New substitute material for botulinum neurotoxin and its manufacturing method

Abstract

The present invention relates to a novel alternative material to botulinum neurotoxin and a method for producing the same, specifically, to a myricetin acetyl derivative that has a dramatically improved stability of the substance, including stability in the body, and has the ability to inhibit the formation of the SNARE complex and thereby inhibit acetylcholine secretion, and a method for producing the same.

Description

[Technical Field]

[0001] The present invention relates to a novel material that can replace botulinum neurotoxin and a method for producing the same, and more particularly to an acetyl derivative of myricetin that has dramatically improved stability, including in vivo stability, and the ability to inhibit acetylcholine secretion, and a method for producing the same. [Background technology]

[0002] Botulinum neurotoxin hydrolyzes SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which are involved in the docking of the presynaptic membrane and synaptic vesicles in nerve cells, thereby inhibiting the secretion of acetylcholine and ultimately blocking neurotransmission. As a result, it can be used in cosmetic procedures such as treating wrinkles, as well as in the treatment of over 100 diseases caused by excessive secretion of acetylcholine, such as crossed eyes and excessive sweating.

[0003] Because botulinum neurotoxin itself is a toxin, misuse can be fatal. It is also known to induce allergic reactions and cause side effects such as slurred speech, awkward facial expressions, and eyelid inability to close. These side effects are generally caused by improper dosage control or the spread of botulinum neurotoxin to unwanted muscles. These side effects resolve once the botulinum neurotoxin is broken down in the body or new nerves are formed, restoring normal function. This process can take several months. For these reasons, considerable care must be taken when using botulinum neurotoxin for cosmetic or medical purposes. Botulinum neurotoxin is produced by culturing the anaerobic microorganism Clostridium botulinum. Because this bacterium is highly lethal, strict control is required throughout the entire process, from cultivation to isolation and purification, disposal, and sterilization.

[0004] For these reasons, attempts are being made to develop various materials that can replace botulinum neurotoxins. Examples include (Patent Documents 1-8), when attempting to use proteins or polypeptides (Korean Patent Publication No. 10-2012-0001964; Korean Patent Publication No. 10-2012-0122999), when attempting to use natural polyphenols (Korean Patent Publication No. 10-2008-0083438; Korean Patent Publication No. 10-2008-0091735; Korean Patent Publication No. 10-2011-0017599; Korean Patent Publication No. 10-2016-0058739), and when attempting to use myricetin derivatives (Korean Patent Publication No. 10-2017-0015845; Korean Patent Publication No. 10-2017-0091554). Among these, pseudo-botulinum neurotoxin materials using peptides have been developed and commercialized overseas. One example is Argireline, developed by Lipotec. However, the commercial success of botulinum neurotoxin substitutes has been limited due to various drawbacks.

[0005] Therefore, there is a need to develop a new material that can overcome the drawbacks of existing alternative materials to botulinum neurotoxin while also solving the side effects and problems of botulinum neurotoxin. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Republic of Korea Patent Publication No. 10-2012-0001964 [Patent Document 2] Republic of Korea Patent Publication No. 10-2012-0122999 [Patent Document 3] Republic of Korea Patent Publication No. 10-2008-0083438 [Patent Document 4] Republic of Korea Patent Publication No. 10-2008-0091735 [Patent Document 5] Republic of Korea Patent Publication No. 10-2011-0017599 [Patent Document 6] Republic of Korea Patent Publication No. 10-2016-0058739 [Patent Document 7] Republic of Korea Patent Publication No. 10-2017-0015845 [Patent Document 8] Republic of Korea Patent Publication No. 10-2017-0091554 Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, the inventors of the present invention have focused on the fact that, among existing botulinum neurotoxin alternative materials, myricetin derivatives have reduced stability, including in vivo stability, and that solving this problem would allow the development of more valuable botulinum neurotoxin alternative materials. As a result of diligent efforts to solve the stability problems of existing myricetin derivatives, the present invention has been completed by developing a material and a method for producing the same that have dramatically improved stability compared to existing publicly available myricetin derivatives.

[0008] Therefore, the object of the present invention is to provide a myricetin acetyl derivative (3-acetyl-myricetin) of [Chemical Formula 1] that can be used as an alternative material to botulinum neurotoxin, has dramatically improved stability, and has the ability to inhibit the formation of SNARE complexes and thereby inhibit acetylcholine secretion.

[0009] [ka]

[0010] Another object of the present invention is to provide a method for producing the myricetin acetyl derivative represented by [Chemical Formula 1].

[0011] Another object of the present invention is to provide a cosmetic composition for cosmetic purposes as an alternative to botulinum neurotoxin, which contains the myricetin acetyl derivative represented by the above [chemical formula 1] as an active ingredient.

[0012] Another object of the present invention is to provide a pharmaceutical composition for treating diseases caused by acetylcholine hypersecretion, comprising the myricetin acetyl derivative represented by the formula 1 as an active ingredient. [Means for solving the problem]

[0013] Therefore, according to one aspect of the present invention, the present invention provides a myricetin acetyl derivative represented by [Chemical Formula 1], which has the ability to inhibit the formation of SNARE complexes and thereby inhibit acetylcholine secretion.

[0014] Of course, it is obvious within the scope of the present invention that the effects of the present invention can be obtained even if additional derivatization is applied to the basic skeleton of [Chemical Formula 1].

[0015] According to another aspect of the present invention, there is provided a cosmetic composition for cosmetic purposes, comprising an acetyl myricetin derivative represented by [Chemical Formula 1] as an active ingredient.

[0016] The cosmetic purposes may include, but are not limited to, wrinkle reduction, pore size reduction, and skin elasticity improvement. Examples of wrinkles include brow droop, crow's feet, nasolabial folds, lip frown, and neck wrinkles. It is possible to present, but is not limited to, wrinkles, forehead lines, frown lines, tear troughs, jowls, and the like.

[0017] In one aspect of the present invention, the term "cosmetic composition" is a concept that includes all compositions that beautify the appearance of the human body, and the cosmetic composition for cosmetic purposes of the present disclosure is a concept that refers to a composition that beautifies the appearance of the human body by suppressing adverse effects on the skin caused by physiological phenomena associated with biological rhythms, but is not limited thereto.

[0018] In one aspect of the present invention, the cosmetic composition is essentially intended for application to the skin, and therefore may be prepared in any formulation commonly used in cosmetic compositions in the art. Examples include, but are not limited to, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, oils, powder foundations, emulsion foundations, wax foundations, and sprays. More specifically, the cosmetic composition may be prepared in the form of a softening lotion, nourishing lotion, nourishing cream, massage cream, essence, eye cream, spray, or powder.

[0019] In one aspect of the present invention, when the dosage form of the present invention is a paste, cream or gel, the carrier component may include animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc.

[0020] In one aspect of the present invention, when the dosage form of the present invention is a powder or spray, carrier components may include lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc., and particularly when the dosage form is a spray, a propellant such as chlorofluorohydrocarbon, propane / butane, dimethyl ether, etc. may be further included.

[0021] In one aspect of the present invention, when the dosage form of the present invention is a solution or emulsion, the carrier component may include a solvent, a solubilizer, an emulsifier, etc., and specifically, may include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, fatty acid esters of sorbitan, etc.

[0022] In one aspect of the present invention, when the dosage form of the present invention is a suspension, the carrier component may include a liquid diluent such as water, ethanol, propylene glycol, etc.; a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, polyoxyethylene sorbitan ester, etc.; finely divided cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, etc.

[0023] In one aspect of the present invention, when the dosage form of the present invention is a surfactant-containing cleansing agent, the carrier component may be a fatty alcohol sulfate, a fatty alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyl taurate, a sarcosinate, a fatty acid amide ether sulfate, an alkylamidobetaine, a fatty alcohol, a fatty acid glyceride, a fatty acid diethanolamide, a vegetable oil, a lanolin derivative, or an ethoxylated glycerol fatty acid ester.

[0024] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for treating diseases caused by acetylcholine hypersecretion, comprising an acetyl myricetin derivative represented by [Chemical Formula 1] as an active ingredient.

[0025] Diseases caused by the hypersecretion of acetylcholine include, but are not limited to, strabismus, inner ear disorder, teeth grinding, keloid scarring, hyperhidrosis, back pain, tremors, anal fissure, buttock deformity, muscle injuries (twitching), chronic migraine, depression, facial nerve disorder, neck pain (spasms), thyroid disorder, cardiac muscle disorder, upper limb spasticity, pancreas disorders, irritable bowel syndrome, stretch marks, and juvenile cerebral palsy.

[0026] Pharmaceutically acceptable carriers contained in the pharmaceutical composition of the present invention are those commonly used in pharmaceutical formulations, including, but not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the pharmaceutical composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0027] The pharmaceutical composition containing the myricetin acetyl derivative represented by [Chemical Formula 1] of the present invention as an active ingredient can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous administration, intraperitoneal administration, intramuscular administration, subcutaneous administration, intranasal administration or local administration, but is not limited thereto.

[0028] Pharmaceutical compositions containing the myricetin acetyl derivative represented by [Chemical Formula 1] as an active ingredient may be prepared in unit dose form or in multi-dose containers by formulating them with pharmaceutically acceptable carriers and / or excipients according to a method easily implemented by those skilled in the art to which the present invention pertains. In this case, the dosage form may be a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granules, tablets, or capsules, and may further contain a dispersant or stabilizer.

[0029] In addition, the suitable application, spraying and dosage of the pharmaceutical composition varies depending on factors such as formulation method, administration mode, age, body weight, sex, severity of disease symptoms, diet, administration time, administration route, excretion rate and reaction sensitivity, and an ordinarily skilled physician can easily determine and prescribe an effective dosage for the desired treatment.

[0030] According to another aspect of the present invention, the present invention provides a method for producing an acetyl myricetin derivative represented by [Chemical Formula 1].

[0031] Specifically, the method includes the steps of (A) adding acetic anhydride to myricetin dissolved in anhydrous pyridine in a nitrogen atmosphere that prevents the addition of external air and moisture, and reacting the mixture; (B) extracting the mixture with ethyl acetate (EA); (C) drying the ethyl acetate (EA) extract under reduced pressure; (D) adding dimethyl sulfoxide (DMSO) to the dried sample, and then diluting it with phosphate-buffered saline (PBS; pH 7.0); (E) incubating the diluted sample to decompose by-products; and (F) freeze-drying the sample. The method provides a method for producing a myricetin acetyl derivative represented by [Chemical Formula 1]. [Effects of the Invention]

[0032] The myricetin acetyl derivative represented by [Chemical Formula 1] according to the present invention can provide improved substance stability, so that a composition containing it as an active ingredient is easier to store and handle, and more importantly, it can also provide high stability in the body after administration, so it can provide improved and sustained efficacy. [Brief explanation of the drawings]

[0033] [Figure 1] 1 is a schematic diagram showing the synthesis process of 3-acetyl-myricetin represented by [Chemical Formula 1] of the present invention. [Figure 2] 1 shows the results of reversed phase-high performance liquid chromatography (RP-HPLC) analysis of a freeze-dried sample in the process of producing 3-acetyl-myricetin represented by [Chemical Formula 1] of the present invention. [Figure 3] This is the RP-HPLC analysis result of the final purified 3-acetyl-myricetin represented by [Chemical Formula 1]. [Figure 4] This is the liquid chromatography-mass spectrometry (LC / MS) analysis result of the final purified 3-acetyl-myricetin represented by [Chemical Formula 1]. [Figure 5] This is the result of nuclear magnetic resonance (NMR) analysis of the final purified 3-acetyl-myricetin represented by [Chemical Formula 1]. [Figure 6] 1 shows the results of measuring the degree of acetylcholine secretion inhibition of 3-acetyl-myricetin represented by [Chemical Formula 1] according to the present invention. 1 is the result of the control group, 2 is the result of applying a 0.5 μM sample, 3 is the result of applying a 2 μM sample, 4 is the result of applying a 10 μM sample, and 5 is the result of applying a 20 μM sample. [Figure 7] 1 shows the results of a comparative experiment on the stability of 3-acetyl-myricetin, myricetin, and myricetin-C4 (Myricetin-C4) represented by [Chemical Formula 1] according to the present invention. [Figure 8]These are the results of an efficacy comparison experiment. 1 is the control group (PBS-treated group; untreated group), 2 is the 3-acetyl-myricetin-treated group, 3 is the myricetin-treated group, 4 is the laricitrin-treated group, 5 is the Combretol-treated group, 6 is the syringetin-treated group, 7 is the myricetin-C4-treated group, 8 is the myricetin-C8-treated group, 9 is the myricetin-C12-treated group, 10 is the myricetin-C16-treated group, 11 is the myricetin-C20-treated group, 12 is the derivative E4-2-treated group, 13 is the derivative E4-4-treated group, and 14 is the derivative The groups were the Derivative E4-8 (Derivative E4-8) treatment group, 15 the Derivative E4-12 (Derivative E4-12) treatment group, 16 the Derivative E4-16 (Derivative E4-16) treatment group, 17 the Derivative E4-18 (Derivative E4-18) treatment group, and 18 the Derivative E4-20 (Derivative E4-20) treatment group. In the efficacy comparison experiment, the sweating area of ​​the control group (untreated group) was set as 100%, and the results were presented as relative values. DETAILED DESCRIPTION OF THE INVENTION

[0034] The present invention will be described in more detail below based on examples. However, these examples are merely illustrative of the present invention, and the scope of the present invention is not limited to these examples.

[0035] Example 1: Preparation of control substances and myricetin acetyl derivatives In this example, control substances used in subsequent comparative experiments were prepared. The control substances were myricetin, laricitrin of [Chemical Formula 2], combretol of [Chemical Formula 3], syringetin of [Chemical Formula 4], myricetin derivatives (Myricetin-C4) prepared by acylation with vinyl butyrate under lipase catalysis, myricetin derivatives (Myricetin-C8) prepared by acylation with vinyl octanoate under lipase catalysis, myricetin derivatives (Myricetin-C12) prepared by acylation with vinyl laurate under lipase catalysis, and vinyl palmitate under lipase catalysis. Myricetin derivatives (Myricetin-C16) produced by acylation with vinyl eicosanoate under lipase catalysis (Myricetin-C20), myricetin derivatives (Derivative E4-2) produced by acylation with acetic acid using a base and oxalyl chloride, myricetin derivatives (Derivative E4-4) produced by acylation with butyric acid using a base and oxalyl chloride, myricetin derivatives (Derivative E4-8) produced by acylation with octanoic acid using a base and oxalyl chloride, myricetin derivatives (Derivative E4-12) produced by acylation with lauric acid using a base and oxalyl chloride, myricetin derivatives (Derivative E4-12) produced by acylation with palmitic acid using a base and oxalyl chloride E4-16, a myricetin derivative prepared by acylation with stearic acid using a base and oxalyl chloride.E4-18), and myricetin derivative (Derivative E4-20) prepared by acylation with arachidic acid using a base and oxalyl chloride were selected.

[0036] [ka]

[0037] [ka]

[0038] [ka]

[0039] Myricetin, laricitrin, combretol, and syringetin were purchased commercially and used. The remaining derivatives used as control substances were prepared according to the methods presented in the examples of Korean Patent Publication Nos. 10-2017-0015845 and 10-2017-0091554. The myricetin acetyl derivative (3-acetyl-myricetin) of [Chemical Formula 1] developed by the present inventors was prepared according to the following method. A stirring bar and myricetin (1 equivalent) were placed in a round-bottom flask (RBF), and the atmosphere was purged with nitrogen. The flask was then sealed with a rubber stopper to prevent the addition of external air and moisture. Anhydrous pyridine (0.5 equivalents) was added to the RBF, and the myricetin was completely dissolved in an ice bath. Acetic anhydride was added dropwise to the dissolved myricetin using a syringe. Stir overnight in an ice bath. Add cold distilled water to the overnight reaction solution to terminate the reaction. Place all reactants in a separating funnel and add ethyl acetate (EA). Add EA to the RBF so that no reactants remain in the RBF, then transfer to the separatory funnel. Shake the separatory funnel multiple times to ensure that the synthesized 3-acetyl-myricetin dissolves well in the EA, then place it on a stand to separate the water and EA layers. Once the water and EA layers are separated, remove only the water layer (EA's density is 0.9, lower than that of water, so the water layer is at the bottom). Add saturated brine to the separatory funnel and shake the separatory funnel multiple times to ensure that the synthesized 3-acetyl-myricetin dissolves well in the EA, then place it on a stand to separate the water and EA layers. Once the water and EA layers are separated, remove only the water layer. After adding distilled water to the separatory funnel, shake the separatory funnel several times to ensure that the synthesized 3-acetyl-myricetin dissolves well in EA. Then, place the separatory funnel on a stand to separate the water and EA layers, and once the water and EA layers have separated, remove only the water layer. This process is repeated three times. The EA layer is transferred to a new RBF and dried under reduced pressure.The sample dried under reduced pressure through this process is dissolved in DMSO at a concentration of 10 mg / ml. PBS (pH 7.0) is added to the 10 mg / ml synthetic mixture to dilute it to a concentration of 1 mg / ml. The sample thus prepared is incubated overnight at 37°C with stirring at 200 rpm. The sample after overnight incubation is then freeze-dried. The freeze-dried sample is dissolved in DMSO. The sample thus prepared is subjected to RP-HPLC under the conditions in Table 1, and only the peak fraction in the 16-minute band is collected.

[0040] [Table 1]

[0041] The synthesis process of 3-acetyl-myricetin represented by [Chemical Formula 1] is summarized in Figure 1, and the RP-HPLC results are shown in Figure 2. The RP-HPLC analysis results for the final purified 3-acetyl-myricetin represented by [Chemical Formula 1] (purity 99% or more) are shown in Figure 3. To confirm the structure, LC / MS analysis and 1 H-NMR analysis was performed [the NMR analysis paper on myricetin (Molecules 2014;19:13643) was referred to for the NMR analysis], and the results are presented in Figures 4 and 5.

[0042] Example 2: Investigation of neurotransmission suppression efficacy In this example, we investigated whether 3-acetyl-myricetin represented by [Formula 1] prepared in Example 1 can provide the expected neurotransmitter inhibitory effect. The investigation was carried out using an acetylcholine release assay, a conventional cell-based efficacy evaluation method, to determine whether it can inhibit the secretion of the neurotransmitter acetylcholine. The acetylcholine release assay is a test method that quantitatively analyzes acetylcholine secreted from acetylcholinergic neurons at the cellular level. It is a test method that can confirm the secretion of neurotransmitters by quantitatively analyzing acetylcholine, a messenger that transmits signals for skeletal muscle contraction and relaxation.

[0043] Specifically, PC12 cells were subcultured in T75 flasks every three days using full serum medium [84% Dulbecco's modified eagle's medium (Hyclone), 10% horse serum (Gibco), 5% fetal bovine serum (Gibco), 1% antibiotic antimycotic solution (Hyclone)]. Between subcultures, the medium was replaced with fresh full serum medium every day. On the first day of the experiment, collagen-coated plates were prepared as follows: A 20 mM acetic acid solution was prepared using sterilized water filtered through a 0.2 μm filter. This solution was used to prepare 50 ml of collagen solution at a concentration of 50 μg / ml. One ml of the prepared collagen solution was added per well and allowed to incubate at room temperature for 3 hours. After standing, the collagen solution was carefully removed from each well. The plates were then thoroughly washed with 1 ml of PBS. The plates were then air-dried on a clean bench to complete the preparation of collagen-coated plates. On the second day of the experiment, cells were seeded onto the prepared collagen-coated plates. Cell seeding was performed as follows: After removing the medium from PC12 cells cultured in a T75 flask, 2 ml of serum-free medium (99% Dulbecco's modified Eagle's media (Hyclone) and 1% Antibiotic Antimycotic Solution (Hyclone)) was added. The cells were gently pipetted to separate them, then transferred to a 15 ml conical tube and centrifuged (2,500 rpm, 3 minutes). After centrifugation, the supernatant was removed, and the cells were resuspended in 1 ml of serum-free medium and counted.Then, 2 x 10 cells were cultured using serum-free medium. 5 A PC12 cell suspension at 1000 cells / ml was prepared and dispensed into the collagen-coated plates at 1 ml per well. The plates were then incubated for 24 hours in a CO2 incubator at 37°C. On day 3 of the experiment, the medium was removed from the plates and replaced with NGF medium [98.6% Dulbecco's modified eagle's media (Hyclone), 1% antibiotic antimycotic solution (Hyclone), 0.4% (25 μg / ml) NGF (nerve growth factor r-2.5S from murine submaxillary gland; Sigma)] for NGF treatment. After NGF treatment, the plates were incubated for an additional 5 days in a CO2 incubator at 37°C. On day 8 of the experiment, samples were processed and analyzed as follows. After 5 days of culture, the medium was removed from the PC12 cell plates, and 995 μl of serum-free medium was added to each well. 5 μl of 0.5 μM, 2 μM, 10 μM, or 20 μM 3-acetyl-myricetin samples prepared in DMSO were added to each well and then cultured for 18 hours in a CO2 incubator at 37°C. 5 μl of DMSO was added to the control group. After 18 hours of culture, quantitative analysis was performed using an acetylcholine release assay kit (Cell Biolabs) according to the manufacturer's instructions. The results are shown in Figure 6.

[0044] From these results, it was confirmed that the myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1] of the present invention can also inhibit the formation of SNARE complexes and consequently inhibit acetylcholine secretion. This indicates that the myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1] of the present invention can provide a neurotransmitter blocking effect.

[0045] Example 3: Comparison of material stability In this example, a stability comparison experiment was conducted. To compare extreme conditions, a one-day incubation was performed. Incidentally, existing myricetin or myricetin derivatives are largely decomposed and disappear even after being left for approximately two hours. The stability experiment was conducted as follows: A sample prepared using DMSO to a concentration of 10 mg / ml was diluted to a concentration of 1 mg / ml by adding PBS (pH 7.0) (1 / 10 dilution). The prepared sample was incubated at 37°C for one day while stirring at 200 rpm. The incubated sample was lyophilized, dissolved in DMSO, and analyzed by RP-HPLC under the same conditions as previously described to compare stability. The results are shown in Table 2.

[0046] [Table 2]

[0047] To aid understanding, representative experimental results (myricetin, myricetin-C4, and 3-acetyl-myricetin) are presented in Figure 7.

[0048] From the above results, it was confirmed that the myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1] of the present invention can provide improved stability to a degree that previously reported myricetin derivatives cannot provide. Such improved stability can act as a significant advantage in the storage and distribution of final products manufactured using the myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1]. In addition, since the experimental conditions of this example are similar to the conditions in the body, it can be inferred that the myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1] of the present invention can also provide improved in vivo stability, and as a result, provide improved efficacy.

[0049] Example 4: Preparation of the composition In this example, nanoparticles incorporating various myricetin derivatives, including myricetin, were prepared. To conduct the efficacy comparison experiment of Example 5 using the method presented in the examples of Korean Patent Publication No. 10-2017-0091554, nanoparticles that can be used for transdermal drug delivery were prepared. Specifically, the following steps were carried out in order: (1) lipid phase preparation, (2) water phase preparation, and (3) lipid-nanoparticle formation. The detailed preparation method is as follows.

[0050] 4.1 Preparation of lipid layers First, the lipid layer was prepared as follows: 70 mg of tristearin and 30 mg of tricaprylin were weighed and mixed in a glass reaction vial, then stored in a water bath at 85°C until the next step. 2 mg of myricetin or myricetin derivative was dissolved in 1 ml of acetone, and this was mixed with the lipid mixture prepared and stored previously and stirred for 30 minutes while maintaining the temperature at 85°C. After 30 minutes of stirring, the mixture was evaporated under reduced pressure to remove the acetone, and the lipid layer was then stored in a water bath at 85°C.

[0051] 4.2 Preparation of aqueous layer The aqueous solution layer was prepared by adding Brij (Brij® S100, 0.9735 g) and Pluronic (Pluronic® P-123, 0.9735 g) to a glass reaction vessel, adding 10 ml of distilled water, and then stirring for 12 hours while maintaining the temperature at 85°C.

[0052] 4.3 Preparation of lipid-nanoparticles The lipid layer (volume ratio 0.5) and aqueous solution layer (volume ratio 9.5) were placed in a 50ml conical tube and mixed. The lipid and aqueous solution layers were maintained at 85°C. The mixture was homogenized at 8,000 rpm for 1 minute using a high-speed blender while maintaining the temperature at 85°C, followed by an additional 1 minute at 10,000 rpm. Next, the mixture was sonicated at 60% amplitude for 4 minutes using a tip-type sonicator with a 1-second on / 1-second off cycle while maintaining the temperature at 85°C. The conical tube containing the sample was immersed in a beaker filled with water at 25°C and cooled to 45°C while stirring. The mixture was then sonicated at 60% amplitude for 4 minutes using a tip-type sonicator with a 1-second on / 1-second off cycle. The produced samples should be stored refrigerated (4°C).

[0053] Example 5: Efficacy Comparison Experiment 1 An efficacy comparison experiment was conducted using the composition prepared in Example 4. The efficacy comparison experiment was conducted by measuring the effect on hyperhidrosis according to the method described in the examples of Korean Patent Publication No. 10-2017-0091554. Specifically, the composition prepared in Example 4 was mixed at a concentration of 1 mg with 1 ml of commercially available sunscreen cream to prepare a test sample. The control group (untreated group) was treated with sunscreen cream containing no composition, while the treatment groups were treated with sunscreen cream containing each composition. After applying the test sample to both hands, the subjects were instructed to hold weighed cosmetic cotton puffs in both hands for 5 minutes, then removed and reweighed the cosmetic cotton puffs to calculate the weight gain. The average values ​​for 10 subjects per treatment group are presented in Table 3. The average sweat weight of the control group (untreated group) was set to 100%, and the proportional values ​​are presented as results.

[0054] [Table 3]

[0055] From the above results, it can be seen that the myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1] of the present invention can provide improved effects that previously reported myricetin derivatives cannot provide. This can be interpreted as being due to improved stability in the body.

[0056] Example 6: Efficacy comparison experiment 2 An additional efficacy comparison experiment was conducted using mice. The composition prepared in Example 4 was used. The specific experimental method is as follows: 180 6-week-old male ICR mice (weight: 25-35 g) were randomly divided into 18 groups (10 mice per group) and treated by skin application. Specifically, a mixture of 80 mg / kg Alfaxan and 10 mg / kg Rompun was intraperitoneally injected into the mice to anesthetize them for at least one hour. The anesthetized mice were then treated with the sample on the sole of their right hind paw. The negative control group received PBS (pH 7.4) at a dose of 0.8 ml / kg, while the treatment groups received each of the compositions prepared in Example 4 (prepared by dissolving in PBS) at a dose of 0.8 mg / kg. The mice were left untreated for 30 minutes to allow the applied sample to be fully absorbed. The soles of the mice were then wiped clean and a 3.5% iodine solution (w / v) in ethanol was applied again. After drying for 1 minute, a 10% starch solution (w / v) in castor oil was applied. Immediately after applying the starch solution, the soles of all mice were photographed under the same conditions (distance, magnification, and exposure) using a 12-megapixel digital camera. The photographs were then analyzed for sweating area using image analysis. The results are shown in Figure 8.

[0057] From these results, it was confirmed that the myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1] of the present invention can provide improved effects to a degree that previously reported myricetin derivatives cannot provide. This can also be attributed to improved stability in the body.

[0058] Example 7: Efficacy Comparison Experiment 3 A comparative experiment on the efficacy of skin wrinkle improvement was conducted. Seventy-two women aged 30 to 50 were divided into groups of four. Prior to the experiment, replicas were collected in the usual manner. One hour after the replicas were collected, each experimental group was assigned to apply 5 ml of a test sample prepared using PBS at a concentration of 1 mg / ml from the composition prepared in Example 4 to the face. PBS was applied to the control group. Six hours after application, replicas were collected from the same areas of the face. Two-dimensional analysis of skin wrinkles was performed using image analysis of the replicas, and the skin wrinkle density was measured. The results of the skin wrinkle density measurement using image analysis were calculated by averaging the ratio of the skin wrinkle density before application to the skin wrinkle density after application, and the results are shown in Table 4 below.

[0059] [Table 4]

[0060] From these results, it was confirmed that the myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1] of the present invention can provide improved effects that cannot be provided by previously reported myricetin derivatives.

[0061] Examples of dosage forms and formulations for the cosmetic composition of the present invention are shown below. However, the following dosage forms and formulations are merely illustrative of the present invention, and the content of the present invention is not limited to the following dosage forms and formulations.

[0062] Dosage example 1: Manufacturing of softening lotion (skin) A softening lotion is prepared in the usual manner by mixing 0.1 wt% of nanoparticles of myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1], 5.2 wt% of 1,3-butylene glycol, 1.5 wt% of oleyl alcohol, 3.2 wt% of ethanol, 3.2 wt% of polysorbate 20, 2.0 wt% of benzophenone-9, 1.0 wt% of carboxyl vinyl polymer, 3.5 wt% of glycerin, a trace amount of fragrance, a trace amount of preservative, and the remaining amount of purified water.

[0063] Dosage example 2: Manufacture of milk lotion A milk lotion is prepared in a conventional manner by mixing 0.1% by weight of nanoparticles of myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1], 5.1% by weight of glycerin, 4.2% by weight of propylene glycol, 3.0% by weight of tocopheryl acetate, 4.6% by weight of liquid paraffin, 1.0% by weight of triethanolamine, 3.1% by weight of squalane, 2.5% by weight of macadamia nut oil, 1.6% by weight of polysorbate 60, 1.6% by weight of sorbitan sesquioleate, 0.6% by weight of propylparaben, 1.5% by weight of carboxyl vinyl polymer, a trace amount of fragrance, a trace amount of preservative, and the remaining amount of purified water.

[0064] Dosage Form Example 3: Manufacture of nutritional cream A nourishing cream is prepared in the usual way by mixing 0.5 wt% of nanoparticles of myricetin derivative (3-acetyl-myricetin) represented by [Chemical Formula 1], 4.0 wt% of glycerin, 3.5 wt% of petrolatum, 2.1 wt% of triethanolamine, 5.3 wt% of liquid paraffin, 3.0 wt% of squalane, 2.6 wt% of beeswax, 5.4 wt% of tocopheryl acetate, 3.2 wt% of polysorbate 60, 1.0 wt% of carboxyl vinyl polymer, 3.1 wt% of sorbitan sesquioleate, a trace of fragrance, a trace of preservatives and the remaining amount of purified water.

[0065] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. The following [Chemical Formula 1]: 【Chemistry 1】 The active ingredient is 3-acetyl-myricetin, a myricetin derivative represented by the formula: A composition that provides an acetylcholine secretion inhibitory effect, characterized by:

2. The composition is a cosmetic composition for beauty purposes or a pharmaceutical composition for the treatment of diseases caused by acetylcholine hypersecretion. A composition that provides the acetylcholine secretion inhibitory effect described in claim 1.

3. The cosmetic purpose is any one selected from the group consisting of treating wrinkles, reducing pore size, and improving skin elasticity. A composition that provides the acetylcholine secretion inhibitory effect described in claim 2.

4. The disease caused by the hypersecretion of acetylcholine is excessive sweating. A composition that provides the acetylcholine secretion inhibitory effect described in claim 2.

5. The following [Chemical Formula 1]: 【Chemistry 2】 A method for producing 3-acetyl-myricetin, a myricetin derivative represented by the formula: (A) adding acetic anhydride to myricetin dissolved in anhydrous pyridine in a nitrogen atmosphere that prevents the addition of external air and moisture, and reacting; (B) extracting with ethyl acetate (EA); (C) drying the ethyl acetate (EA) extract under reduced pressure; (D) adding dimethyl sulfoxide (DMSO) to the vacuum-dried sample, and then diluting it with phosphate-buffered saline (PBS; pH 7.0); (E) Incubating the diluted sample to decompose by-products; (F) freeze-drying. A method characterized by:

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