WO2021073132A1 - 高纯度神经酸在美白方面的应用及其制备方法 - Google Patents
高纯度神经酸在美白方面的应用及其制备方法 Download PDFInfo
- Publication number
- WO2021073132A1 WO2021073132A1 PCT/CN2020/095723 CN2020095723W WO2021073132A1 WO 2021073132 A1 WO2021073132 A1 WO 2021073132A1 CN 2020095723 W CN2020095723 W CN 2020095723W WO 2021073132 A1 WO2021073132 A1 WO 2021073132A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- nervonic acid
- acid
- preparation
- formula
- crude
- Prior art date
Links
- GWHCXVQVJPWHRF-KTKRTIGZSA-N (15Z)-tetracosenoic acid Chemical compound CCCCCCCC\C=C/CCCCCCCCCCCCCC(O)=O GWHCXVQVJPWHRF-KTKRTIGZSA-N 0.000 title claims abstract description 49
- XJXROGWVRIJYMO-SJDLZYGOSA-N Nervonic acid Natural products O=C(O)[C@@H](/C=C/CCCCCCCC)CCCCCCCCCCCC XJXROGWVRIJYMO-SJDLZYGOSA-N 0.000 title claims abstract description 49
- GWHCXVQVJPWHRF-UHFFFAOYSA-N cis-tetracosenoic acid Natural products CCCCCCCCC=CCCCCCCCCCCCCCC(O)=O GWHCXVQVJPWHRF-UHFFFAOYSA-N 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 230000002087 whitening effect Effects 0.000 title claims abstract description 17
- 238000002425 crystallisation Methods 0.000 claims abstract description 17
- 230000008025 crystallization Effects 0.000 claims abstract description 16
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 14
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 10
- 239000002537 cosmetic Substances 0.000 claims abstract description 8
- 239000000203 mixture Substances 0.000 claims abstract description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 24
- 239000002253 acid Substances 0.000 claims description 11
- 239000011347 resin Substances 0.000 claims description 10
- 229920005989 resin Polymers 0.000 claims description 10
- PPBRXRYQALVLMV-UHFFFAOYSA-N Styrene Natural products C=CC1=CC=CC=C1 PPBRXRYQALVLMV-UHFFFAOYSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 230000008099 melanin synthesis Effects 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 8
- 241000219226 Acer truncatum Species 0.000 claims description 5
- NIXOWILDQLNWCW-UHFFFAOYSA-N Acrylic acid Chemical class OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 5
- 239000003957 anion exchange resin Substances 0.000 claims description 5
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 239000013078 crystal Substances 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 5
- 235000013399 edible fruits Nutrition 0.000 claims description 5
- 239000002245 particle Substances 0.000 claims description 5
- 240000002234 Allium sativum Species 0.000 claims description 4
- 240000002791 Brassica napus Species 0.000 claims description 4
- 235000004977 Brassica sinapistrum Nutrition 0.000 claims description 4
- 239000003937 drug carrier Substances 0.000 claims description 4
- 235000004611 garlic Nutrition 0.000 claims description 4
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 4
- 238000009472 formulation Methods 0.000 claims description 2
- 125000003011 styrenyl group Chemical group [H]\C(*)=C(/[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims 1
- XUMBMVFBXHLACL-UHFFFAOYSA-N Melanin Chemical compound O=C1C(=O)C(C2=CNC3=C(C(C(=O)C4=C32)=O)C)=C2C4=CNC2=C1C XUMBMVFBXHLACL-UHFFFAOYSA-N 0.000 abstract description 24
- 230000000694 effects Effects 0.000 abstract description 16
- 230000005764 inhibitory process Effects 0.000 abstract description 12
- 150000001875 compounds Chemical class 0.000 abstract description 7
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 238000004440 column chromatography Methods 0.000 abstract description 4
- 239000000178 monomer Substances 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 238000005325 percolation Methods 0.000 abstract 1
- 102000003425 Tyrosinase Human genes 0.000 description 13
- 108060008724 Tyrosinase Proteins 0.000 description 13
- 238000002835 absorbance Methods 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- 239000000523 sample Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 7
- 241000196324 Embryophyta Species 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 239000006285 cell suspension Substances 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000000199 molecular distillation Methods 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 241000251730 Chondrichthyes Species 0.000 description 2
- 102000004195 Isomerases Human genes 0.000 description 2
- 108090000769 Isomerases Proteins 0.000 description 2
- WTDRDQBEARUVNC-LURJTMIESA-N L-DOPA Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-LURJTMIESA-N 0.000 description 2
- WTDRDQBEARUVNC-UHFFFAOYSA-N L-Dopa Natural products OC(=O)C(N)CC1=CC=C(O)C(O)=C1 WTDRDQBEARUVNC-UHFFFAOYSA-N 0.000 description 2
- 102000004316 Oxidoreductases Human genes 0.000 description 2
- 108090000854 Oxidoreductases Proteins 0.000 description 2
- 230000019522 cellular metabolic process Effects 0.000 description 2
- 229930183167 cerebroside Natural products 0.000 description 2
- 150000001784 cerebrosides Chemical class 0.000 description 2
- 238000000354 decomposition reaction Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000004945 emulsification Methods 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- BJRNKVDFDLYUGJ-RMPHRYRLSA-N hydroquinone O-beta-D-glucopyranoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-RMPHRYRLSA-N 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 210000002510 keratinocyte Anatomy 0.000 description 2
- 230000036564 melanin content Effects 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 230000007433 nerve pathway Effects 0.000 description 2
- 210000000944 nerve tissue Anatomy 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 102000002045 Endothelin Human genes 0.000 description 1
- 108050009340 Endothelin Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 230000037374 absorbed through the skin Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 229960000271 arbutin Drugs 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000036995 brain health Effects 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012059 conventional drug carrier Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005008 domestic process Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- ZUBDGKVDJUIMQQ-UBFCDGJISA-N endothelin-1 Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)NC(=O)[C@H]1NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@@H](CC=2C=CC(O)=CC=2)NC(=O)[C@H](C(C)C)NC(=O)[C@H]2CSSC[C@@H](C(N[C@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N2)=O)NC(=O)[C@@H](CO)NC(=O)[C@H](N)CSSC1)C1=CNC=N1 ZUBDGKVDJUIMQQ-UBFCDGJISA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- -1 fatty acid salt Chemical class 0.000 description 1
- 229940087603 grape seed extract Drugs 0.000 description 1
- 235000002532 grape seed extract Nutrition 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- BEJNERDRQOWKJM-UHFFFAOYSA-N kojic acid Chemical compound OCC1=CC(=O)C(O)=CO1 BEJNERDRQOWKJM-UHFFFAOYSA-N 0.000 description 1
- 229960004705 kojic acid Drugs 0.000 description 1
- WZNJWVWKTVETCG-UHFFFAOYSA-N kojic acid Natural products OC(=O)C(N)CN1C=CC(=O)C(O)=C1 WZNJWVWKTVETCG-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000000874 microwave-assisted extraction Methods 0.000 description 1
- BJRNKVDFDLYUGJ-UHFFFAOYSA-N p-hydroxyphenyl beta-D-alloside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC=C(O)C=C1 BJRNKVDFDLYUGJ-UHFFFAOYSA-N 0.000 description 1
- 229940124531 pharmaceutical excipient Drugs 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229940051841 polyoxyethylene ether Drugs 0.000 description 1
- 229920000056 polyoxyethylene ether Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000000194 supercritical-fluid extraction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
- 150000004670 unsaturated fatty acids Chemical class 0.000 description 1
- 235000021122 unsaturated fatty acids Nutrition 0.000 description 1
- 239000001717 vitis vinifera seed extract Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
- A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
- A61K8/36—Carboxylic acids; Salts or anhydrides thereof
- A61K8/361—Carboxylic acids having more than seven carbon atoms in an unbroken chain; Salts or anhydrides thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/43—Separation; Purification; Stabilisation; Use of additives by change of the physical state, e.g. crystallisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
- C07C51/42—Separation; Purification; Stabilisation; Use of additives
- C07C51/47—Separation; Purification; Stabilisation; Use of additives by solid-liquid treatment; by chemisorption
Definitions
- the invention relates to the fields of whitening agent products and melanin suppression products, in particular to the application of high-purity nervonic acid in whitening and a preparation method thereof.
- Nervonic acid (NA for short) was first discovered in the nerve tissue of mammals. It is an important component of the biofilm in the nerve tissue and a symbolic component of the medulla in cerebrosides.
- Professor Klenk first isolated unsaturated fatty acids with a melting point of 4l°C from the cerebrosides of human and cattle brains, and introduced its molecular formula as C 24 H 46 O 2 .
- Japanese scholars extracted nervonic acid from shark oil and confirmed it as a cis structure for the first time. For this reason, it is also called shark acid. Nerve acid is recognized worldwide as a health food that can repair and dredge the damaged brain nerve pathways. For this reason, the development and utilization of nerve acid products are gradually developed.
- domestic methods for extracting and separating nervonic acid from plants include metal salt precipitation, emulsification separation, fatty acid salt column chromatography, solvent crystallization, molecular distillation, multi-stage molecular distillation purification, and molecular distillation and urea inclusion.
- metal salt precipitation method, emulsification separation method, solvent crystallization method to separate nervonic acid mostly use fats with high nervonic acid content as raw materials, while the content of nervonic acid in plants such as Acer truncatum and Lancai is low, so it is not suitable to directly use these methods.
- Kind of method At present, methods with advanced technology, high extraction rate, and short time are used, such as: extraction enzyme extraction, CO2 supercritical extraction, ultrasonic-assisted extraction, and microwave-assisted extraction.
- Common whitening ingredients include Vc, kojic acid, fruit acid, arbutin, placenta, natural plants and herbs, and endothelin antagonists.
- the mechanism of action includes reduction of melanin; inhibition of melanin formation, promotion of epidermal melanin shedding, and inhibition of TYR , DH ICA oxidase and dopa isomerase activity and other methods inhibit melanin production, block DH I polymerization, repair damaged melanocytes, and maintain their metabolic balance; revitalize and promote cell metabolism, accelerate the decomposition of keratinocytes, etc., However, other health care aspects are not strong.
- the plant-derived nervonic acid of the present invention overcomes many technical problems such as traditional source deficiency, complex process flow and low content, and at the same time, it takes into account the whitening of the product and increases the effect of brain health care at the same time.
- Neural acid inhibits the formation of melanin, promotes the shedding of epidermal melanin, inhibits the production of melanin by inhibiting the activities of TYR, DH ICA oxidase and dopa isomerase, and also has the function of promoting cell metabolism and accelerating the decomposition of keratinocytes, and it is absorbed through the skin Korean dramas have the potential to repair and unblock the damaged brain nerve pathways, and have a broad market prospect.
- the present invention discloses the application of high-purity nervonic acid in whitening and its preparation method.
- the compound has a good inhibitory effect on the synthesis of black in the human body, and has a good penetration effect on the human epidermis.
- This product is derived from plants and is prepared by column chromatography to crystallize monomers with a purity of greater than 95%. It has high safety and no toxic side effects. It is a natural whitening product.
- the pharmaceutical composition comprises a pharmaceutically effective dose of nervonic acid and a pharmaceutically acceptable carrier.
- the pharmaceutically acceptable carrier is an excipient used in the formulation of the composition.
- the pharmaceutical composition or whitening cosmetics is used to eliminate dark spots and spots.
- the content of the nervonic acid in the pharmaceutical composition or whitening cosmetics is 0.1%-99.9%.
- the above-mentioned preparation method of nervonic acid includes the following steps:
- step b) After concentrating the post-column liquid of step a), crystallize at low temperature to obtain high-purity crystals.
- the concentration of dissolved ethanol in step a) is 60-80 v/v%
- the amount of ethanol used is 2-8 times the amount of crude nervonic acid
- the dissolution temperature is 60-80°C.
- the resin in step a) is an anion exchange resin
- the molecular structure is basic skeleton particles of styrene or acrylic acid series.
- the concentration in step b) is 3-4 times the amount of crude proneuronic acid, the crystallization temperature is 0-4°C, and the crystallization time is 6-12 hours.
- the compound of the present invention can be used alone as a melanin production inhibitor, or can be mixed with other compounds, and contains pharmaceutically acceptable pharmaceutical excipients or conventional pharmaceutical carriers. Conventional excipients in the art can be used to discourage
- the active ingredient of the present invention can be used as a melanin production-inhibiting medicine or whitening cosmetics on the premise that it reacts or does not affect the curative effect of the drug of the present invention.
- the preparation method of mixing the inhibitor with other compounds can be prepared by conventional preparation methods in the art.
- the preparation method for mixing inhibitors and other compounds is not limited.
- Neural acid is directly prepared into preparations, or separately or/and adjuvants are mixed to prepare preparations, and then prepared into preparations according to conventional methods in the art.
- the dosage of the pharmaceutical composition of the present invention varies according to the subject of administration and the stage of the disease.
- the compound of the present invention has a good inhibitory effect on the synthesis of black in the human body, and has a good penetration effect when used in the human epidermis.
- the product is derived from plants and has a purity greater than that prepared by column chromatography crystallization. 95% monomer, high safety, no toxic side effects, at the same time, nervonic acid has obvious inhibitory effect on melanin, it is a natural whitening product.
- the preparation of extracting high-purity nervonic acid from crude garlic fruit includes the following steps:
- step b) After concentrating the post-column solution of step a), it is concentrated to 3 times the amount of crude pronervonic acid, the crystallization temperature is 4°C, the crystallization time is 6 hours, the low-temperature crystallization obtains high-purity crystals, and the purity of the obtained nervonic acid is 95.1 %.
- step b) After concentrating the post-column liquid of step a), it is concentrated to 3 times the amount of crude pronervonic acid, the crystallization temperature is 2°C, the crystallization time is 9 hours, the low-temperature crystallization obtains high-purity crystals, and the purity of the obtained nervonic acid is 95.4 %.
- the preparation of extracting high-purity nervonic acid from crude rapeseed includes the following steps:
- step b) After concentrating the post-column liquid of step a), it is concentrated to 4 times the amount of crude pronervonic acid, the crystallization temperature is 0°C, the crystallization time is 12 hours, and the low-temperature crystallization obtains high-purity crystals, and the purity of the obtained nervonic acid is 95.3 %.
- the cultured B16 cells were inoculated at a density of 1 ⁇ 10 4 cells/mL, and the medium was changed after 24 hours. Add medium containing samples of different mass concentrations, and inoculate three copies of each mass concentration.
- the control group replaced the sample solution with RPMI-1640 complete medium. After incubating at 37°C and 5% CO 2 for 72 hours, the supernatant was discarded. Add 1 mL of trypsin digestion solution with a mass concentration of 2.5 g ⁇ L -1 to each well and digest for 5 min at room temperature. Add 4mL RPMI-1640 complete medium to stop the digestion, and pipette into a single cell suspension.
- Inhibition rate [1-(absorbance of sample well/cell density of sample well)/(absorbance of control well/cell density of control well)] ⁇ 100%
- inhibition rate [(AB)-(CD)]/(AB) ⁇ 100%; where A is the mixture of no sample and tyrosinase at 475nm Measured absorbance; B is the absorbance measured at 475nm for the mixed solution without sample and tyrosinase; C is the absorbance measured at 475nm for the mixed solution with sample and tyrosinase; D is the sample with added sample The absorbance measured at 475nm for the mixed solution without tyrosinase.
- the concentration of the cell suspension is (7 ⁇ 8) ⁇ 10 3 cells/well. After being attached for 24 hours, add medium containing samples of different mass concentrations. Set 3 multiple holes for one mass concentration. The control group replaced the sample solution with RPMI-1640 complete medium. After incubating at 37°C and 5% CO 2 for 72 hours, the supernatant was discarded.
- Inhibition rate (1-average absorbance of sample group/average absorbance of control group) ⁇ 100%
- nervonic acid exhibits a strong inhibitory effect on intracellular tyrosinase activity, and it is mass concentration dependent. It can be seen that the inhibitory effect of grape seed extract on melanin synthesis is achieved by reducing the catalytic activity of tyrosinase.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Animal Behavior & Ethology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Emergency Medicine (AREA)
- Birds (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Cosmetics (AREA)
- Medicines Containing Plant Substances (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
一种神经酸在制备用于抑制黑色素生成的药物组合物或抑制黑色素生成的美白化妆品中的应用，所述神经酸的纯度达到95％以上，神经酸的结构式如式(1)所示， (I) 上式为式(1)。该化合物对人体的黑色的合成具有很好的抑制作用，而且用于人体表皮具有很好的渗透作用，其来源于植物，通过柱层析结晶的方式制备出纯度大于95％的单体，安全性高，无毒副作用，是一种天然的美白产品。
Description
本发明涉及美白剂产品、黑色素抑制产品领域，具体涉及高纯度神经酸在美白方面的应用及其制备方法。
神经酸(Nervonic acid，简称NA)最早发现于哺乳动物的神经组织，是神经组织中生物膜的重要组成成分，是脑甙中髓质的标志性成分。1925年，Klenk教授首次从人和牛大脑的脑苷中分离出熔点为4l℃的不饱和脂肪酸，并推出其分子式为C
24H
46O
2。1926年日本学者从鲨鱼油中提取出神经酸，并首次确认为顺式结构，为此又称为鲨鱼酸。神经酸是世界公认的能修复疏通受损大脑神经通路的保健食品，为此逐步开拓神经酸的产品的开发和利用。
过去，国内从植物中提取分离神经酸的方法有金属盐沉淀法、乳化分离法、脂肪酸盐柱层析法、溶剂结晶法、分子蒸馏法及多级分子蒸馏提纯和分子蒸馏与尿素包合法相结合等。其中，金属盐沉淀法、乳化分离法、溶剂结晶法分离神经酸多以神经酸含量高的油脂为原料，而元宝枫、遏蓝菜等植物中神经酸含量较低，不适宜直接采用这几种方法。目前，采用技术先进、提取率高、时间短等优点的方法，如：提取酶法提取、CO2超临界萃取法、超声波辅助提取法、微波辅助萃取法等。
美白常见的成分包括Vc、曲酸、果酸、熊果苷、胎盘素、天然植物及草药、内皮素拮抗剂，其作用机理包括，还原黑色素；抑制黑色素形成、促进表皮黑色素脱落，通过抑制TYR、DH ICA氧化酶及多巴异构酶活性等方式抑制黑色素生成，阻断DH I聚合，修复受损黑素细胞，维持其代谢平衡；赋活并促进细胞新陈代谢，加速角质细胞分解等方面，然而其他保健方面不强。
本发明植物来源的神经酸克服了传统的来源不足，工艺流程复杂及含量低等诸多工艺问题，同时在产品功能性问题上兼顾了美白的同时增加了脑部保健的作用。神经酸通过抑制黑色素形成、促进表皮黑色素脱落，通过抑制TYR、DH ICA氧化酶及多巴异构酶活性等方式抑制黑色素生成，同时具有促进细胞新陈代谢,加速角质细胞分解的功能，而且经皮肤吸收韩剧有具有修复疏通受损大脑神经通路，市场前景广阔。
发明内容
要解决的技术问题：本发明公开了高纯度神经酸在美白方面的应用及其制备方法，该化合物对人体的黑色的合成具有很好的抑制作用，而且用于人体表皮具有很好的渗透作用， 本品来源于植物，通过柱层析结晶的方式制备出纯度大于95％的单体，安全性高，无毒副作用，是一种天然的美白产品。
技术方案：神经酸在制备用于抑制黑色素生成的药物组合物或抑制黑色素生成的美白化妆品中的应用，所述神经酸的纯度达到95％以上，神经酸的结构式如式(1)所示，
上式为式(1)。
优选的，所述药物组合物包含药学上有效剂量的神经酸和药学上可接受的载体。
优选的，所述药学上可接受的载体为组合物中制剂使用的辅料。
优选的，所述药物组合物或美白化妆品用于消除面黑、面斑。
优选的，所述神经酸在药物组合物或美白化妆品中的含量为0.1％-99.9％。
上述的神经酸的制备方法，包括以下步骤：
a)将来源于蒜头果、元宝枫或油菜籽的含量在60-90％的神经酸粗品经过乙醇溶解通过树脂层析，收集柱后液；
b)将步骤a)的柱后液浓缩后，低温结晶获得高纯度晶体。
优选的，步骤a)所述的溶解乙醇的浓度为60-80v/v％，乙醇使用量为神经酸粗品的2-8倍量，溶解温度为60-80℃。
优选的，步骤a)所述的树脂为阴离子交换树脂，分子结构为苯乙烯或者丙烯酸系列的碱性骨架颗粒。
优选的，步骤b)所述的浓缩为原神经酸粗品的3-4倍量，结晶温度为0-4℃，结晶时间为6-12小时。
本发明中的化合物作为黑色素生产抑制剂可以单独使用，也可以与其它化合物混合使用，并含有药学上可接受的药用辅料或常规的药用载体，可采用本领域常规的辅料，以不和本发明活性成分发生反应或不影响本发明药物的疗效为前提，用作抑制黑色素生成的药品或美白化妆品，所述抑制剂与其它化合物混合的制备方法可采用本领域常规的制备方法进行制备。
本发明中，抑制剂与其它化合物混合的制备方法没有限制，神经酸直接做成制剂，或者分别或/和辅料混合后做成制剂，然后按照本领域常规的方法制备成剂。本发明中的药物组合物的 给药剂量根据给药对象及疾病时期不同而改变。
有益效果：本发明中的化合物对人体的黑色的合成具有很好的抑制作用，而且用于人体表皮具有很好的渗透作用，本品来源于植物，通过柱层析结晶的方式制备出纯度大于95％的单体，安全性高，无毒副作用，同时，神经酸对黑色素具有明显的抑制作用，是一种天然的美白产品。
1、样品的制备
1.1从蒜头果粗品中提取高纯度神经酸的制备，包括以下步骤：
a)将来源于蒜头果的含量在60％的神经酸粗品经过80v/v％乙醇溶解通过树脂层析，乙醇使用量为神经酸粗品的8倍量，溶解温度为60℃，收集柱后液，其中树脂为含有苯乙烯或者丙烯酸系列的碱性骨架颗粒的阴离子交换树脂；
b)将步骤a)的柱后液浓缩后，浓缩为原神经酸粗品的3倍量，结晶温度为4℃，结晶时间为6小时，低温结晶获得高纯度晶体，得到神经酸的纯度为95.1％。
1.2从元宝枫粗品中提取高纯度神经酸的制备，包括以下步骤：
a)将来源于元宝枫的含量在70％的神经酸粗品经过70v/v％乙醇溶解通过树脂层析，乙醇使用量为神经酸粗品的6倍量，溶解温度为70℃，收集柱后液，其中树脂为含有苯乙烯或者丙烯酸系列的碱性骨架颗粒的阴离子交换树脂；
b)将步骤a)的柱后液浓缩后，浓缩为原神经酸粗品的3倍量，结晶温度为2℃，结晶时间为9小时，低温结晶获得高纯度晶体，得到神经酸的纯度为95.4％。
1.3从油菜籽粗品中提取高纯度神经酸的制备，包括以下步骤：
a)将来源于油菜籽的含量在90％的神经酸粗品经过60v/v％乙醇溶解通过树脂层析，乙醇使用量为神经酸粗品的2倍量，溶解温度为80℃，收集柱后液，其中树脂为含有苯乙烯或者丙烯酸系列的碱性骨架颗粒的阴离子交换树脂；
b)将步骤a)的柱后液浓缩后，浓缩为原神经酸粗品的4倍量，结晶温度为0℃，结晶时间为12小时，低温结晶获得高纯度晶体，得到神经酸的纯度为95.3％。
2.黑色素含量的测试：
2.1 B16细胞内黑色素含量的测定：
将培养的B16细胞以密度1×10
4个/mL进行接种，24h后换液。添加含有不同质量浓度样品的培养基，每一质量浓度接种三份。对照组以RPMI-1640完全培养基代替样品溶液。37℃，5％CO
2条件下孵育72h后，弃去上清液。每孔加入质量浓度2.5g·L
-1胰蛋白酶消化液1mL于室温下消化5min。加入4mL RPMI-1640完全培养基中止消化，并吹打成单细胞悬液。取0.5 mL作细胞计数，其余细胞悬液于1 500r·min
-1离心10min，弃上清液，加入1mL lmol·L
-1的NaOH溶液，并在90℃水浴中孵育2h后，在酶标仪490nm处测其吸光度。按下列公式计算样品对黑色素合成的抑制率：
抑制率＝[1-(样品孔吸光度/样品孔细胞密度)/(对照孔吸光度/对照孔细胞密度)]×100％
从上表可知，采用神经酸处理细胞后，对细胞内黑色素的抑制率达到45.56％，且随着浓度的升高，抑制能力变强。
2.2体外酪氨酸酶活性测定：
取不同体积的样品(对照组以磷酸缓冲液(PBS)代替)与酪氨酸酶(100U)的混合液，37℃孵育10min，加入0.003mol·L-多巴反应液1mL，继续在37℃孵育2min，立即在475nm处测吸光度。
酪氨酸酶活性抑制率按下列公式计算：抑制率＝[(A-B)-(C-D)]/(A-B)×100％；式中A为未加样品加酪氨酸酶的混合液在475nm处测得的吸光度；B为未加样品和酪氨酸酶的混合液在475nm处测得的吸光度；C为加样品和酪氨酸酶的混合液在475nm处测得的吸光度；D为加样品而未加酪氨酸酶的混合液在475nm处测得的吸光度。
从上表可知，采用酪氨酸酶进行分析，对酪氨酸酶的抑制率达到53.23％，且随着用量的变多的升高，抑制能力变强。
2.3 B16细胞内酪氨酸酶活性的测定：
将培养的B16单细胞悬液将入24孔板中，细胞悬液的浓度为(7～8)×10
3个/孔，待贴壁24h后，添加含有不同质量浓度样品的培养基，每一质量浓度设3个复孔。对照组以RPMI一1640完全培养基代替样品溶液。37℃，5％CO
2条件下孵育72h后，弃去上清液。用pH＝7.4的PBS洗涤2次，每孔加质量分数为1％的辛基酚聚氧乙烯醚(TritonX-100)溶液50uL，迅速置于一80℃冻存30min，随后室温融化使细胞完全破裂，37℃预温5min后加入质量分数为1％的L-DOPA溶液10uL，37℃反应2h，在酶标仪490nm处测其吸光度。按下列公式计算络氨酸 酶活性抑制率：
抑制率＝(1-样品组平均吸光度/对照组平均吸光度)×100％
由上表可知，神经酸表现出较强的细胞内酪氨酸酶活性抑制作用，而且呈质量浓度依赖性。由此可见，葡萄籽提取物对黑色素合成的抑制作用是通过降低酪氨酸酶的催化活性实现的。
Claims (9)
- 神经酸在制备用于抑制黑色素生成的药物组合物或抑制黑色素生成的美白化妆品中的应用，所述神经酸的纯度达到95％以上，神经酸的结构式如式(1)所示，
上式为式(1)。 - 根据权利要求1所述的应用，其特征在于：所述药物组合物包含药学上有效剂量的神经酸和药学上可接受的载体。
- 根据权利要求2所述的应用，其特征在于：所述药学上可接受的载体为组合物中制剂使用的辅料。
- 根据权利要求1所述的应用，其特征在于：所述药物组合物或美白化妆品用于消除面黑、面斑。
- 根据权利要求1所述的应用，其特征在于：所述神经酸在药物组合物或美白化妆品中的含量为0.1％-99.9％。
- 如权利要求1中所述的神经酸的制备方法，其特征在于，包括以下步骤：a)将来源于蒜头果、元宝枫或油菜籽的含量在60-90％的神经酸粗品经过乙醇溶解通过树脂层析，收集柱后液；b)将步骤a)的柱后液浓缩后，低温结晶获得高纯度晶体。
- 根据权利要求4中所述的神经酸的制备方法，其特征在于：步骤a)所述的溶解乙醇的浓度为60-80v/v％，乙醇使用量为神经酸粗品的2-8倍量，溶解温度为60-80℃。
- 根据权利要求4中所述的神经酸的制备方法，其特征在于：步骤a)所述的树脂为阴离子交换树脂，分子结构为苯乙烯或者丙烯酸系列的碱性骨架颗粒。
- 根据权利要求4中所述的神经酸的制备方法，其特征在于：步骤b)所述的浓缩为原神经酸粗品的3-4倍量，结晶温度为0-4℃，结晶时间为6-12小时。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910988526.8A CN110801447A (zh) | 2019-10-17 | 2019-10-17 | 高纯度神经酸在美白方面的应用及其制备方法 |
| CN201910988526.8 | 2019-10-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2021073132A1 true WO2021073132A1 (zh) | 2021-04-22 |
Family
ID=69488717
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2020/095723 WO2021073132A1 (zh) | 2019-10-17 | 2020-06-12 | 高纯度神经酸在美白方面的应用及其制备方法 |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN110801447A (zh) |
| WO (1) | WO2021073132A1 (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110801447A (zh) * | 2019-10-17 | 2020-02-18 | 南京康齐生物科技有限公司 | 高纯度神经酸在美白方面的应用及其制备方法 |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5571503A (en) * | 1995-08-01 | 1996-11-05 | Mausner; Jack | Anti-pollution cosmetic composition |
| JP2007161591A (ja) * | 2005-12-09 | 2007-06-28 | Naris Cosmetics Co Ltd | メラニン産生阻害皮膚外用剤 |
| WO2015019193A2 (en) * | 2013-08-08 | 2015-02-12 | Dalhousie University | Acylated derivatives of phloridzin and isoquercetrin as anticancer therapeutics and methods of use thereof |
| JP2016088857A (ja) * | 2014-10-31 | 2016-05-23 | 二村 芳弘 | アミロイド形成阻害作用を呈するネルボン酸誘導体及びその製造方法 |
| CN108467345A (zh) * | 2018-04-10 | 2018-08-31 | 大理大学 | 一种从蒜头果中提取神经酸的方法及神经酸包合物 |
| CN110801447A (zh) * | 2019-10-17 | 2020-02-18 | 南京康齐生物科技有限公司 | 高纯度神经酸在美白方面的应用及其制备方法 |
-
2019
- 2019-10-17 CN CN201910988526.8A patent/CN110801447A/zh active Pending
-
2020
- 2020-06-12 WO PCT/CN2020/095723 patent/WO2021073132A1/zh active Application Filing
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5571503A (en) * | 1995-08-01 | 1996-11-05 | Mausner; Jack | Anti-pollution cosmetic composition |
| JP2007161591A (ja) * | 2005-12-09 | 2007-06-28 | Naris Cosmetics Co Ltd | メラニン産生阻害皮膚外用剤 |
| WO2015019193A2 (en) * | 2013-08-08 | 2015-02-12 | Dalhousie University | Acylated derivatives of phloridzin and isoquercetrin as anticancer therapeutics and methods of use thereof |
| JP2016088857A (ja) * | 2014-10-31 | 2016-05-23 | 二村 芳弘 | アミロイド形成阻害作用を呈するネルボン酸誘導体及びその製造方法 |
| CN108467345A (zh) * | 2018-04-10 | 2018-08-31 | 大理大学 | 一种从蒜头果中提取神经酸的方法及神经酸包合物 |
| CN110801447A (zh) * | 2019-10-17 | 2020-02-18 | 南京康齐生物科技有限公司 | 高纯度神经酸在美白方面的应用及其制备方法 |
Non-Patent Citations (2)
| Title |
|---|
| WU, LIRONG: "Press Conference (Kunming) on Nervonic Acid Content of Natural Acer Truncatum Oil Product", CHINA OILS AND FATS, vol. 39, no. 8, 31 December 2014 (2014-12-31), pages 68, XP009527442 * |
| YU QING SEY: "Nervonic Acid Content in Acer Palmatum Edible Oil", 13 June 2018 (2018-06-13), XP009527461, Retrieved from the Internet <URL:http://wenda.tianya.cn/question/1cfrssgo02081dlg7vug1a2067c4qibgoai0d> * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110801447A (zh) | 2020-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110433114B (zh) | 抑制酪氨酸酶活性的植物提取物及其应用 | |
| CN109528615B (zh) | 一种凝胶型白及美白面膜及其制备方法 | |
| US20240052390A1 (en) | Fermented supernatant of bangia fusco-purpure and lactobacillus with alpha-glucosidase inhibitory activity and use thereof | |
| CN113384506A (zh) | 一种低刺激性植物酵素美白纳米组合物和制备方法及其应用 | |
| CN115400061A (zh) | 一种人参愈伤组织提取物及其制备方法和应用 | |
| WO2021073132A1 (zh) | 高纯度神经酸在美白方面的应用及其制备方法 | |
| KR101771695B1 (ko) | 스피루리나 추출물을 유효성분으로 함유하는 비만 예방 및 치료용 약학적 조성물 | |
| CN109749941B (zh) | 蝉拟青霉发酵培养基、蝉拟青霉发酵制备麦角甾醇的方法 | |
| KR20140100117A (ko) | 오리나무 추출물 또는 그로부터 분리된 화합물을 포함하는 지방분화 억제용 조성물 | |
| CN111135159B (zh) | 二萜化合物在制备酪氨酸酶抑制剂中的应用 | |
| CN102113993B (zh) | 琼玉方及其制备方法和应用 | |
| KR102218529B1 (ko) | 단삼 생물전환 추출물 및 홍삼 생물전환 추출물을 포함하는 심혈관계 질환 개선, 예방 또는 치료용 조성물 | |
| CN106822331A (zh) | 以锁状多聚体多酚为主的荔枝皮提取物在制备治疗高尿酸血症药物或保健品中的应用 | |
| CN107141330A (zh) | 一组α‑葡萄糖苷酶抑制剂及其应用 | |
| CN111494231B (zh) | 甘草中三种天然成分用作酪氨酸酶抑制剂及用于美白的用途 | |
| CN112022766A (zh) | 一种美白祛痘组合物及其制备方法和应用 | |
| CN112386543A (zh) | 白梅花总黄酮提取物的制备方法及应用 | |
| CN114224800B (zh) | 一种蔷薇属植物提取物及其制备方法、组合物及应用 | |
| WO2020170388A1 (ja) | Ｉ型コラーゲン又はエラスチンの産生促進剤 | |
| KR20070121193A (ko) | 청주 순미주 농축물 및 청주박 추출물 중 어느 하나 이상을함유하는 피부 보호 또는 염증 완화용 조성물 | |
| CN116650380B (zh) | 一种改善皮肤微循环的抗衰老茶叶发酵产品及其制备方法和应用 | |
| CN104771456A (zh) | 拳参提取物在抑制酪氨酸酶活性中的应用 | |
| CN108685782B (zh) | 一种栀子叶提取物的用途 | |
| CN108434047B (zh) | 一种甜罗勒提取物的用途 | |
| Kim et al. | Inhibitory effects of marine algae extract on adipocyte differentiation and pancreatic lipase activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20876519 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 20876519 Country of ref document: EP Kind code of ref document: A1 |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 20876519 Country of ref document: EP Kind code of ref document: A1 |
|
| 32PN | Ep: public notification in the ep bulletin as address of the adressee cannot be established |
Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 17.05.2023) |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 20876519 Country of ref document: EP Kind code of ref document: A1 |