Purification method and application of vitamin K2

Abstract

The invention provides a purification method of vitamin K2, which comprises the following steps: dissolving vitamin K2 to be purified in a mixed solvent, and cooling and crystallizing, so that the purity of the obtained vitamin K2 is greater than 99.9%. The high-purity vitamin K2 is used for preparing medicines or cosmetics with a skin protecting or repairing function, such as skin care products for fading pigmentation, and compared with the low-purity vitamin K2, the high-purity vitamin K2 has a remarkable effect.

Description

Technical Field

[0001] The invention relates to a method for purifying vitamin K2 and application thereof, and belongs to the technical field of vitamin K2 purification. Background Art

[0002] The vitamin K family includes several fat-soluble 2-methyl-1,4-naphthoquinone derivatives. Natural vitamin K includes vitamin K1 (i.e. phylloquinone) and vitamin K2 (i.e. menaquinones), which are characterized by a lipophilic side chain at position 3 of the 2-methyl-1,4-naphthoquinone ring structure. The side chain of phylloquinone is an unsaturated hydrocarbon chain of a certain length, while the side chain of menaquinone is an unsaturated hydrocarbon chain of an uncertain length (vitamin K1, i.e. phylloquinone, is a compound, and vitamin K2 is a series of compounds). Vitamin K2 (menaquinone-7) is a type of vitamin K2, i.e., vitamin K2 contains 7 isoprene units on its side chain. It is a fat-soluble vitamin and one of the important vitamins that are indispensable to the human body.

[0003] Effects of trace impurities on drug efficacy: Li, N. (2014). Overview of the research on drug impurity. The Journal of Pharmaceutical Practice ., Thakur, A., Mishra, B., & Mahata, P. (2015). Pharmaceutical impurities: A Review. International Journal of Pharmaceutical Chemistry, 5, 232-239. and Swain, S., Sahu, P., & Manoharbabu, S. (2015). An Overview of Pharmaceutical Impurities and Degradation Products. Trace impurities in drugs have a significant impact on their efficacy and safety. With the emphasis on impurity control in drug development, researchers are paying more and more attention to the analysis and control of trace impurities. Impurities affect drug stability and efficacy: Impurities can affect the stability and efficacy of drugs, so controlling impurities is crucial for drug quality, safety and efficacy. Sahu, P., Swain, S., & Manoharbabu, S. (2015). Pharmaceutical Impurities and Degradation Products: An Overview. Literature shows that impurity residues may cause side effects and adverse effects of drug treatment.

[0004] The limits of impurities are formulated according to the relevant requirements of ICH (International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use). For example, ICH requires that for new APIs, when the maximum daily dose is greater than 2 g, the threshold for the definition of a single impurity is 0.05%; when the maximum daily dose is not greater than 2 g, the threshold for the definition of impurities is 0.1% or 1 mg (determined according to strict requirements). When the maximum daily dose of an API is 1.5 g, if calculated according to the definition threshold of 0.1%, the absolute intake of impurities is 1.5 g × 0.1% = 1.5 mg, which is greater than the definition threshold of 1 mg; if calculated according to the threshold of 1 mg, the limit value of impurities is 1 mg ÷ 1.5 g × 100% = 0.07%, which is inconsistent with the definition threshold (0.1%). In this case, the principle of formulating the limits of impurities is "determined according to strict requirements", that is, the limit of impurities should be set at 0.07%. Of course, ICH is only a general requirement. For special impurities (with certain activity) or when the impurity limits exceed the requirements of ICH, relevant safety studies should be conducted or based on literature data to judge the rationality of the limits.

[0005] Therefore, it is particularly important to develop high-purity vitamin K2. In the prior art, Ma Guoliang et al. established a high-purity VK2 green separation and purification process. VK2 in the dried bacteria of Bacillus natto was extracted using organic reagents such as 0, methanol, and ethanol. The results showed that the extraction effect of medium-chain triglycerides was the best, with a maximum extraction amount of 2.7 mg / g. By using 7 different types of macroporous resins to separate VK2 in the medium-chain triglyceride solvent phase, and using static adsorption and desorption experiments, the optimal macroporous resin HC-200S was screened out. The dynamic adsorption and desorption process of the macroporous resin was further optimized, and the dynamic desorption curve of the macroporous resin was drawn. The results showed that when the adsorption flow rate of the sample solution was 2.0 mL / min, the desorbent was a mixed solution of anhydrous ethanol / butyl acetate (1:2, volume ratio), and the desorbent flow rate was 1.0 mL / min, the HC-200S macroporous resin had the best adsorption and desorption effect on VK2; the purity of VK2 increased by 4 times compared with that before purification, reaching 87.8%, and the recovery rate reached 93.1%. The VK2 desorption liquid obtained by resin separation and purification was subjected to frozen crystallization and solvent evaporation crystallization to obtain VK2 crystals with purities of 96.7% and 97.1%, respectively. CN114315549 invention provides a method for extracting vitamin K2, which obtains high-purity vitamin K2 through a carbon dioxide supercritical extraction process, and the purity of the extracted vitamin K2 reaches up to 95%. Chinese invention patent CN113308500 relates to a method for extracting vitamin K2 from a fermentation broth, wherein the crude vitamin K2 product is mixed with acetone again for extraction, and then clean water is added. After the precipitation is completed, the precipitate is separated to obtain a vitamin K2 product with a maximum content of 86.3%. Patent CN110041184B discloses a method for purifying vitamin menaquinone-7, which comprises: extracting MK-7 from dry bacteria with a solvent, purifying the extract by adsorption with a macroporous resin, purifying with a chromatographic filler, and crystallizing with a solvent to obtain a high-purity MK-7 pure product (the purity reaches 99%, but there is still a large gap with 99.9%). The method of the present invention solves the problem that the existing process fillers cannot be reused and the production cost is high, and is convenient for industrial production. Patent CN106631748A discloses a method for separating and purifying vitamin K2 from Bacillus natto. The natto Bacillus fermentation liquid is centrifuged to obtain the bacterial cells, dried, and then extracted with solvents. After the extract is evaporated to dryness, it is redissolved with a polar solvent, purified by adsorption through a macroporous resin, purified by a molecular weight exclusion chromatography column, and finally eluted with solvents, and then separated by a reversed-phase silica gel column. Finally, high-purity vitamin K2 is obtained by crystallization. The above patents all require silica gel purification technology to obtain high-purity MK-7 (purity 98%). Since vitamin K2 contains a large amount of impurities, its content still does not reach 99%, and contains other impurities such as trans-MK-7, MK-9, MK-6 and MK-8, which affect the product effect and performance.

[0006] Therefore, there is an urgent need for a purification method that can increase the content of vitamin K2 to more than 99.9%. Summary of the invention

[0007] In order to solve the above-mentioned defects and shortcomings in the prior art, the present invention provides a method for purifying vitamin K2, which can make the purity of vitamin K2 reach 99.9% or above, and the obtained high-purity vitamin K2 is used in the preparation of medicines and cosmetics that stimulate the expression of keratinocyte heme oxygenase-1 gene.

[0008] To solve the above technical problems: The first object of the present invention is to provide a method for purifying vitamin K2, comprising the following steps: Add the vitamin K2 to be purified into the mixed solvent under light-proof conditions, heat to 55-60°C until the vitamin K2 to be purified is completely dissolved, cool to a certain temperature, recrystallize, and filter to obtain preliminarily purified vitamin K2; Repeat the above steps to obtain vitamin K2 with a purity of not less than 99.9%; The mixed solvent includes normal alkanes, amino acid type oils and lower alcohols.

[0009] Furthermore, the volume ratio of the n-alkane to the amino acid type oil is 9:1 to 1:9, preferably, the volume ratio of the n-alkane to the amino acid type oil is 4:1 to 3:2, more preferably, the volume ratio of the n-alkane to the amino acid type oil is 3:1; the volume ratio of the n-alkane to the lower alcohol is 1:9 to 1:1, preferably, the volume ratio of the n-alkane to the lower alcohol is 1:4 to 3:7, and optimally, the volume ratio of the n-alkane to the lower alcohol is 1:3.

[0010] Furthermore, the certain temperature is -5°C to 20°C, preferably, 0 to 15°C, more preferably, 5 to 10°C, and most preferably, 10°C.

[0011] Furthermore, the n-hexane is any one of n-pentane, n-hexane, n-heptane or n-octane.

[0012] Furthermore, the amino acid oil is any one of lauroyl sarcosine methyl ester, lauroyl sarcosine ethyl ester, lauroyl sarcosine isopropyl ester, lauroyl alanine methyl ester, lauroyl alanine ethyl ester or lauroyl alanine isopropyl ester, preferably, lauroyl sarcosine methyl ester, lauroyl sarcosine ethyl ester or lauroyl sarcosine isopropyl ester, more preferably, lauroyl sarcosine isopropyl ester.

[0013] Furthermore, the lower alcohol is any one of methanol, ethanol, n-propanol, isopropanol, n-butanol or tert-butanol.

[0014] Furthermore, the purity of the vitamin K2 to be purified is >99.0% after purification.

[0015] The second object of the present invention is to provide the use of vitamin K2 obtained by the above-mentioned purification method in the preparation of medicines and cosmetics that stimulate the expression of keratinocyte heme oxygenase-1 gene. Apply to the skin locally to improve local blood circulation and protect skin cells. As a scheme, it can be used for personal care or local cosmetics, i.e. the outer surface of human skin. It can be classified as wash-free or rinse-off, and includes any form of product applied to the human body to improve appearance, cleaning, odor control or general aesthetics. The product form can be liquid, emulsion, cream, foam, stick, serum, essence or gel, etc., preferably, a long-acting gel, emulsion, essence or cream, more preferably in the form of an emulsion or cream.

[0016] Furthermore, the medicine or cosmetic can be delivered in the form of cream, lotion, gel or aqueous formulation, preferably in the form of cream, and used in combination with other raw materials of the medicine or cosmetic.

[0017] The beneficial technical effects achieved by the present invention are as follows: the present invention provides a method for purifying vitamin K2, wherein the purity of the obtained vitamin K2 is greater than 99.9% by dissolving the vitamin K2 to be purified in a mixed solvent and then cooling and crystallizing, and the purification process is simple and mild. High-purity vitamin K2 is used in the preparation of medicines or cosmetics with protective or repairing functions for the skin, such as skin care products for lightening pigmentation, and has a significant effect compared with vitamin K2 with lower purity.

[0018] It should be noted that the "skin" mentioned in the present invention refers to the skin of the neck, chest, back, arms, armpits, hands, legs and scalp, especially the exposed parts thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The effects of the embodiments of the present invention on the expression of heme oxygenase-1 gene in keratinocytes. DETAILED DESCRIPTION

[0020] The present invention will be further described below in conjunction with specific examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention.

[0021] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0022] The reagents used in this application are as follows: Vitamin K2 (MK-7, produced by Nanjing Shengde Baitai); n-hexane (analytical grade, 98%, Nanjing Chemical Reagent); lauroyl sarcosine isopropyl ester (Evertouch™ LPS-100, produced); lauroyl sarcosine methyl ester (self-made), lauroyl alanine ethyl ester (self-made); methanol (analytical grade, 99%, Nanjing Chemical Reagent); ethanol (analytical grade, 99%, Nanjing Chemical Reagent).

[0023] Lauroyl sarcosine methyl ester can be prepared by the existing preparation method. As a preferred embodiment, the specific process is as follows: 10g of lauroyl sarcosine, 200mL of methanol and 2g of concentrated sulfuric acid are mixed, heated to reflux for 6 hours, neutralized with sodium bicarbonate, concentrated, washed with water, and dried to obtain 9.5g of oily lauroyl sarcosine methyl ester.

[0024] Lauroyl alanine ethyl ester can be prepared by the existing preparation method. As a preferred embodiment, the specific process is as follows: 10g of lauroyl alanine, 100mL of anhydrous ethanol and 30mL of cyclohexane are mixed, 1g of concentrated sulfuric acid is heated to reflux for dehydration for 8 hours, neutralized with sodium bicarbonate, concentrated, washed with water, and dried to obtain 10.5g of oily lauroyl alanine ethyl ester.

[0025] Example 1

[0026] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, isopropyl lauroyl sarcosinate, and ethanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 8.4 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.99%.

[0027] Example 2

[0028] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, lauroyl sarcosine methyl ester, and ethanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 8.7 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.95%.

[0029] Example 3

[0030] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, isopropyl lauroyl alanine, and ethanol (volume ratio of 10:1:30), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 7.9 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.96%.

[0031] Example 4

[0032] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, ethyl lauroyl alanine, and ethanol (volume ratio of 10:1:30), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 5° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 9.1 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.91%.

[0033] Example 5

[0034] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, isopropyl lauroyl sarcosinate, and isopropyl alcohol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 7.8 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.99%.

[0035] Example 6

[0036] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, isopropyl lauroyl sarcosinate and methanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until the solution is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 7.2 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.93%.

[0037] Example 7

[0038] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-pentane, isopropyl lauroyl sarcosinate and ethanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 8.6 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.90%.

[0039] Example 8

[0040] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-heptane, isopropyl lauroyl sarcosinate and ethanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 9.0 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.93%.

[0041] Example 9

[0042] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-pentane, isopropyl lauroyl sarcosinate, and ethanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 7.5 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.94%.

[0043] Example 10

[0044] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-octane, isopropyl lauroyl sarcosinate, and ethanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 8.2 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.96%.

[0045] Embodiment 11

[0046] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, isopropyl lauroyl sarcosinate, and n-propanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 0° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 7.1 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.91%.

[0047] Example 12

[0048] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, isopropyl lauroyl sarcosinate and n-butanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 20° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 7.6 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.93%.

[0049] Embodiment 13

[0050] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, isopropyl lauroyl sarcosinate, and tert-butyl alcohol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to -5° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 8.3 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.93%.

[0051] Embodiment 14

[0052] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, ethyl lauroyl alanine, and ethanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 7.8 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.96%.

[0053] Embodiment 15

[0054] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, lauroyl alanine methyl ester, and ethanol (volume ratio of 3:1:9), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 8.7 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.96%.

[0055] Example 16

[0056] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, lauroyl alanine methyl ester, and ethanol (volume ratio of 9:1:81), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 6.9 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.89%.

[0057] Embodiment 17

[0058] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, lauroyl alanine methyl ester, and ethanol (volume ratio of 1:9:1), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 7.9 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.98%.

[0059] Embodiment 18

[0060] A method for extracting vitamin K2: under light-proof conditions, 10 g of vitamin K2 (MK-7 content of 95.21%) is added to 60 mL of a mixed solution of n-hexane, lauroyl alanine methyl ester, and ethanol (volume ratio of 4:1:16), the temperature is raised to 55-60° C. until it is completely dissolved, the temperature is lowered to 10° C. for crystallization, the solution is filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 8.5 g of vitamin K2. The MK-7 content in the vitamin K2 is detected by referring to the method recorded in the United States Pharmacopoeia USP42: 99.95%.

[0061] Test Example 1 Stability Test of High Purity Vitamin K2 1. Room temperature stability test: The vitamin K2 obtained in Examples 1-6 and Comparative Examples 1-4 was subjected to a room temperature stability study. The test conditions were: temperature 25°C, humidity 60%, and the test period was 30 days, 90 days, and 180 days. Sampling was performed for content testing. The test method was based on the United States Pharmacopoeia USP42 (the same below). The results are shown in Table 1.

[0062] Among them, Comparative Example 1: Under light-proof conditions, 10g of vitamin K2 (MK-7 content of 95.21%) was added to 60mL of a mixed solution of n-hexane and ethanol (volume ratio of 1:3), the temperature was raised to 55-60°C until it was completely dissolved, the temperature was lowered to 10°C for crystallization, the solution was filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 8.7g of vitamin K2, with a content of 97.51%.

[0063] Comparative Example 2: Under light-proof conditions, 10 g of vitamin K2 (MK-7 content: 95.21%) was added to 60 mL of a mixed solution of n-hexane and ethanol (volume ratio: 1:4), heated to 55-60°C until completely dissolved, cooled to 10°C for crystallization, filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 9.0 g of vitamin K2, content: 96.78%.

[0064] Comparative Example 3: Under light-proof conditions, 10 g of vitamin K2 (MK-7 content: 95.21%) was added to 60 mL of a mixed solution of n-hexane and isopropanol (volume ratio: 1:3), heated to 55-60°C until completely dissolved, cooled to 10°C for crystallization, filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 8.6 g of vitamin K2, content: 97.68%.

[0065] Comparative Example 4: Under light-proof conditions, 10 g of vitamin K2 (MK-7 content: 95.21%) was added to 60 mL of a mixed solution of n-hexane and methanol (volume ratio: 1:3), heated to 55-60°C until completely dissolved, cooled to 10°C for crystallization, filtered, washed with a small amount of crystallization solvent, and vacuum dried to obtain 7.2 g of vitamin K2, content: 97.79%.

[0066] Table 1

[0067] 2. Accelerated stability testing The investigation conditions were: temperature 40°C, humidity 75%, sampling and testing were carried out at 30 days, 90 days and 180 days respectively. The results are shown in Table 2.

[0068] Table 2

[0069] It can be seen from Table 1 and Table 2 that Examples 1-6 have high stability and no significant changes within 180 days, while Comparative Examples 1-4 and vitamin K2 with an initial yield of 95.21% have obvious changes after 180 days, especially vitamin K2 with the lowest initial yield of 95.21%, in which the content of MK-7 decreased by 7.5%, seriously affecting the stability of the efficacy.

[0070] Experimental Example 2 Effect on heme oxygenase-1 gene expression Keratinocytes were inoculated in a 6-well cell culture plate, and the culture medium was a keratinocyte growth medium containing 0.2% bovine pituitary hormone, 5 μg / mL bovine insulin, 0.18 μg / mL cortisone, 5 μg / ml bovine transferrin and 0.2 ng / mL epidermal growth factor, and cultured for two days at 37°C and 5% CO2. The experiment was divided into a control group and an experimental group, and the culture medium in the control group was replaced with a fresh culture medium (the same as the culture medium of keratinocytes), and the culture medium in the experimental group was replaced with a fresh culture medium containing a DMSO solution of 0.02% of the mass fraction of vitamin K2 MK-7 of Examples 1-6 and Comparative Examples 1-4 (the same as the culture medium of keratinocytes, in which vitamin K2 MK-7 was added), and the culture was continued for 24 hours. Then wash with PBS solution, extract total RNA from cells according to the instructions of the genome extraction kit of Beijing Solebow Technology Co., Ltd., and use it as a template to reverse transcribe into cDNA using reverse transcriptase, and then use cDNA as a template for polymerase chain amplification (PCR amplification kit, Nanjing Novogene Biotech Co., Ltd.), and detect HO-1 gene expression according to the instructions of Human Heme Oxygenase 1 (HO-1) ELISA Kit (Jianglai Biology, catalog number JL10932-96T). Experimental data processing: Calculate the expression of HO-1 mRNA in the experimental group relative to the control group. The larger the value, the more HO-1 gene is expressed. Among them, the expression of the control group is normalized to 1 by the instrument, that is, the expression of the control group is 1. The calculation results are as follows: Figure 1 As shown. Figure 1 It can be seen that the high-purity vitamin K2 of Examples 1-6 has a more significant effect of stimulating the expression of the heme oxygenase-1 gene in keratinocytes than vitamin K2 of normal purity.

[0071] Test Example 3: Effect Detection of Cream Taking eye cream as an example, the efficacy of vitamin K2 obtained in the present application is explained. The ingredient list of the eye cream is shown in Table 3.

[0072] Table 3

[0073] The configuration process of eye cream is as follows: Item A: Add water-soluble raw material preservatives, MGDA, potassium cetyl phosphate, etc. to water and disperse. Using a homogenizer, add carbomer and hydrate it. Then heat the water phase to 70°C.

[0074] Item B: Mix butylene glycol, caprylic / decanoic acid glyceryl, liquid paraffin, dimethicone, isopropyl lauroyl sarcosinate, hexyl alcohol, etc. and heat to 70°C to melt the wax.

[0075] Homogenization: Add item B to item A and mix until emulsified and uniform. Then cool the emulsion to below 35°C with stirring, then add the vitamin K2 prepared in Examples 1-6 and Comparative Examples 1-4, then weigh the product with purified water and stir until uniform.

[0076] The eye skin color difference value test was conducted on different experimental groups. The test principle is as follows: the depth of skin color is determined according to the size of the skin color difference value L*. The larger the L* value, the lighter the skin color, that is, the better the effect of removing dark circles.

[0077] Test method: People with obvious dark circles under the eyes, aged 20-40 years old, were selected and divided into 10 groups, each with 10 people, one of which was a group of subjects using an eye cream that did not contain the dark circle removing composition of the present invention, as the control group. The subjects applied the eye creams of application examples 1-6 and comparative examples 1-4, respectively, once in the morning and evening every day, and continued to use them for 28 days. A facial imager was used to take pictures, and the skin color difference values ​​L* around the eyes on the 0th day and the 28th day were tested and recorded, and the average value of each group was taken. The test results are shown in Table 4 below.

[0078] Table 4

[0079] As can be seen from Table 4, the eye creams of Examples 1-6 have significant color lightening effects, which are about twice the effects of Comparative Examples 1-4.

[0080] In summary, the high-purity vitamin K2 obtained in the present application can stimulate the expression of heme oxygenase-1, thereby improving skin blood circulation and further playing a role in reducing pigmentation.

[0081] The present invention has been disclosed above with preferred embodiments, but they are not intended to limit the present invention. Any technical solutions obtained by adopting equivalent replacement or equivalent transformation solutions fall within the protection scope of the present invention.

Claims

1. A method for purifying vitamin K2, characterized in that: The following steps are involved: Add the vitamin K2 to be purified into the mixed solvent under light-proof conditions, heat to 55-60°C until the vitamin K2 to be purified is completely dissolved, cool to a certain temperature, recrystallize, and filter to obtain preliminarily purified vitamin K2; Repeat the above steps to obtain vitamin K2 with a purity of not less than 99.9%; The mixed solvent includes normal alkanes, amino acid type oils and lower alcohols.

2. The method for purifying vitamin K2 according to claim 1, characterized in that: The volume ratio of the normal alkane to the amino acid type oil is 9:1 to 1:9; the volume ratio of the normal alkane to the lower alcohol is 1:9 to 1:

1.

3. The method for purifying vitamin K2 according to claim 1, characterized in that: The certain temperature is -5℃~20℃.

4. The method for purifying vitamin K2 according to any one of claims 1 to 3, characterized in that: The n-hexane is any one of n-pentane, n-hexane, n-heptane or n-octane.

5. The method for purifying vitamin K2 according to any one of claims 1 to 3, characterized in that: The amino acid oil is any one of lauroyl sarcosine methyl ester, lauroyl sarcosine ethyl ester, lauroyl sarcosine isopropyl ester, lauroyl alanine methyl ester, lauroyl alanine ethyl ester or lauroyl alanine isopropyl ester.

6. The method for purifying vitamin K2 according to any one of claims 1 to 3, characterized in that: The lower alcohol is any one of methanol, ethanol, n-propanol, isopropanol, n-butanol or tert-butanol.

7. The method for purifying vitamin K2 according to any one of claims 1 to 3, characterized in that: The purity of the vitamin K2 to be purified is >99.9% after purification.

8. Use of vitamin K2 obtained by the purification method according to claim 1 in the preparation of medicines and cosmetics for stimulating the expression of keratinocyte heme oxygenase-1 gene.

9. The use according to claim 8, characterized in that: The dosage form of the medicine or cosmetic is a cream, lotion, gel or water dosage form.

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