% or more. In other words, it is possible to suppress a decrease of 15% or more, preferably 10% or more, based on the HPLC peak area. This maintains stability at a very high level compared to the serotonin content of black wasp venom, which generally decreases by about 50% or more after about 4 weeks at room temperature.

The aqueous solution formulation of black wasp venom provided by the present invention,

Black-backed wasp venom; Sterile distilled water or sterile saline solution; and one or two or more stabilizers selected from Methyl ρ-Hydroxybenzoate, Cresol, and Chlorocresol, and the content of serotonin contained in the black wasp venom is determined by HPLC. Suppress a decrease of more than 15% based on peak area for at least 4 weeks.

The description of each component is the same as the description of the stability maintaining composition.

The stability-maintaining composition and the aqueous solution formulation of black wasp venom can be usefully applied to pharmaceutical compositions, health foods, cosmetics, etc. containing black wasp venom as an active ingredient.

The present invention will be described in more detail through examples below. These examples illustrate the present invention and the scope of the present invention is not limited thereto.

<Example 1>

An aqueous solution formulation of black wasp venom was prepared by dissolving black wasp venom powder in sterilized distilled water at a concentration of 5.0 mg/ml and adding methyl paraoxybenzoate at a concentration of 0.18% (w/v).

<Example 2>

Black wasp venom powder was dissolved in sterilized distilled water at a concentration of 5.0 mg/ml and cresol was added at a concentration of 0.4% (w/v) to prepare an aqueous solution formulation of black wasp venom.

<Example 3>

Black wasp venom powder was dissolved in sterilized distilled water at a concentration of 5.0 mg/ml and chlorocresol was added at a concentration of 0.2% (w/v) to prepare an aqueous solution formulation of black wasp venom.

<Example 4>

Black wasp venom powder was dissolved in sterile saline solution (0.9% NaCl) at a concentration of 5.0 mg/ml and methyl paraoxybenzoate was added at a concentration of 0.18% (w/v) to prepare an aqueous solution formulation of black wasp venom.

<Example 5>

Black wasp venom powder was dissolved in sterile saline solution (0.9% NaCl) at a concentration of 5.0 mg/ml and cresol was added at a concentration of 0.4% (w/v) to prepare an aqueous solution formulation of black wasp venom.

<Example 6>

Black wasp venom powder was dissolved in sterile saline solution (0.9% NaCl) at a concentration of 5.0 mg/ml and chlorocresol was added at a concentration of 0.2% (w/v) to prepare an aqueous solution formulation of black wasp venom.

<Experimental Example 1>

Selection of stabilizers and preparation of compositions for maintaining stability

In order to select a stabilizer capable of stabilizing black wasp venom, a composition for maintaining stability under experimental conditions was prepared using the following allowable additives.

1. Selection of acceptable additives

In order to select the stabilizer for the purpose of the present invention within the range of additives and use permitted by the Ministry of Food and Drug Safety in accordance with the guidelines for pharmaceutical additives presented by the Ministry of Food and Drug Safety (MFDS), the test subjects were first methyl paraoxybenzoate, A total of 9 types of additives were initially selected: propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzalkonium chloride, benzethonium chloride, cresol, chlorocresol, and benzyl alcohol.

2. Preparation of composition for maintaining stability

The first selected methyl paraoxybenzoate, propyl paraoxybenzoate, butyl paraoxybenzoate, chlorobutanol, benzalkonium chloride, benzethonium chloride, cresol, chlorocresol and benzyl alcohol were purchased from Sigma-aldrich (St. Louis, MO, USA). Purchased and used from .

Each additive was dissolved in sterilized distilled water or sterilized saline solution within the maximum concentration range permitted by the Ministry of Food and Drug Safety, then stored in a refrigerator, protected from light.

The types, concentrations, and usage conditions of each additive used are shown in Table 1 below.

No. additive density(%) Terms of use black wasp venom Sterile distilled water (DW) / Sterilized saline solution (0.9% NaCl) 5mg/mL One Methyl ρ-Hydroxybenzoate 0.18 pH3~7 2 Propyl ρ-Hydroxybenzoate 0.02 pH3~7 3 Butyl ρ-Hydroxybenzoate 0.01 pH3~7 4 Chlorobutanol 0.5 5 Benzalkonium chloride 0.02 pH9 6 Benzethonium chloride 0.02 pH9 7 Cresol 0.4 Neutral~Acidic 8 Chlorocresol 0.2 Neutral~Acidic 9 Benzyl alcohol 0.1

<Experimental Example 2>

stability test

1. Preparation of stability test samples

For the black wasp venom, wasp venom powder extracted from black wasps in June 2020 and dried and powdered was used.

5.0 mg of wasp venom powder was precisely weighed and completely dissolved in sterilized distilled water, sterilized saline solution, and the stability-maintaining composition prepared in Experimental Example 1 to obtain a concentration of 5.0 mg/ml, and 3 lots were prepared.

Each sample was filtered once through a 0.2㎛ PVDF syringe filter immediately after sample preparation, and stability data at week 0 was obtained by HPLC. Afterwards, the stability test samples were sealed in amber vials and stored in refrigeration (approximately 4°C), and stability was evaluated by comparing the content and composition of wasp venom using HPLC chromatograms at 2-week intervals for up to 4 weeks.

2. HPLC analysis conditions

HPLC analysis was performed using a Waters ALLIANCE e2695 HPLC and 2998 PDA detector.

A YMC C ₁₈ column (4.6 × 150 mm, 5 μm) was used as the stationary phase, and the column temperature was maintained at 30°C.

As the analysis time was long due to the large number of samples, the sample temperature was maintained at 4°C to minimize the effect of temperature on the analysis sample.

To increase separation performance, water with 0.1% TFA (Trifluoroacetic acid) (Solvent A) and acetonitrile with 0.1% TFA (Solvent B) were used as mobile phases.

The detection wavelength was set to 215 nm, which is the optimal detection wavelength for detection with the highest sensitivity by comparing chromatograms according to UV wavelengths of 190 to 400 nm. HPLC analysis conditions, including solvent gradient composition, are shown in Table 2 below.

3. Stability test results

Changes in the content of serotonin, the largest peak, in the HPLC chromatogram of black wasp venom on day 0 were evaluated.

The HPLC chromatogram resulting from testing the stability of a composition obtained by dissolving black wasp venom in sterilized distilled water and adding a stabilizer is shown in Figure 1, and shows the results of testing the stability of a composition obtained by dissolving black wasp venom in sterilized distilled water and adding a stabilizer. The HPLC chromatogram resulting from the stability test is shown in Figure 2.

In addition, the HPLC chromatogram showing the results of testing the stability of a composition in which black wasp venom was dissolved in sterilized distilled water and no stabilizer was added is shown in Figure 3, and the black wasp venom was dissolved in sterilized saline solution and no stabilizer was added. The HPLC chromatogram resulting from testing the stability of the composition is shown in Figure 4.

From the results in Figures 1 and 2, it was confirmed that when black wasp venom was dissolved in sterile saline and methyl paraoxybenzoate, cresol, or chlorocresol was added, the change in composition was maintained without significant difference for 0, 2, and 4 weeks. Even when black wasp venom was dissolved in sterile distilled water and methyl paraoxybenzoate, cresol, or chlorocresol was added, there was little change in composition.

On the other hand, in the results of Figures 3 and 4, when black wasp venom was dissolved in sterile distilled water or sterile saline and no stabilizer was added, it was confirmed that serotonin decreased significantly after 4 weeks.

The results of confirming the change in serotonin content (%) over 4 weeks after dissolving black wasp venom in sterilized distilled water or sterilized saline solution and preparing a composition with or without adding a stabilizer are shown in Figure 5. After dissolving the poison in sterilized distilled water or sterilized saline solution and preparing a composition with or without adding a stabilizer, the results of confirming the change in serotonin content (%) at week 0 and week 4 are shown in Figure 6.

From the results in Figures 5 and 6, the serotonin peak of the black wasp venom dissolved in sterilized saline solution 4 weeks after preparing the aqueous solution formulation without using a stabilizer was 43.9% on average, and in sterilized distilled water, the serotonin peak was 43.9% on average. The serotonin peak of dissolved black wasp venom decreased by an average of 28.7%, and the by-product peaks also tended to increase qualitatively and quantitatively. On the other hand, when methyl paraoxybenzoate, cresol, or chlorocresol was added as a stabilizer, it was confirmed that the serotonin content was at least 85% based on peak area even after 4 weeks, showing a very low decrease in serotonin.

From the above results, methyl paraoxybenzoate, cresol, and chlorocresol could be selected as stabilizers that inhibit the decrease of serotonin contained in black wasp venom. In addition, among the three stabilizers, cresol and chlorocresol were found to be more preferable as they showed a relatively small increase in by-product peaks (Figures 1 and 2).

The stability-maintaining composition of the present invention and the aqueous solution formulation of black wasp venom containing the same described above are exemplary, and those skilled in the art will be able to make various modifications and other equivalent implementations thereof. You can see that an example is possible. Therefore, it will be well understood that the present invention is not limited to the forms mentioned in the description of the invention above. The true technical protection scope of the present invention shall be determined by the technical spirit of the claims, and is understood to include all modifications, equivalents and substitutes within the spirit and scope of the present invention defined by the claims of the present invention. It has to be. In addition, terms or words used in this specification and claims should not be construed as limited to their usual or dictionary meanings, and the inventor appropriately defines the concept of terms in order to explain his or her invention in the best way. It should be interpreted with meaning and concept consistent with the technical idea of the present invention based on the principle that it can be done.

Claims (8)

A composition for maintaining stability that inhibits the decrease in the content of serotonin contained in black wasp venom in an aqueous solution,
Sterile distilled water or sterile saline solution; and
A composition comprising one or two or more stabilizers selected from Methyl ρ-Hydroxybenzoate, Cresol, and Chlorocresol as an active ingredient. According to paragraph 1,
The methyl paraoxybenzoate is contained in the composition at a concentration of 0.05 to 0.18% (w/v),
The cresol is contained in the composition at a concentration of 0.15 to 0.4% (w/v),
A composition characterized in that the chlorocresol is contained in the composition at a concentration of 0.1 to 0.2% (w/v). According to claim 1 or 2,
A composition characterized in that it inhibits the serotonin content from being reduced by more than 15% for at least 4 weeks based on HPLC peak area. According to claim 1 or 2,
A composition characterized in that it is used by dissolving black wasp venom at a predetermined concentration in the composition. According to clause 4,
A composition characterized in that black wasp venom is dissolved in the composition at a concentration of 2 to 8 mg/ml. Black-backed wasp venom;
Sterile distilled water or sterile saline solution; and
An aqueous solution formulation containing one or two or more stabilizers selected from Methyl ρ-Hydroxybenzoate, Cresol, and Chlorocresol,
An aqueous solution formulation of black wasp venom, characterized in that it inhibits the content of serotonin contained in the black wasp venom from being reduced by more than 15% for at least 4 weeks based on HPLC peak area. According to clause 6,
The methyl paraoxybenzoate is included in the formulation at a concentration of 0.05 to 0.18% (w/v),
The cresol is included in the formulation at a concentration of 0.15 to 0.4% (w/v),
An aqueous solution formulation of black wasp venom, characterized in that the chlorocresol is included in the formulation at a concentration of 0.1 to 0.2% (w/v). According to clause 6 or 7,
An aqueous solution formulation of black wasp venom, characterized in that the black wasp venom is included in the formulation at a concentration of 2 to 8 mg/ml.