KR102801893B1 - Composition for treating acne containing nanoized bee pollen extract as an active ingredient - Google Patents

Abstract

The present invention relates to an acne treatment composition comprising a nano-sized bee pollen extract as an active ingredient, and more specifically, to an acne treatment composition comprising a nano-sized bee pollen extract as an active ingredient, which has a distinct acne improvement effect and can be administered for a long period of time, by combining purified bee venom with excellent anti-inflammatory efficacy and nano-sized bee pollen.

Description

{Composition for treating acne containing nanoized bee pollen extract as an active ingredient}

The present invention relates to a composition for treating acne derived from natural products, and more specifically, to a composition for treating acne comprising a nano-sized bee pollen extract as an effective ingredient.

Acne is a chronic sebaceous gland inflammatory disease caused by retention hyperkeratosis in pores due to blockage of sebum secretion. 70-80% of patients are concentrated in the age group of 11-25 years, and mild acne can be said to be a characteristic of the skin in the growth period rather than a skin disease. The cause of acne can be explained by the degree of development of acne symptoms, such as increased sebum secretion, narrowing of hair follicles due to hyperkeratosis, proliferation of acne bacteria, and inflammatory reactions, and various mechanisms can work in combination. Representative factors that cause acne include excessive sebum secretion, keratinization of hair follicles, and proliferation of acne bacteria, but hypertrophy of sebaceous glands, hyperkeratosis of hair follicles, and the influence of bacteria work in a complex manner to worsen acne, and each of these can cause acne on its own. In addition, genetic factors that make acne prone, external factors such as seasons and climate, food influences, fatigue and stress are also related to acne. The cause of acne has not yet been clearly identified, but if genetic and environmental factors are excluded, it has been confirmed through research reports to date that the three important causes mentioned above are directly involved in the development of acne. There is a method of using oral or external medicines to treat such acne. However, the above treatment agents have side effects such as hormone abuse, skin irritation, and contact dermatitis, so there is a constant need for the development of products that are safe for the human body and have excellent effects. In this regard, Korean Patent No. 1459742 discloses a cosmetic composition for improving acne containing a natural product extract.

However, in the case of the above prior art, there is still no research on a composition for treating acne that contains purified bee venom and nano-sized bee pollen as active ingredients, with extracts of century plant, poplar and cinnamon as active ingredients.

The present invention is intended to solve various problems including the above-mentioned problems, and aims to provide an acne treatment composition containing a nano-sized bee pollen extract as an effective ingredient, which has a distinct acne improvement effect and can be administered for a long period of time, by combining purified bee venom with excellent anti-inflammatory efficacy and nano-sized bee pollen. However, this task is exemplary and the scope of the present invention is not limited thereby.

According to one aspect of the present invention, a composition for treating acne is provided, which comprises a nano-sized bee pollen composition and purified bee venom as active ingredients and a pharmaceutically acceptable carrier.

According to another aspect of the present invention, an external preparation for treating acne, comprising the composition, is provided.

According to another aspect of the present invention, a cosmetic composition for improving acne, comprising the composition, is provided.

As described above, the composition for treating acne comprising the nano-sized bee pollen extract of the present invention as an effective ingredient can implement an excellent acne treatment effect according to the anti-inflammatory effect by combining the significant effects of the nano-sized bee pollen derived from natural products and bee venom. Therefore, it can be used in product groups related to acne skin treatment and all industrial fields. Of course, the scope of the present invention is not limited by these effects.

Figure 1 is a graph analyzing the NO production inhibition effect according to the composition treatment of the present invention.
Figure 2 is a graph analyzing changes in the content of proinflammatory cytokine TNF-α according to treatment with the composition of the present invention.
Figure 3 is a graph analyzing the effect on the proinflammatory cytokine IL-6 profile according to treatment with the composition of the present invention.
Figure 4 is a graph analyzing the inhibitory effect of β-hexaminidase activity according to treatment with the composition of the present invention.
Figure 5 is a graph analyzing the effect of inhibiting leukotriene (LT) production according to treatment with the composition of the present invention.

Definition of terms:

The term "bee pollen" used in this document refers to a mixture of nectar and saliva collected by worker bees when they collect nectar and pollen, which the worker bees attach to their hind legs and transport to the hive to use as food. The bee pollen is known to be a nutrient containing various minerals, vitamins, and essential amino acids, and many European athletes take bee pollen collected by foragers as a long-term nutritional supplement to improve their athletic ability.

The term "bee venom" used in this document refers to the venom contained in the venom sac of bees, which consists of about 40 different substances and has been used to treat human diseases since BC. The components of bee venom vary depending on the type of bee, and even within the same bee, the components vary depending on the age. Currently, research is being reported on the actions and mechanisms of bee venom components, hypersensitivity and toxicity of bee venom, immunotherapy, and treatment of diseases such as arthritis, herpes simplex, multiple sclerosis, and tumors, and in particular, it is known to have antibacterial effects and cell damage improvement effects. The bee venom of domestic honeybees ( Apis mellifera ) can be collected in large quantities using a bee venom collection device that uses an electric shock method without harming the bees, and then purified bee venom can be obtained by removing foreign substances and freeze-drying.

Detailed description of the invention:

According to one aspect of the present invention, a composition for treating acne is provided, which comprises a nano-sized bee pollen composition and purified bee venom as active ingredients and a pharmaceutically acceptable carrier.

In the above composition, the nano-sized bee pollen composition may include 1 to 20 wt%, and purified bee venom 0.000001 to 1 wt%, and may be mixed to comprise 0.1 to 4 wt% of a dispersant, 0.1 to 4 wt% of a preservative, 1 to 10 wt% of a thickener, 0.05 to 0.3% of an antioxidant, and 0.1 to 1 wt% of a neutralizer, and the remainder being distilled water.

In the above composition, the nano-sized bee pollen can be manufactured by mixing distilled water into the bee pollen and then using a wet nano-grinding process, the nano-sized bee pollen can have a particle size of 50 to 500 nm, the nano-sized bee pollen can have the exine removed, and the bee venom can be isolated from the venom sac of worker bees of the western honeybee ( Apis mellifera L.) that are 15 days old or older.

According to another aspect of the present invention, an external preparation for treating acne, comprising the composition, is provided.

According to another aspect of the present invention, a cosmetic composition for improving acne, comprising the composition, is provided.

The composition of the present invention can be applied to any form such as liquid, cream, paste or solid, and can be applied to not only creams, skin lotions, packs and makeup bases, but also face-washing and cleansing agents such as soaps, facial foams and body cleansers, and hair products such as shampoos and rinses, and, if necessary, can be mixed with ingredients generally used in external skin preparations, for example, aqueous ingredients, oily ingredients, surfactants, moisturizers, thickeners, preservatives, fragrances and pigments. In addition, it can be applied by manufacturing it in the form of powder, granules, capsules or injections by mixing it with a pharmaceutically acceptable carrier, forming agent, dilute, etc., and can also be applied as a body cleansing or cleaning agent such as wet tissues.

The composition of the present invention can exhibit an excellent effect that is superior to the effect of each component in preventing or treating acne due to the effects of nano-sized bee pollen and purified bee venom. In the composition of the present invention, it is preferable that the nano-sized bee pollen is added in an amount of 1 to 20 wt% and the purified bee venom is added in an amount of 0.00001 to 1 wt% of the composition in order to exhibit an effect in preventing or treating acne, and in the case of the bee venom, it is more preferable that it is added in an amount of 0.0001 to 0.5 wt%. When the content of the purified bee venom is less than 0.00001 part by weight, it is difficult to obtain the acne treatment effect, which is the intended effect of the present invention, and when it is included in an amount exceeding 1 wt%, there is a disadvantage in that it is uneconomical in terms of effect.

The composition of the present invention additionally contains at least one component selected from the group consisting of green tea extract, collagen, moistin, grape seed oil, licorice extract, allantoin, soybean oil, and tocopherol, and when these components are additionally contained, the acne improvement effect can be improved. It is preferable that each of these components is contained in an amount of 1 to 20 wt% based on the total weight of the composition. However, there is no significant limitation on the weight.

The composition of the present invention can prepare an acne treatment composition by adding 1 to 20 wt% of nano-sized bee pollen, 0.000001 to 1 wt% of purified bee venom, and mixing the remainder with distilled water based on the composition of Patent No. 10-1572067, which has already been evaluated for antiseptic effect, spreading effect, cytotoxicity, and stability, and can further comprise 0.1 to 4 wt% of a dispersant, 0.1 to 4 wt% of a preservative, 1 to 10 wt% of a thickener, 0.05 to 0.3% of an antioxidant, and 0.1 to 1 wt% of a neutralizer, and mixing the remainder with distilled water.

The composition of the present invention can be applied in any form such as liquid, cream, paste or solid, and can be applied not only to creams, skin lotions, packs and makeup bases, but also to washing and cleansing agents such as soaps, facial foams and body cleansers, and hair products such as shampoos and rinses, and can also be applied in the form of patches such as mask packs. In addition, it is possible to mix with ingredients generally used in external skin preparations, such as aqueous ingredients, oily ingredients, surfactants, moisturizers, thickeners, preservatives, fragrance pigments, etc., as needed.

The composition of the present invention may use a natural preservative, MSE (Multi-Scutellaria Extracts), as a preservative. MSE is extracted from the root of the goldenrod, a plant of the Lamiaceae family, and has an antibacterial effect on various skin inflammations and skin troubles, and helps with antioxidant effects, whitening effects, and anti-aging. In addition, the composition of the present invention may be manufactured as a semi-solid preparation, such as a cream, lotion, emulsifier, liniment, etc., but is not limited thereto, and may be manufactured by adding an appropriate colorant, flavoring agent, stabilizer, viscosifier, surfactant, etc. In addition, the skin external preparation composition according to the present invention may be manufactured as a liquid preparation, and may include a solution such as water, alcohol, or propylene glycol solution, a suspension, an emulsion, etc., but is not limited thereto, and may be manufactured by adding an appropriate colorant, flavoring agent, stabilizer, viscosifier, etc. Specifically, the skin external preparation may contain an appropriate pharmaceutically acceptable base such as petrolatum or stearyl alcohol; It can be manufactured by a conventional method for manufacturing external skin preparations, which comprises homogeneously mixing a pharmaceutically acceptable suitable surfactant such as polysorbate, sorbitan sesquioleate; a pharmaceutically acceptable suitable moisturizer such as glycerin; a pharmaceutically acceptable suitable solvent; and a flavoring agent, a coloring agent, a stabilizer, a viscosifier, etc.

The composition of the present invention can be administered daily at a dose of about 0.0001 mg/cm ² of skin area to about 5 g/cm ² of skin area, and a daily dosage of about 0.001 mg/cm ² of skin area to about 1 g/cm ² of skin area is preferred. However, the dosage may vary depending on the route of administration and formulation, the patient's condition (age, sex, weight, etc.), the severity of the condition being treated, etc. If necessary, the total daily dosage may be divided for convenience and administered several times during the day.

Up to now, there are methods of using oral or external medications to treat acne. Specifically, there are methods of suppressing sebum production using antiandrogens, treatments using the anti-inflammatory action of steroid hormones and nonsteroidal anti-inflammatory analgesics, antibacterial agents such as resorcinol, sulfur, salicylic acid, benzoyl peroxide, retinoic acid, or isotretinoin, exfoliating agents, and methods of suppressing the activity of Propionibacterium acnes using antibiotics such as tetracycline, erythromycin, and macrocycline. Meanwhile, treatment methods using azelaic acid have also been widely used recently. However, although the methods described above have achieved a certain degree of success in the treatment of acne, they have several problems in terms of effectiveness, side effects, and usability. The use of hormonal agents has been reported to have side effects of hormone abuse, such as suppression of epidermal growth, and exfoliating agents such as retinoic acid and benzoyl peroxide have concerns about skin irritation and contact dermatitis due to exfoliation, and antibiotics including tetracycline have the possibility of bleeding and photosensitivity by resistant bacteria. In particular, isotretinoin is known to have a teratogenic effect on the fetus. Therefore, the need for the development of products that are safe for the human body and effective for acne skin is constantly emerging. Meanwhile, purified bee venom has been proven to have anti-inflammatory and antibacterial effects, and there is a high possibility that it can be developed as a treatment for acne bacteria, and it is being studied in this regard. In this regard, there have been many studies, including Korean Patent No. 10-1459742, a cosmetic composition for improving acne containing a natural product extract, and Korean Patent No. 10-1835397, a composition for preventing and treating inflammation and allergic diseases containing a composite extract of Naebaekja, Nahan-gwa, and Dongsimcho as effective ingredients. However, acne improvement compositions made mainly of herbal medicine compositions and natural material compositions have complicated manufacturing processes due to the many processes and substances contained. Therefore, the inventors of the present invention removed allergic components from nano-sized bee pollen by crushing the outer skin, and the extract has excellent antibacterial, anti-inflammatory, and antiviral properties, and is recognized for its value as a cosmetic composition. Based on this, the inventors completed an acne treatment composition containing two natural materials. Although purified bee venom and nano-sized bee pollen are excellent in antibacterial properties and as cosmetic compositions, they have not yet been used as a composition limited to acne, and thus are thought to be suitable as a convergence material. Accordingly, in the present invention, a composition containing purified bee venom melatin and nano-sized bee pollen suppresses inflammatory responses through the regulation of inflammation and apoptosis in keratinocytes and monocytes induced by acne bacteria, and the excellent anti-inflammatory effects of bee venom and bee pollen were confirmed through comparative antibacterial tests with commercially available drugs. In addition, safety, antigenicity, histamine test, phototoxicity, photosensitivity, and skin irritation evaluations were performed to confirm safety for improving acne, and therefore, the composition of the present invention will be useful as a formulation for various cosmetics and therapeutic agents.

The present invention relates to a composition for improving and treating acne, which is a fusion biomaterial of purified bee venom and nano-sized bee pollen, and which contains two natural complex extracts as active ingredients for the purpose of treating anti-inflammation and allergic symptoms. The properties of the extract are as follows: 1) LPS stimulation suppresses the production mechanisms of NO, TNF-α, and IL-6 cytokines secreted by macrophages, and 2) also suppresses the production mechanisms of β-Hex release and LTC4 secreted by mast cells, thereby suppressing the production of IgE, IL-4, IL-5, and IL-13 in the bronchoalveolar lavage fluid of an asthma animal model induced by ovalbumin, and suppressing the production of IL-4, IL-5, and IL-13 in the lungs, thereby significantly exhibiting anti-inflammation and anti-allergic effects. Therefore, through application thereof, the complex extract of the two natural substances can be utilized for the prevention and treatment of inflammatory and allergic diseases.

In relation to the above composition, research is being actively conducted on the extraction of specialized components from the corresponding substances, but with the development of technologies focusing on the skin cleansing effect, technologies that open closed pores while minimizing skin irritation with the exfoliating effect of cleansing and moisturizing ingredients, technologies for developing enzymes that suppress excessive secretion of male hormones and produce free fatty acids with the excessive sebum control effect, technologies for preventing acne by removing excessive sebum with fine particles that can absorb sebum, technologies for preventing acne by performing antibacterial action on a wide spectrum including Propionbacterium ancness and various bacteria and fungi harmful to the skin, and technologies for acne treatment through PDT (photodynamic therapy) such as ALA and MAL with simultaneous skin sebum control and antibacterial soothing effects are being developed. In this regard, although the purified bee venom and nano-sized bee pollen of the present invention have excellent antibacterial properties as a cosmetic composition, they have not yet been used as a composition limited to acne, and therefore are considered to be suitable as fusion materials. In addition, since the development of the bee venom collection device, domestic bee venom production has been actively carried out, and this purified bee venom has been proven to have anti-inflammatory and antibacterial effects, and there is a high possibility that it can be developed as a treatment for acne bacteria.

Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the examples disclosed below, but can be implemented in various different forms. The following examples are provided to ensure that the disclosure of the present invention is complete, and to fully inform a person having ordinary skill in the art of the scope of the invention.

Experimental materials

The bee pollen used in the present invention was purchased from bee pollen farms in Namwon, Jeollabuk-do and Sancheong, Gyeongsangnam-do, and the cell culture reagents, such as RPMI-1640, MEM (Modifed Eagle Medium), non-essential amino acids solution, FBS (fetal bovine serum), streptomycine, and penicillin, were purchased from Hyclone (Logan, UT, USA). In addition, among the reagents used in the experiment, SCF (Stem cell factor) was purchased from STEMCELL (Vancouver, Canada), LPS (Lipopolysaccharides) and ovalbumin (Albumin from chickenegg white) were purchased from Sigma (St. Louis, MO, USA), the LTC4 measurement kit was purchased from Cayman (Ann Arbor, MI, USA), alum was purchased from Thermo (Hanover Park, IL, USA), the IgE measurement product was purchased from BD Biosciences (San Jose, CA, USA), and the IL measurement kit was purchased from R&D System (Minneapolis, MN, USA).

Preparation of wet nano-pulverized pollen

The wet nano-pulverization process of bee pollen was performed using a wet pulverizer (Rotate Mill; Ilsin Nanotechnology, Japan) according to the conventional technology (Choi et al., Journal of Life Science. 26(6): 651-656, 2016). Specifically, 10 g of bee pollen was mixed per 100 ml of distilled water to prepare a bee pollen mixture, and the mixture was wet-pulverized for 3 hours. Afterwards, the particle size distribution of the wet-pulverized bee pollen was checked using a particle size analyzer (Mastersizer 2000; Malvern Instruments, UK). As a result, it was confirmed that most of the particles were less than 500 nm. The wet-pulverized nano-sized bee pollen was freeze-dried to obtain 300 g of the final nano-sized bee pollen.

Purified bee venom manufacturing

The present inventors isolated bee venom from the venom sac of Western honeybee ( Apis mellifera L.) worker bees and collected a large amount of pure bee venom without harming the bees by using a venom collection device using an electric shock method. Thereafter, foreign substances were removed from the bee venom, and the collected bee venom was dissolved in distilled water, centrifuged, filtered, and then freeze-dried to obtain 25 μg of pure purified bee venom.

Preparation of mixtures for cell experiments

According to one embodiment of the present invention, the freeze-dried nano-sized bee pollen obtained was mixed in distilled water at concentrations of 3 μg/mL, 4 μg/mL, 5 μg/mL, and 10 μg/mL, and bee venom was added at 2 ng/mL.

Preparation of mixture for skin testing

According to one embodiment of the present invention, a mixture for skin testing was prepared. Specifically, 1 ml of a bee venom solution prepared by adding 0.1 g of purified bee venom to 1 ml of distilled water and 0.5 g of the nano-sized bee pollen prepared above were added to 99 ml of a mixture composed of 0.1 to 4 wt% of a dispersant, 0.1 to 4 wt% of a preservative, 1 to 10 wt% of a thickener, 0.05 to 0.3% of an antioxidant, and 0.1 to 1 wt% of a neutralizer, the remainder being distilled water, based on the composition of Patent No. 10-1572067, which has already been evaluated for antiseptic effect, spreading effect, cytotoxicity, and stability, and the mixture was stirred to prepare an external skin composition for acne treatment.

Cell culture

The RAW 264.7 cells, a mouse macrophage cell line used in the present invention, were cultured at 37°C under 5% _CO2 conditions in DMEM medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. In addition, mouse bone marrow-derived mast cells (BMMC) were isolated from the femur of BALB/C mice, and cultured in a culture medium containing RPMI-1640 medium containing 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin, with PWM-SCM (containing pokeweed mitogen-stimulated spleen cell-conditioned medium) as an IL-3 source at a final concentration of 20%, to obtain more than 90% homogeneous BMMCs. The above cells were cultured in a 37°C, 5% CO ₂ incubator, and a certain number of BMMC cells were dispensed into 96-well plates for experiments.

Experimental Example 1: Inhibitory Effect on NO (Nitric Oxide) Production

The inventors of the present invention confirmed the NO production inhibitory effect by treating RAW 264.7 cells with the composition of the present invention. Specifically, the experiment was performed as follows using the method described in the existing literature (Yang, JH et al., Immunopharmacol Immunotoxicol. 33(2):373-379, 2011). First, RAW 264.7 cells (1.5×10 ⁵ cells/well) were pre-cultured in a 24-well plate, replaced with DMEM medium, and pre-treated with the composition of the present invention at various concentrations for 1 hour. Then, 200 ng/mL of LPS was treated to each well, and cultured for 24 hours under 37°C, 5% CO ₂ conditions. After that, the supernatant was collected, 100 μL was mixed with an equal volume of Griess reagent (G4410, Sigma), and the reaction was performed. The absorbance at 570 nm was measured using an ELISA reader (ELISA reader, Sunrise-Basic Tecan, TECAN), and the amount of NO produced was analyzed using a standard curve for each concentration of sodium nitrite.

As a result, when the composition of the present invention was treated at concentrations of 4 μg/mL, 5 μg/mL, and 10 μg/mL, the production of nitrite was suppressed to 11 μM, 9 μM, and 4 μM, respectively (Fig. 1).

Experimental Example 2: Inhibitory Effect on Proinflammatory Cytokine Production

The inventors of the present invention performed the inhibitory effect of the composition of the present invention on the production of proinflammatory cytokines TNF-α and IL-6 by referring to the previous research literature (Yang JH et al., Immunopharmacol Immunotoxicol, 33(2):373-379, 2011). Specifically, RAW 264.7 cells (1.5×10 ⁵ cells/well) were pre-cultured in a 24-well plate, replaced with DMEM medium, and the cells were pre-treated with the composition of the present invention for 1 hour. After 200 ng/㎖ of LPS was treated to each well, the cells were cultured for 24 hours under 37°C, 5% CO ₂ conditions. Thereafter, the supernatant was collected and the amounts of proinflammatory cytokines TNF-α and IL-6 were measured using an ELISA kit (R&D System, USA).

As a result, in the cell supernatant treated with LPS, the production of TNF-α significantly increased to 114 ng/mL, but when the composition of the present invention was pretreated at concentrations of 4 μg/mL, 5 μg/mL, and 10 μg/mL, the production of TNF-α was found to decrease in a concentration-dependent manner to 80 ng/mL, 76 ng/mL, and 68 ng/mL (Fig. 2). In addition, in the cell supernatant treated with LPS, the production of IL-6 significantly increased to 1125 pg/mL, but when the composition of the present invention was pretreated at concentrations of 4 μg/mL, 5 μg/mL, and 10 μg/mL, the production was found to decrease in a concentration-dependent manner to 601 pg/mL, 503 pg/mL, and 450 pg/mL (Fig. 3). The above results suggest that the composition of the present invention exhibits excellent anti-inflammatory activity.

Experimental Example 2: Inhibition of β-Hex Release

The inventors of the present invention performed the inhibition effect of β-hexaminidase (β-Hex), a degranulation marker substance of BMMC, by referring to the previous research literature according to the treatment with the composition of the present invention (Son JK et al, Biol Pharm Bull, 28(12):2181-84, 2005). Specifically, 2× ¹⁰⁵ cells/well of BMMC were treated with the composition of the present invention at various concentrations (3 to 5 μg/mL) and pre-cultured for 30 minutes under 37°C, 5% _CO2 conditions, and then stimulated with SCF (stem cell factor) (100 ng/mL) (STEMCELL Technologies, Vancouver, Canada), cultured for 15 minutes, and centrifuged at 3000 rpm, 4°C for 5 minutes. After that, the supernatant was mixed with β-Hex substrate {100 mM citrate buffer (citric acid 0.955%, sodium citrate dihydrate 1.478%, pH 4.5), 1.3 mg/mL p-nitrophenyl-N-acetyl-b-D-glucosaminide)} in a mixing ratio of 1:2 (w/v) and reacted at 37°C for 1 hour. After stopping the reaction with 0.2 M glycine (pH 10.7) (Sigma), the absorbance at a wavelength of 405 nm was measured using an ELISA reader (Tecan System, San Jose, CA, USA), and the measured value was converted into inhibition ratio (%).

As a result, the composition of the present invention was found to inhibit the release of β-hexaminidase by 25%, 30%, and 43% at treatment concentrations of 3 μg/mL, 4 μg/mL, and 5 μg/mL, respectively (Fig. 4).

Experimental Example 3: Inhibitory Effect on Leukotriene Production

The present inventors performed the inhibitory effect of leukotriene (LT) production according to the treatment of the composition of the present invention with reference to previous research literature (Hua JM et al., Arch Pharm Res , 31:617-621, 2008). Specifically, in order to confirm LT production, 1×10 ⁵ cells/mL BMMC were treated with the composition of the present invention at various concentrations (3 to 5 μg/mL), cultured for 1 hour under 37°C, 5% CO ₂ conditions, and stimulated with SCF (stem cell factor) at a concentration of 100 ng/mL for 15 minutes. Then, the supernatant was separated using a centrifuge (Centrifuge 5415R, Eppendorf) at 3000 rpm and 4°C for 5 minutes. Thereafter, the LTC ₄ content in the supernatant was measured using an LTC ₄ -EIA kit (Cayman). As a result, the composition of the present invention was found to inhibit the production of LTC ₄ by 30%, 51%, and 90% at treatment concentrations of 3 μg/mL, 4 μg/mL, and 5 μg/mL, respectively (Fig. 5).

Experimental Example 4: Antibacterial activity against multidrug-resistant bacteria

When infected with multidrug-resistant bacteria (MRSA), symptoms of skin and soft tissue infection include seizures on the skin as if struck, overall swelling, pus, and pain. In addition, the infected area appears as acne, mild burns, or insect bites. As a supplementary experiment for this, the inventors of the present invention performed antibacterial activity according to treatment with the composition of the present invention by referring to previous research literature (Kwak, MK et al., Food Control 85: 223-234, 2018). Specifically, the antibacterial activity against multidrug-resistant strains such as Staphylococcus aureus 11471, Streptococcus pneumoniae 14596, and Salmonella typhimurium 12219 was determined in terms of minimum inhibitory concentration (MIC) and activity concentration against fungi using Candida albicans , a human opportunistic/pathogenic fungus, and Ganoderma boninense , a plant pathogenic fungus, respectively, in 6-well plates. As a result, the mixture comprising nano-sized bee pollen and bee venom of the present invention containing natural ingredients was found to have high antibacterial and antifungal activity against multidrug-resistant bacteria. The results of the antibacterial activity evaluation of the composition of the present invention against the multidrug-resistant bacteria are summarized in Table 1 below.

Antibacterial activity effect multidrug resistant strains Active concentration of bee pollen-venom mixture (MIC, μg/L)* Staphylococcus aureus 114711 7.3 ± 0.77 Streptococcus pneumoniae 145961 8.5 ± 0.32 Salmonella typhimurium 122192 5.1 ± 0.54 Fungus Active concentration of the mixture (μg complex in 3 mL agar assay)* Plant pathogen Ganoderma boninense 110.9 ± 3.56 Human pathogen Candida albicans 101.4 ± 3.26

*Data represent ± SD of three independent experiments, expressed as mg/L of each test sample.

Experimental Example 5: Formulation Stability Evaluation

The inventors of the present invention confirmed phase stability by storing the composition of the present invention at temperatures of 40°C, room temperature (25°C), and -10°C for 25 days to experimentally evaluate the degree of stabilization, and found that there was no change in formulation under all of the above temperature conditions.

Experimental Example 6: Measurement of acne treatment effect

The inventors of the present invention manufactured an external preparation containing the composition of the present invention and observed the acne treatment effect. Specifically, the skin test mixture was manufactured according to the above-described manufacturing method and then refrigerated. Then, 42 female students aged 17 to 20 with acne skin were divided into 6 groups, with 7 subjects per group, and were instructed to apply an appropriate amount of the external preparation containing the composition of the present invention to the entire face once a day for 7 days after washing their faces in the evening, and the acne treatment effect was observed for 7 days.

In terms of evaluation criteria, if the acne treatment effect is remarkable, that is, if the degree of acne inflammation is clearly reduced, 5 points are given, if the degree of inflammation is improved, 4 points are given, if the degree of inflammation remains but shows improvement, 3 points are given, if it is unchanged, 2 points are given, and if it gets worse, 1 point is given, and a comprehensive judgment is made based on the total score. Distilled water without bee venom or pollen and distilled water containing 5% alcohol were used as comparison groups.

As a result, the excellent acne treatment effect of the experimental group that applied the external preparation containing the composition of the present invention was confirmed. The acne treatment effect measurement results are summarized in Table 2 below.

Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 Subject 7 Total evaluation score Composition 5 4 5 5 4 4 5 32 Distilled water 1 2 2 1 3 2 1 12 Alcohol distillate 3 2 1 3 2 2 1 14

Experimental Example 7: Antibacterial activity evaluation experiment

The present inventors performed an antibacterial activity evaluation experiment of an external preparation including the composition of the present invention using Propionibacterium acnes , an acne bacterium, as a test bacteria. Specifically, the acne bacterium was inoculated into BH1 broth, pre-cultured, and then inoculated at a concentration of 1% into 100 ml of sterilized new BHI broth containing 5% of the composition of the present invention. After culturing for 2 days in an anaerobic incubator, the number of bacteria was measured. Since the pathogenic agent of the strain is involved in sebum secretion and abnormal keratinization of hair follicles for acne, thereby forming a bacterial colony of the strain, the growth of the strain was observed when alleviating a localized chronic inflammatory disease caused by an inflammatory reaction, etc.

As a result, it was confirmed that the growth of acne bacteria was significantly reduced by treatment with the composition of the present invention. The above results suggest the excellent antibacterial activity of the composition of the present invention. The results of the antibacterial activity evaluation experiment are summarized in Table 3 below.

Antibacterial activity evaluation test results division acnes ( Propionibacterium acnes ) Inoculum concentration (cfu/mL) Concentration of bacteria after culture (cfu/mL) Example 1 2.32 × 10 ⁶ 1.3 × 10 ² Comparative Example 1 2.32 × 10 ⁶ 2.0 × 10 ⁵

In conclusion, the composition for treating acne comprising the nano-sized bee pollen extract of the present invention as an active ingredient contains bee pollen wet-pulverized into nano-sized particles and purified bee venom as active ingredients, and therefore has excellent anti-inflammatory, antibacterial, and antiviral effects, and therefore can be utilized as a pharmaceutical composition or food composition for improving skin diseases such as acne treatment, and prevention and treatment of inflammation and allergic diseases.

Although the present invention has been described with reference to the above-described embodiments, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical idea of the appended claims.

Claims (9)

A composition for treating acne, comprising a nano-sized bee pollen composition and purified bee venom as active ingredients and a pharmaceutically acceptable carrier. In the first paragraph,
A composition comprising 1 to 20 wt% of a nano-sized bee pollen composition and 0.000001 to 1 wt% of purified bee venom. In the first paragraph,
A composition further comprising 0.1 to 4 wt% of a dispersant, 0.1 to 4 wt% of a preservative, 1 to 10 wt% of a thickener, 0.05 to 0.3 wt% of an antioxidant, and 0.1 to 1 wt% of a neutralizer. In the first paragraph,
The above nano-sized bee pollen is a composition manufactured by mixing distilled water into bee pollen and then using a wet nano-grinding process. In the first paragraph,
A composition wherein the nano-sized pollen has a particle size of 100 to 500 nm. In the first paragraph,
The above nano-sized bee pollen is a composition from which the exine has been removed. In the first paragraph,
The above bee venom is a composition isolated from the venom sac of worker bees of the Western honeybee ( Apis mellifera L.) that are 15 days old or older. An external preparation for treating acne, comprising a composition according to any one of claims 1 to 7. A cosmetic composition for improving acne, comprising a composition according to any one of claims 1 to 7.