KR102213858B1 - composition comprising the compound isolated from an extract of Leonurus japonicas as an active ingredient for skin whitening activity - Google Patents

Abstract

The present invention relates to a composition containing a compound isolated from a Leonurus japonicas extract as an active component. Specifically, since a strong whitening activity is confirmed through a tyrosinase inhibitory activity test (experimental example 1), compounds of the present invention having a whitening effect can be usefully used as a pharmaceutical composition for external use on the skin and a cosmetic composition.

Description

Composition comprising the compound isolated from an extract of Leonurus japonicas as an active ingredient for skin whitening activity

The present invention is a compound isolated from motherwort extract, specifically, 10-methoxy-leonurine (10-methoxy-leonurine, compound 1), leonurine (compound 2) or leonurusoid E (leonurusoide E, It relates to a composition for skin whitening containing a compound selected from compound 5) as an active ingredient.

[Document 1] Voegeli, R. 1996. Elastase and typtase determination on human skin surface. Cosmetic &Toiletries. 111, 51-58.

[Document 2] Aroca, P., et al. 1993. Melanin biosynthesis patterns of following hormonal stimulation. J. Biol Chem 268, 25650-25655

[Document 3] Fan, M.; Ding, H.; Zhang, G.; Hu, X.; Gong, D. Relationships of dietary flavonoid structure with its tyrosinase inhibitory activity and affinity. LWT-Food Science and Technology. 2019, 107, 25-34

[Document 4] Gui, L.; Lee, J.h.; Hao, H.; Park, Y.-D.; Zhan, Y.; Lu, Z.-R. The effect of oxaloacetic acid on tyrosinase activity and structure: Integration of inhibition kinetics with docking simulation. International Journal of Biological Macromolecules. 2017, 101, 59-66.

[Document 5] Carcelli, M.; Rogolino, D.; Bartoli, J.; Pala, N.; Compari, C.; Ronda, N.; Bacciottini, F.; Incerti, M.; Fisicaro, E. Hydroxyphenyl thiosemicarbazones as inhibitors of mushroom tyrosinase and antibrowning agents. Food Chemistry. 2020, 303, 125310

[Document 6] Zeng, H.-j.; Liu, Z.; Hu, G.-z.; Qu, L.-b.; Yang, R. Investigation on the binding of aloe-emodin with tyrosinase by spectral analysis and molecular docking. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2019, 211, 79-85.

[Document 7] Dehghani, Z.; Khoshneviszadeh, M.; Khoshneviszadeh, M.; Ranjbar, S. Veratric acid derivatives containing benzylidene-hydrazine moieties as promising tyrosinase inhibitors and free radical scavengers. Bioorganic & Medicinal Chemistry. 2019, 27, 2644-2651

[Document 8] Tian, J.-L.; Liu, T.-L.; Xue, J.-J.; Hong, W.; Zhang, Y.; Zhang, D.-X.; Cui, C.-C.; Liu, M.-C.; Liu, S.-L. Flavanoids derivatives from the root bark of Broussonetia papyrifera as a tyrosinase inhibitor. Industrial Crops & Products. 2019, 138, 111445.

[Document 9] Wagle, D.; Seong, SH; Jung, HA; Choi, JS Identifying an isoflavone from the root of Pueraria lobata as a potent tyrosinase inhibitor. Food Chemistry, 2019, 276, 383-389.

[Document 10] Tan, Y.-J.; Xu, D.-Q.; Yue, S.-J.; Tang, Y.-P.; Guo, S.; Yan, H.; Zhang, J.; Zhu, Z.-H.; Shi, X.-Q.; Chen, Y.-Y.; Gu, Y.; Ding, X.-R.; Huang, S.-L.; Peng, G.-P.; Zhou, G.-S.; Duan, J.-A. Comparative analysis of the main active constituents from different parts of Leonurus japonicus Houtt. and from different regions in China by ultra-high performance liquid chromatography with triple quadrupole tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. 2020, 177, 112873.

[Document 11] Zhong, W.-M.; Cui, Z.-M.; Liu, Z.-K.; Yang, Y.; Wu, D.-R.; Liu, S.-H.; Long, H.; Sun, H.-D.; Dang, Y.-J.; Xiao, W.-L. Three minor new compounds from the aerial parts of Leonurus japonicas . Chinese Chemical Letters, 2015, 26, 1000-1003.

[Document 12] Li, H.-Y.; Peng, X.; Jin, Xj; Wei, W.-J.; Ma, K.-L.; Li, Y.; Chen, J.-J.; Gao, K. Labadane-type diterpenoids from Leonurus japonicus and their plant-growth regulatory activity. Journal of Natural Products. 2019, 82(9), 2568-2579

[Document 13] Li, Y.; Chen, Z.; Feng, Z.; Yang, Y.; Jiang, Js; Zhang, Pc Hepatoprotective glycosides from Leonurus japonicas Houtt. Carbohydrate Research. 2012. 348, 42-46.

[Document 14] Zhou, Q.-m.; Peng, C.; Yang, H.; Liu, L.-s.; Yang, Y.-t.; Xie, X.-f.; Guo, L.; Liu, Z.-h.; Xiong, L. Steroids from the aerial parts of Leonurus japonicus . Phytochemistry Letters. 2015, 12, 287-290.

[Document 15] Liu, J.; Peng, C.; Zhou, Q.-M.; Guo, L.; Liu, Z.-H.; Xiong, L. Alkaloids and flavonoid glycosides from the aerial parts of Leonurus japonicas and their opposite effects on uterine smooth muscle. Phytochemistry. 2018, 145, 128-136.

[Document 16] Lai, K.-Y.; Hu, H.-C.; Chiang, H.-M.; Liu, Y.-J.; Yang, J.-C.; Lin, Y.-A.; Chen, C.-J.; Chang, Y.-S.; Lee, C.-L. New diterpenes leojaponins GL from Leonurus japonicas . Fitoterapia. 2018, 130, 125-133

[Document 17] Leem, HH; Lee, GY; Lee, JS; Lee, Hn; Kim, JH; Kim, YH Soluble epoxide hydrolase inhibitory activity of components from Leonurus japonicas . International Journal of Biological Macromolecules. 2017, 103, 451-457..]

[Document 18] Wang, L.; Yang, X.; Qin, P.; Shan, F.; Ren, G. Flavonoid composition, antibacterial and antioxidant properties of tartary buckwheat bran extract. Industrial Crops and Products. 2013, 49, 312-317

[Document 19] Santi, MD; Bouzidi, C.; Gorod, NS; Puiatti, M.; Michel, S.; Grougnet, R.; Ortega, MG In vitro biological evaluation and molecular docking studies of natural and semisynthetic flavones from Gardenia oudiepe (Rubiaceae) as tyrosinase inhibitors. Bioorganic Chemistry. 2019, 82, 241-245.

Modern people promote skin aging phenomena such as spots, freckles, and skin pigmentation by causing various skin troubles by various internal and external factors such as ultraviolet rays and stress (1.Voegeli, R. 1996. Elastase and typtase determination on human skin) surface.Cosmetic & Toiletries. 111, 51-58.). Skin pigmentation is known that melanin pigment regulates the initial reaction from DOPA to DOPA quinone from L-tyrosine such as DHICA oxidase (TRP-1) to DOPA (3,4-dihydroxyphenyla-lanine) in biosynthesis. (2. Aroca, P., et al. 1993. Melanin biosynthesis patterns of following hormonal stimulation. J. Biol Chem 268, 25650-25655.).

Tyrosinase is known to be present in bacteria, fungi, plants and mammals [Fan, M.; Ding, H.; Zhang, G.; Hu, X.; Gong, D. Relationships of dietary flavonoid structure with its tyrosinase inhibitory activity and affinity. LWT-Food Science and Technology. 2019, 107, 25-34]. This is a metalloenzyme containing copper that has a catalytic reaction such as the hydroxylation of monophenols involved in the production of melanin pigments and the oxidation of diphenols [2.Gui] , L.; Lee, Jh; Hao, H.; Park, Y.-D.; Zhan, Y.; Lu, Z.-R. The effect of oxaloacetic acid on tyrosinase activity and structure: Integration of inhibition kinetics with docking simulation. International Journal of Biological Macromolecules. 2017, 101, 59-66.]. Melanin overaccumulation affects not only skin pigment disorders, but also enzyme browning of cut fresh fruits and vegetables [3. Carcelli, M.; Rogolino, D.; Bartoli, J.; Pala, N.; Compari, C.; Ronda, N.; Bacciottini, F.; Incerti, M.; Fisicaro, E. Hydroxyphenyl thiosemicarbazones as inhibitors of mushroom tyrosinase and antibrowning agents. Food Chemistry. 2020, 303, 125310]. Moreover, tyrosinase was found to be involved in the formation of the epidermis of insects [4. Zeng, H.-j.; Liu, Z.; Hu, G.-z.; Qu, L.-b.; Yang, R. Investigation on the binding of aloe-emodin with tyrosinase by spectral analysis and molecular docking. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy. 2019, 211, 79-85.]. In order to solve the unintended effects of melanin, inhibition of the production of melanin precursors has been considered as an important strategy [4]. As a result, it is considered as a target enzyme for the development of whitening agents, food preservatives, and pesticides in agriculture [5 Dehghani, Z.; Khoshneviszadeh, M.; Khoshneviszadeh, M.; Ranjbar, S. Veratric acid derivatives containing benzylidene-hydrazine moieties as promising tyrosinase inhibitors and free radical scavengers. Bioorganic & Medicinal Chemistry. 2019, 27, 2644-2651]. Recently, aloe-emodin, broussoflavonols J, broussoflavonol H [6Tian, J.-L.; Liu, T.-L.; Xue, J.-J.; Hong, W.; Zhang, Y.; Zhang, D.-X.; Cui, C.-C.; Liu, M.-C.; Liu, S.-L. Flavanoids derivatives from the root bark of Broussonetia papyrifera as a tyrosinase inhibitor. Industrial Crops & Products. 2019, 138, 111445.], and calycosin [7. Wagle, D.; Seong, SH; Jung, HA; Choi, JS Identifying an isoflavone from the root of Pueraria lobata as a potent tyrosinase inhibitor. Food Chemistry, 2019, 276, 383-389.], secondary metabolites from natural products have been studied for the development of tyrosinase inhibitors.

Motherwort ( Leonurus japonicas ) is a perennial herb of the family Labiatae distributed throughout Asia [8.Tan, Y.-J.; Xu, D.-Q.; Yue, S.-J.; Tang, Y.-P.; Guo, S.; Yan, H.; Zhang, J.; Zhu, Z.-H.; Shi, X.-Q.; Chen, Y.-Y.; Gu, Y.; Ding, X.-R.; Huang, S.-L.; Peng, G.-P.; Zhou, G.-S.; Duan, J.-A. Comparative analysis of the main active constituents from different parts of Leonurus japonicus Houtt. and from different regions in China by ultra-high performance liquid chromatography with triple quadrupole tandem mass spectrometry. Journal of Pharmaceutical and Biomedical Analysis. 2020, 177, 112873.].

Called “Yi Mu Cao” in China, this plant has been used to treat diseases such as menoxenia, dysmenorrheal, amenorrhea and ulcerations [9. Zhong, W.-M .; Cui, Z.-M.; Liu, Z.-K.; Yang, Y.; Wu, D.-R.; Liu, S.-H.; Long, H.; Sun, H.-D .; Dang, Y.-J.; Xiao, W.-L. Three minor new compounds from the aerial parts of Leonurus japonicas . Chinese Chemical Letters, 2015, 26, 1000-1003.].

The study of plant chemistry of motherwort was conducted using column chromatography (ladbane diterpenes) [10 Li, H.-Y.; Peng, X.; Jin, Xj; Wei, W.-J.; Ma, K.-L.; Li, Y.; Chen, J.-J.; Gao, K. Labadane-type diterpenoids from Leonurus japonicus and their plant-growth regulatory activity. Journal of Natural Products. 2019, 82(9), 2568-2579], propenylethanoid glycosides [11Li, Y.; Chen, Z.; Feng, Z.; Yang, Y.; Jiang, Js; Zhang, Pc Hepatoprotective glycosides from Leonurus japonicas Houtt. Carbohydrate Research. 2012. 348, 42-46.], steroids [12. Zhou, Q.-m.; Peng, C.; Yang, H.; Liu, L.-s.; Yang, Y.-t.; Xie, X.-f.; Guo, L.; Liu, Z.-h.; Xiong, L. Steroids from the aerial parts of Leonurus japonicus . Phytochemistry Letters. 2015, 12, 287-290.], alkaloids [13. Liu, J.; Peng, C.; Zhou, Q.-M.; Guo, L.; Liu, Z.-H.; Xiong, L. Alkaloids and flavonoid glycosides from the aerial parts of Leonurus japonicas and their opposite effects on uterine smooth muscle. Phytochemistry. 2018, 145, 128-136.] and flamonoid glycosides. Phenylethanoid glycosides were found to have a hepatocellular protective effect similar to bicyclol in HL-7702 cells.

However, in any of the prior documents cited only by reference herein, 10-methoxy-leonurine (10-methoxy-leonurine, compound 1), leonurine (compound 2), and syringin isolated from the motherwort extract of the present invention There is no teaching or disclosure regarding the whitening effect of the acid (compound 3), isoquercitrin (compound 4) or leonurusoide E (compound 5) compounds.

Accordingly, the present inventors confirmed strong whitening activity through tyrosinase inhibitory activity test (Experimental Example 1) for the compounds of the present invention derived from motherwort extract. The present invention was completed by confirming its usefulness as a skin whitening composition.

In order to achieve the above object, the present invention is 10-methoxy-leonurine (10-methoxy-leonurine, compound 1), leonurine (compound 2 ), or leonurusoid E (leonurusoide) isolated from motherwort extract It provides a skin whitening pharmaceutical composition for external use for skin containing a compound selected from E, compound 5), or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention is 10-methoxy-leonurine (10-methoxy-leonurine, compound 1), leonurine (compound 2 ), or leonurusoid E (leonurusoide E, compound 5) isolated from motherwort extract It provides a cosmetic composition for skin whitening containing a compound selected from, or a pharmaceutically acceptable salt thereof, as an active ingredient.

Motherwort extract as defined herein is one or more solvents selected from the group consisting of water, ethanol, methanol, propanol, butanol, acetone, ethyl acetate, hexane, butylene glycol, propylene glycol, hydrous butylene glycol, hydrous propylene glycol, and hydrous glycerin. , Preferably water or a mixed solvent of water and methanol, most preferably 60% to 100% methanol-soluble extract.

The compound is characterized in that it contains 0.1 to 50% by weight of the external pharmaceutical composition for skin.

The compounds of the present invention can be prepared as pharmaceutically acceptable salts and solvates according to conventional methods in the art.

As a pharmaceutically acceptable salt, an acid addition salt formed by a free acid is useful. Acid addition salts are prepared by conventional methods, for example, by dissolving a compound in an excess acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and an acid or alcohol (eg glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be suction filtered.

At this time, organic acids and inorganic acids can be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. can be used as inorganic acids, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, and the like can be used as organic acids. Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like can be used.

In addition, a pharmaceutically acceptable metal salt can be made using a base. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. In this case, as the metal salt, it is particularly suitable for pharmaceutical use to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (eg, silver nitrate).

Pharmaceutically acceptable salts of the compounds of the present invention, unless otherwise indicated, include acidic or basic salts that may be present in the compounds of the present invention. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, and dihydrogen There are phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and methods or processes for preparing salts known in the art It can be manufactured through.

Hereinafter, the present invention will be described in more detail.

The extracts and compounds of the present invention can be obtained by the following manufacturing method.

For example, the present invention will be described in detail below.

Motherwort extract of the present invention can be prepared as follows. After washing and shredding the dried motherwort, a solvent selected from water including purified water, lower alcohols having 1 to 4 carbon atoms such as methanol, ethanol, butanol, or a mixed solvent thereof, preferably a mixed solvent of water and methanol, more preferably 60 After mixing ~100% methanol several times, at a temperature of 10°C to 150°C, preferably 20°C to 50°C, for 30 minutes to 7 days, preferably 12 hours to 4 days, ultrasonic extraction, hot water extraction, room temperature extraction, or The extract obtained by repeating the reflux extraction method, preferably the cold needle extraction method, about 1 to 20 times, preferably 2 to 10 times, is filtered, concentrated under reduced pressure, and dried to obtain a crude motherwort extract of the present invention.

In addition, the polar solvent or non-polar solvent-soluble extract of the present invention is about 0.0005 to 50 times the weight of the crude extract obtained above, preferably 60 to 90% methanol crude extract, preferably 0.05 to 5 times volume (v/w% ) After adding water, a non-polar solvent-soluble extract fraction soluble in a non-polar solvent such as n-hexane, methylene chloride, and ethyl acetate by performing a conventional fractionation process using n-hexane, methylene chloride, ethyl acetate and butanol; And a polar solvent-soluble extract fraction soluble in a polar solvent such as butanol and water.

The obtained non-polar solvent-soluble extract fraction soluble in a non-polar solvent such as n-hexane, methylene chloride, ethyl acetate, etc. obtained above, preferably the ethyl acetate soluble fraction, was flashed using a method of increasing the polarity of a normal hexane-acetone inclined mixture. Compounds of the present invention, that is, 10-methoxy-leonurine, by selectively repeating a purification method using chromatography, such as column chromatography, RP C18 column chromatography, or silica gel open column chromatography, is repeatedly performed several times. , Compound 1), leonurine (compound 2 ), syringic acid (compound 3 ), isoquercitrin (compound 4) or leonurusoide E (compound 5 ) compound, respectively Can be purified and obtained.

In addition, motherwort is a medicinal material that has been edible or used as a herbal medicine for a long time, and the compound isolated from the motherwort extract of the present invention also has no problems such as toxicity and side effects.

In the present invention, 10-methoxy-leonurine (10-methoxy-leonurine, compound 1), leonurine, compound 2 , or leonurusoide E, isolated from the motherwort extract obtained in the above production method. Compound 5 ) It provides a skin whitening pharmaceutical composition or cosmetic composition for skin whitening containing the compound as an active ingredient.

The pharmaceutical composition includes a cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta, or cataplasma formulation.

In addition, the cosmetic composition includes a formulation of lotion, skin, lotion, nutrition lotion, nutrition cream, massage cream, essence, and pack.

The present inventors confirmed the strong whitening activity of the compounds of the present invention through a tyrosinase inhibitory activity experiment (Experimental Example 1). It was confirmed to be useful as a composition for skin whitening.

The pharmaceutical composition for external use for skin containing the compound of the present invention can be prepared and used as a pharmaceutical composition for external use for skin such as cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta or cataplasma. However, it is not limited thereto.

The preferred dosage of the compound of the present invention varies depending on the condition and weight of the patient, the degree of disease, the drug form, the route and duration of administration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg/kg per day, preferably 0.001 to 10 mg/kg per day. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.

The compound of the present invention can be variously used in cosmetics and face wash having a whitening effect.

Products to which the present composition can be added include cosmetic products such as lotion, skin, lotion, nutrition lotion, nutrition cream, massage cream, essence, pack, and cleansing, face wash, soap, treatment, essence, etc. have.

The cosmetic composition of the present invention includes a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingo lipids, and seaweed extract.

The water-soluble vitamin may be any one that can be blended in cosmetics, but preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine hydrochloride, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H, etc. And their salts (thiamine hydrochloride, sodium ascorbate, etc.) and derivatives (ascorbic acid-2-phosphate sodium salt, ascorbic acid-2-magnesium salt, etc.) are also included in the water-soluble vitamins that can be used in the present invention. Included. Water-soluble vitamins can be obtained by conventional methods such as a microbial transformation method, a purification method from a culture of microorganisms, an enzyme method or a chemical synthesis method.

As the oil-soluble vitamin, any one may be used as long as it can be blended in cosmetics, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol), etc. are mentioned. , Their derivatives (ascorbine palmitate, ascorbine stearate, ascorbine dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherol vitamin E nicotinic acid, dl-pantotenyl alcohol, D-pantotenyl alcohol, pantotenyl ethyl Ether, etc.) are also included in the oil-soluble vitamin used in the present invention. Oil-soluble vitamins can be obtained by conventional methods such as a microbial transformation method, a purification method from a microorganism culture, an enzyme or a chemical synthesis method.

The polymer peptide may be any one as long as it can be blended into cosmetics. Preferably, collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin, and the like are exemplified. The polymeric peptide can be purified and obtained by a conventional method such as a purification method from a culture medium of a microorganism, an enzyme method, or a chemical synthesis method, or it can be used after being purified from natural products such as dermis of pigs and cattle, silk fibers of silkworms.

As the polymeric polysaccharide, any one may be used as long as it can be blended in cosmetics, but preferably, hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfuric acid or a salt thereof (sodium salt, etc.) may be mentioned. For example, chondroitin sulfate or a salt thereof can be used after being purified from mammals or fish.

As sphingo lipids, any one may be used as long as it can be blended in cosmetics, and ceramides, phytosphingosine, sphingoglycolipids and the like are preferably mentioned. Sphingo lipids are usually purified from mammals, fish, shellfish, yeast, plants, etc. by a conventional method, or can be obtained by chemical synthesis.

The seaweed extract may be anything as long as it can be blended in cosmetics, but preferably brown algae extract, red algae extract, green algae extract, etc. are mentioned.Also, carrageenan, arginic acid, sodium arginate, purified from these seaweed extracts, Potassium arginate and the like are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained by purifying from seaweed by a conventional method.

In the cosmetic of the present invention, in addition to the above essential ingredients, other ingredients that are usually blended in the cosmetic may be blended if necessary.

Other ingredients that may be added include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, fragrances, Blood circulation accelerators, cold sensation agents, restrictors, and purified water.

Examples of oil and fat components include ester oils and fats, hydrocarbon oils, silicone oils, fluorine oils, animal oils and vegetable oils.

Examples of ester oils include glyceryl tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl myristate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, stearic acid. Butyl, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl myristate, isostearyl myristate, isostearyl palmitate, octyldodecyl myristate, isocetyl isostearate, diethyl sebacate, adipine Diisopropyl acid, isoalkyl neopentanoate, tri(capryl, capric acid) glyceryl, tri2-ethylhexanoate trimethylolpropane, triisostearate trimethylolpropane, tetra2-ethylhexanoate pentaelisitol , Cetyl caprylate, decyl laurate, hexyl laurate, decyl myristic acid, myristic myristic acid, cetyl myristate, stearyl stearate, decyl oleate, cetyl ricinooleate, isostea laurate Reel, isotridecyl myristate, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyldodecyl oleate, octyldodecyl linoleate, isopropyl isostearate, 2-ethylhexanoate cetoste Aryl, 2-ethylhexanoate stearyl, isostearate hexyl, dioctanoate ethylene glycol, dioleate ethylene glycol, dicaprylic acid propylene glycol, dicaprylic acid propylene glycol, dicaprylic acid propylene glycol, dica Neopentyl glycol prinate, neopentyl glycol dioctanoate, glyceryl tricaprylate, glyceryl triundecylate, glyceryl triisopalmitate, glyceryl triisostearate, octyldodecyl neopentanoate, isostearyl octanoate , Octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearyl isostearate, octyldecyl isostearate, polyglycerololeic acid ester, polyglycerin isostearate, Triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauryl lactate, myristyl lactate, cetyl lactate, octyldecyl lactate, triethyl citrate, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, di malic acid Isostearyl, 2-ethylhexyl hydroxystearate, di2-ethylhexyl succinate, diisobutyl adipate, diisopropyl sebacate, dioctyl sebacate, Cholesteryl stearate, cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate, dihydrocholesteryl oleate, pitsteryl isostearate, pitsteryl oleate, 12-stealoyl And esters such as isocetyl hydroxystearate, stearyl 12-stealoylhydroxystearate, and isostearyl 12-stealoylhydroxystearate.

Examples of hydrocarbon-based fats and oils include hydrocarbon-based fats such as squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybuden, microcrystalline wax, and petrolatum.

Silicone-based fats and oils include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane/methylcetyloxysiloxane copolymer, dimethylsiloxane/methylsteaoxysiloxane copolymer, alkyl And modified silicone oil and amino-modified silicone oil.

As fluorine-based fats and oils, perfluoropolyether, etc. are mentioned.

Animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, new flower oil, soybean oil, corn oil, rapeseed oil, almond oil, palm kernel oil, palm oil, castor oil, sunflower oil, grape seed oil, Cottonseed Oil, Palm Oil, Cucuine Nut Oil, Wheat Germ Oil, Rice Germ Oil, Shea Butter, Moonshine Colostrum Oil, Marker Demi Anut Oil, Meadow Home Oil, Egg Yolk Oil, Tallow Oil, Horse Oil, Mink Oil, Orange Rape Oil, Jojoba Oil, And animal or plant fats such as canderry wax, carnauba wax, liquid lanolin, and hydrogenated castor oil.

Examples of the moisturizing agent include a water-soluble low-molecular moisturizer, a fat-soluble molecular moisturizer, a water-soluble polymer, and a fat-soluble polymer.

Water-soluble low molecular weight moisturizers include serine, glutamine, sorbitol, mannitol, pyrrolidone-sodium carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n = 2 or more), polypropylene Glycol (polymerization degree n = 2 or more), polyglycerin B (polymerization degree n = 2 or more), lactic acid, lactate, and the like.

As a fat-soluble low molecular weight moisturizer, cholesterol, cholesterol ester, etc. are mentioned.

Examples of the water-soluble polymer include carboxyvinyl polymer, polyaspartic acid salt, tragacanth, xanthan gum, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, carboxymethylcellulose, water-soluble chitin, chitosan, dextrin, and the like. I can.

Examples of the oil-soluble polymer include polyvinylpyrrolidone/eicosene copolymer, polyvinylpyrrolidone/hexadecene copolymer, nitrocellulose, dextrin fatty acid ester, and polymer silicone.

Examples of the emollient agent include long-chain acyl glutamate cholesteryl ester, hydroxystearate cholesteryl, 12-hydroxystearic acid, stearic acid, rosin acid, and lanolin fatty acid cholesteryl ester.

Examples of the surfactant include nonionic surfactants, anionic surfactants, cationic surfactants, and amphoteric surfactants.

Nonionic surfactants include self-emulsifying glycerin monostearate, propylene glycol fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, sorbitan fatty acid ester, POE (polyoxyethylene) sorbitan fatty acid ester, POE sorbit fatty acid ester, POE Glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hydrogenated castor oil, POE castor oil, POE·POP (polyoxyethylene·polyoxypropylene) copolymer, POE·POP alkyl ether, polyether-modified silicone, lauric acid Alkanolamides, alkylamine oxides, hydrogenated soybean phospholipids, and the like.

As anionic surfactants, fatty acid soap, alpha-acyl sulfonate, alkyl sulfonate, alkyl allyl sulfonate, alkyl naphthalene sulfonate, alkyl sulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl phosphate, alkyl amide Phosphate, alkyloylalkyltaurine salt, N-acylamino acid salt, POE alkylether carboxylate, alkyl sulfosuccinate, sodium alkylsulfoacetate, acylated hydrolyzed collagen peptide salt, perfluoroalkyl phosphate, and the like. have.

Cationic surfactants include alkyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium bromide, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyldimethylbenzyl ammonium chloride, behenyl trimethyl ammonium bromide, and chloride. Benzalkonium, diethylaminoethyl amide stearate, dimethylaminopropyl amide stearate, lanolin derivative quaternary ammonium salt, and the like.

Examples of amphoteric surfactants include carboxybetaine type, amidebetaine type, sulfobetaine type, hydroxysulfobetaine type, amide sulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type, amideamine type, etc. Amphoteric surfactants, and the like.

Examples of organic and inorganic pigments include silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium coated mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide. , Inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine, chromium oxide, chromium hydroxide, calamine, and complexes thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene-styrene copolymer, Organic pigments, such as silk powder, cellulose, CI pigment yellow, and CI pigment orange, and complex pigments of these inorganic pigments and organic pigments, etc. are mentioned.

Examples of the organic powder include metal soaps such as calcium stearate; Alkyl phosphate metal salts, such as sodium zinc cetylate, a zinc laurylate, and calcium laurylate; Acylamino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroyl glycine calcium; Amide sulfonic acid polyvalent metal salts such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N, such as N-epsilon-lauroyl-L-lysine, N-epsilon-palmitoyl lizine, N-alpha-paritoylolnitine, N-alpha-lauroylarginine, N-alpha-hardened tallow fatty acid acylarginine, etc. -Acyl basic amino acid; N-acyl polypeptides such as N-lauroylglycylglycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; Polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene/styrene copolymer, ethylene tetrafluoride, and the like.

UV absorbers include paraaminobenzoic acid, ethyl paraaminobenzoate, amyl paraaminobenzoate, octyl paraaminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicate, butyl phenyl salicylate, homomentyl salicylate, benzyl cinnamate , Paramethoxycinnamic acid-2-ethoxyethyl, paramethoxycinnamic acid octyl, diparamethoxycinnamic acid mono-2-ethylhexane glyceryl, paramethoxycinnamic acid isopropyl, diisopropyl·diisopropyl cinnamic acid ester mixture, right Cannic acid, ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and salts thereof, dihydroxymethoxybenzophenone, sodium dihydroxymethoxybenzophenone disulfonate, dihydroxybenzophenone , Tetrahydroxybenzophenone, 4- tert -butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino- p- (carbo-2'-ethylhexyl-1'-oxy)-1 ,3,5-triazine, 2-(2-hydroxy-5-methylphenyl)benzotriazole, etc. are mentioned.

As a disinfectant, hinokitiol, triclosan, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, zinphylithione, benzalkonium chloride, photosensitization Sono No. 301, mononitroguarous sodium, undecylenic acid, and the like.

Examples of the antioxidant include butylhydroxyanisole, propyl gallic acid, and elisorbic acid.

Examples of the pH adjuster include citric acid, sodium citrate, malic acid, sodium malate, pmalic acid, sodium pmarate, succinic acid, sodium succinate, sodium hydroxide, sodium monohydrogen phosphate, and the like.

Examples of alcohol include higher alcohols such as cetyl alcohol.

In addition, the blending components that may be added are not limited thereto, and any of the above components can be blended within a range that does not impair the object and effect of the present invention, but preferably 0.01 to 5% by weight based on the total weight, more It is preferably blended at 0.01-3% by weight.

The cosmetic composition of the present invention may take the shape of a solution, an emulsion, a viscous mixture, or the like.

Ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the complex herbal extract as an active ingredient, for example, stabilizers, solubilizers, vitamins, pigments, and flavors. Phosphorus adjuvants and carriers.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, emulsion, cream, lotion, pack, foundation, lotion, essence, hair cosmetic, and the like.

Specifically, the cosmetic composition of the present invention is a skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, foundation, essence, nutrition essence, It includes formulations of packs, soaps, cleansing foams, cleansing lotions, cleansing creams, body lotions and body cleansers.

When the formulation of the present invention is a paste, cream, or gel, animal fiber, plant fiber, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as carrier components. I can.

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

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

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

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linoline derivatives, or ethoxylated glycerol fatty acid esters may be used.

The present inventors confirmed the strong whitening activity of the compounds of the present invention through a tyrosinase inhibitory activity experiment (Experimental Example 1). It was confirmed to be useful as a composition for skin whitening.

The compounds of the present invention can be usefully used as a composition for skin whitening by confirming strong whitening activity through a tyrosinase inhibitory activity test (Experimental Example 1).

1 is a diagram showing the tyrosinase inhibitory activity of a sample of the present invention.

Below, The present invention will be described in detail by the following examples and experimental examples.

However, the following examples and experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples and experimental examples.

Reference Example 1. Experimental equipment and preparation

ESI-MS spectra were measured with microTOF-QTMII (Bruker, USA). NMR experiments were performed with ECA500 (JEOL, Japan). TLC analysis was performed with Kieselgel 60 F254 (Merck) plates (silicagel, 0.25mm layer thickness). The sample was immersed in 10% (v/v) H ₂ SO ₄ (Aldrich) solvent, and then dried by drying at 300° C. for 30 seconds.

Silica gel (Merck 60A, 70-230 or 230-400 mesh ASTM), Sephadex LH-20 (GE healthcare) and reversed-phase silica gel (YMC Co., ODS-A 12 nm S-150, S-75 μm) Was used for open column chromatography.

Tyrosinase (T3824) and L-tyrosinase (T3754) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Example 1. Preparation of compound from motherwort extract

1-1. Preparation of crude motherwort extract

Motherwort ( Leonurus japonicas , 5 kg, Human Herb Co., Ltd.) was subjected to cold needle extraction in which 80% of 80% ethanol was added to the raw material and leached at 25°C for 72 hours, and this process was repeatedly extracted twice to obtain an extract. Whatman No. 1 Filtered with filter paper and concentrated with a vacuum concentrator (N-1110, Eyela) to obtain about 877.55 g of dried motherwort 80% methanol extract (hereinafter referred to as LJM). Thereafter, the sample was stored in a refrigerator and used as a sample for this experiment.

1-2. Preparation of motherwort non-polar solvent and polar solvent-soluble fraction

Normal hexane (n -hexane) 2,000mL, ethyl acetate (ethyl acetate) and 2,000 mL of n-butanol, methanol concentrate was 877.55 g of the step 1-1 in a conventional fractionation process in the art and then with distilled water turbidity 1,000mL (n - butanol) by performing a fraction (fractionation) was obtained using the 2,000mL normal hexane (n -hexane) fractions, 78.75g, ethyl acetate (ethyl acetate) fraction 73.19g, and n-butanol (n -butanol) 144.97 g of each fraction .

1-3. Preparation of compounds from motherwort

The ethyl acetate fraction (73.19 g) of the step 1-2 was performed by silica gel column chromatography in a normal hexane-acetone inclined mixture (10:1 to 1:100), and then 20 first fractions (E1- 20) was obtained. Fraction E18 of the first fraction was subjected to C-18 column chromatography (AA12S75, YMC) in a methanol elevation gradient (30% to 50%) to obtain compound 3 (syringic acid, 72.1 mg).

Compound 4 (isoquercitrin 19.6 mg) was obtained by performing C-18 column chromatography from the fraction E19 fraction in the first fraction using a 50% methanol isometric method. Among the first fractions, fraction E20 fractions were subjected to C-18 column chromatography using a solvent method such as 50% methanol to obtain six second small fractions (E201-E206). The E203 fraction of the second small fraction was subjected to Sephadex LH-20 column chromatography (17-0090-02, GE healthcare) using a 40% methanol solvent to obtain compound 1 (10-methoxy- leonurine, 15.1 mg), compound 2 (leonurine, 8.5 mg) and compound 5 (leonurusoide E, 51.9 mg) were isolated, respectively,

The structures of compound 1 (10-methoxy-leonurine), compound 2 (leonurine), compound 3 (syringic acid), and compound 5 (leonurusoide E) were identified by comparing the physical properties of the existing literature [Leem, HH; Lee, GY; Lee, JS; Lee, Hn; Kim, JH; Kim, YH Soluble epoxide hydrolase inhibitory activity of components from Leonurus japonicas . International Journal of Biological Macromolecules. 2017, 103, 451-457].

In addition, the structure of compound 4 (isoquercitrin) was identified by comparing it with the physical properties of the existing literature [Wang, L.; Yang, X.; Qin, P.; Shan, F.; Ren, G. Flavonoid composition, antibacterial and antioxidant properties of tartary buckwheat bran extract. Industrial Crops and Products. 2013, 49, 312-317].

compound One (10-methoxy-leonurine)

(I)

ESI-MS: m/z 326.18[M+H] ⁺ , (calcd C ₁₅ H ₂₃ N ₃ O ₅ ). ¹ H-NMR (CD ₃ OD, 500 MHz) δ 7.32 (2H, s, H-2, 6), 4.63 (1H, s, 4-OH), 4.36 (2H, t, J =6.6 Hz, H- 1′), 3.88 (6H, s, 3, 5-OCH ₃ ), 3.82 (2H, s, 4-CH ₂ ), 3.26 (2H, t, J =7.2 Hz, H-4′), 1.85 (2H , p, J =6.6 Hz, H-2'), 1.73 (2H, p, J =7.2 Hz, H-3'). ¹³ C-NMR (CD ₃ OD, 125 MHz) δ 167.7 (C-7), 158.7 (C-6′), 154.5 (C-3), 143.7 (C-4), 126.7 (C-1), 108.0 (C-2, 6), 65.7 (C-1′), 61.3 (4-CH ₂ ), 56.8 (3, 5-OCH ₃ ), 42.2 (C-4′), 27.1 (C-2′), 26.7 (C-3′)

compound 2 (leonurine)

(II)

compound 3 (syringic acid)

(III)

¹ H-NMR (CD ₃ OD, 500 MHz) δ 7.30 (1 H, s, H-2, 6), 3.86 (6H, s, OCH ₃ ). ¹³ C-NMR (CD ₃ OD, 125 MHz) δ 170.0 (COOH), 148.9 (C-3, 5), 141.8 (C-4), 122.0 (C-1), 108.3 (C-2, 6), 56.9 (OCH ₃ )

compound 4 (isoquercitrin)

(Ⅳ)

¹ H-NMR (CD ₃ OD, 500 MHz) δ 7.70 (1H, d, J= 1.8 Hz, H-2 ' ), 7.57 (1H, dd, J =8.5, 1.6 Hz, H-6 ' ), 6.85 (1H, d, J=8.4 Hz, H-5 ' ), 6.38 (1H, d, J= 1.1 Hz, H-8), 6.19 (1H, s, H-6), 5.25 (1H, d, J = 7.5 Hz, H-1 '' ). ¹³ C-NMR (CD ₃ OD, 125 MHz) δ 179.6 (C-4), 166.1 (C-7), 163.1 (C-5), 159.1 (C-2), 158.6 (C-9), 149.9 ( C-4 ' ), 146.0 (C-3 ' ), 135.7 (C-3), 123.3 (C-6 ' ), 123.1 (C-1 ' ), 117.6 (C-2 ' ), 116.1 (C-5 ' ), 105.8 (C-10), 104.3 (C-1 '' ), 100.0 (C-6), 94.8 (C-8), 78.5 (C-3 '' ), 78.2 (C-5 '' ) , 75.8 (C-2 '' ), 71.3 (C-4 '' ), 62.6 (C-6 '' )

compound 5 (leonurusoide E)

(Ⅴ)

¹ H-NMR (CD ₃ OD, 500 MHz) δ 7.65 (1H, s, H-2 ' ), 7.64 (1H, d, J =8.0, H-6 ' ), 7.39 (1H, s, H-2 ''' & H-6 '''' ), 6.87 (1H, dd, J= 2.85, 8.9 Hz, H-5 ' ), 6.36 (1H, d, J= 2.0 Hz, H-8), 6.15 (1H, d, J= 2.0 Hz, H-6), 5.11 (1H, d, J= 7.7 Hz, H-5 '' ), 5.06 (1H, dd, J= 3.4, 9.4 Hz, H-3 ''' ), 4.57 (1H, d, J=1.7 Hz, H-1 ''' ), 3.92 (1H, m, H-2 '' ), 3.89 (6H, s, -OCH ₃ ), 3.87 ( 1H, m, H-6 '' a), 3.63 (1H, m, H-4 ''' ), 3.63 (1H, m, H-5 ''' ), 3.50 (1H, t, J= 7.7 Hz , H-2 '' ), 3.43 (1H, m, H-3 '' ), 3.41 (1H, m, H-5 '' ), 3.41 (1H, m, H-6 '' b), 3.29 ( 1H, m, H-4 '' ), 1.18 (1H, d, J = 5.45 Hz, H-6 ''' ). ¹³ C-NMR (CD ₃ OD, 125 MHz) δ 179.5 (C-4), 167.8 (C-7 ''' ), 166.1 (C-7), 163.0 (C-5), 159.4 (C-2) ), 158.6 (C-9), 149.9 (C-4 ' ), 148.9 (C-3 ''' , C-5 ''' ), 145.9 (C-3 ' ), 141.9 (C-4 ') ''' ), 135.8 (C-3), 123.7 (C-6 ' ), 123.2 (C-1 ' ), 121.7 (C-1 ''' ), 117.8 (C-2 ' ), 116.2 (C -5 ' ), 108.6 (C-2 '''' , C-6 ''' ), 105.7 (C-10), 104.9 (C-1 '' ), 102.6 (C-1 ''' )), 100.0 (C-6), 95.0 (C-8), 78.3 (C-3 '' ), 77.3 (C-5 '' ), 75.9 (C-3 '' ), 75.8 (C-2 '' ) , 71.6 (C-4 '' ), 70.3 (C-4 '' ), 70.1 (C-2 ''' , C-5 '' ), 56.9 (OCH ₃ ), 18.1 (C-6 '') ' )

Experimental Example 1. Whitening activity experiment

1-1. In vitro tyrosinase inhibitory activity

In order to test the tyrosinase inhibitory activity of the samples obtained in the examples, the following experiment was performed by applying the method disclosed in the existing literature [Santi, MD; Bouzidi, C.; Gorod, NS; Puiatti, M.; Michel, S.; Grougnet, R.; Ortega, MG In vitro biological evaluation and molecular docking studies of natural and semisynthetic flavones from Gardenia oudiepe (Rubiaceae) as tyrosinase inhibitors. Bioorganic Chemistry. 2019, 82, 241-245..]

1-1-1. Sample preparation

The tyrosinase of the example was prepared by dissolving in 0.05 mM phosphate buffer at a concentration of 33 units/mL. To screen the compounds, they were dissolved in methanol and prepared at a concentration of 1000 μM and used in the following experiment. To calculate the IC ₅₀ value, 31.2, 62.5, 120.5, 125. 500 and 1000 μM concentrations were prepared and used in the experiment. L-Tyrosine was dissolved in 0.05 mM phosphate buffer and prepared at a concentration of 1.25 mM.

1-1-2. Experiment process

130 μL of tyrosinase (T3824, Sigma-Aldrich) dissolved in 0.05 mM phosphate buffer and 20 μL of inhibitor or methanol were added to a 96 well plate, and then 50 μL of 1.25 mM L-tyrosine (T3754, Sigma-Aldrich) were further mixed. The absorbance was measured at 37° C. for 20 minutes at 30 second intervals using a reader (microplate reader, AMR100, Allsheng, Hangzhou) at 475 nm wavelength. As a positive control, kojic acid (K3125, Sigma-Aldrich) was used. The tyrosinase inhibitory activity was expressed by the decrease in absorbance between the inhibitor and methanol addition groups.

The inhibition rate of this experiment was calculated by Equations 1 to 2.

As a result of this experiment, the isolated compounds 1 and 2 showed a high inhibition rate of 80% or more at a concentration of 100 μM. In order to calculate their IC ₅₀ values, inhibition rates were tested at each concentration from 3.1 to 100 μM. As a result, 10-methoxy-leonurine (1) and leonurine (2) showed concentration-dependent inhibition rates, and IC ₅₀ values of 7.4 ± 0.4 μM and 12.4 ± 0.8 μM, respectively, were derived by equation (2). (Table 1)

Inhibitor Inhibition rate (%) at 100 mM ^a IC ₅₀ value (mM) ^a One 91.8±2.9 7.4±0.4 2 85.6±1.8 12.4±0.8 3 11.6±0.1 NT ^c 4 1.8±5.9 NT ^c 5 8.3±0.6 NT ^c Kojic acid ^b 79.4±2.5 25.7±1.3 ^a 3 times measurement, ^b control group, ^{c no} measurement

In order to find a tyrosinase inhibitor, high purity components (1-5) were isolated from motherwort. The five isolated compounds were evaluated for their enzyme inhibitory activity against tyrosinase. Of the guanidine pseudoalkaloids, 10-Methoxy-leonurine (1) and leonurine (2) were found to have IC ₅₀ values of 7.4±0.4 μM and 12.4±0.8 μM.

Pseudoalkaloids, 10-methoxy-leonurine and leonurine, were found to be potent tyrosinase inhibitors at micromolar concentrations. In particular, they were found to act in a way that interferes with the catalytic reaction by binding to the active site of tyrosinase. This fact confirmed that Psudoalkalodis derived from motherwort is a lead compound for the development of tyrosinase inhibitors.

Finally, it was found that the most effective compound (1) with a K _i value of 1.6±0.7 μM is possible as a tyrosinase inhibitor, and guanidine-derived alkaloids are possible as a lead compound for the development of a new tyrosinase inhibitor.

Hereinafter, as examples of the formulation of the present invention, formulations of cream, massage cream, lotion, skin lotion, essence, pack, and cleansing foam are exemplified, but the formulation including the cosmetic composition of the present invention is not limited thereto.

Formulation Example 1. Cream composition

The oil phase and the aqueous phase were heated and mixed at 75° C., and then cooled to room temperature.

Formulation Example 2. Massage cream composition

The oil phase and the aqueous phase are heated and mixed at 75° C. and then cooled to room temperature.

Formulation Example 3. Lotion composition

After heating and emulsifying the oil phase and the aqueous phase respectively at 75° C., it is cooled to room temperature.

Formulation Example 4. Skin lotion composition

The aqueous phase and the ethanol phase are respectively prepared and mixed and filtered.

Formulation Example 5. Essence Composition

The aqueous phase and the ethanol phase are respectively prepared and mixed and filtered.

Formulation Example 6. Pack composition

After dispersing and mixing the aqueous phase and the ethanol phase, respectively, it is cooled to room temperature.

Formulation Example 7. Cleansing Foam Composition

Disperse and dissolve the aqueous phase and the oil phase, respectively, mix and saponify, and then cool to room temperature.

Claims (5)

An external skin pharmaceutical composition for skin whitening containing a 10-methoxy-leonurine (Compound 1) compound as an active ingredient. delete The pharmaceutical composition for external skin according to claim 1, wherein the pharmaceutical composition is a cream, gel, patch, spray, ointment, warning agent, lotion, liniment, pasta, or cataplasma formulation. A cosmetic composition for skin whitening containing a 10-methoxy-leonurine (Compound 1) compound as an active ingredient. The cosmetic composition of claim 4, wherein the cosmetic composition is a lotion, skin, lotion, nutrition lotion, nutrition cream, massage cream, essence, and pack.

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