Artemisinic acid derivative as well as preparation method and application thereof

Abstract

The invention relates to an arteannuic acid derivative as well as a preparation method and application thereof, the arteannuic acid derivative comprises water-soluble organic salt formed by reaction of arteannuic acid or dihydroarteannuic acid and small molecular alkaline organic matter, or water-soluble complex formed by reaction of arteannuic acid or dihydroarteannuic acid and small molecular peptide, or water-soluble complex formed by reaction of arteannuic acid or dihydroarteannuic acid and small molecular peptide. The arteannuic acid or the dihydroarteannuic acid reacts with an alkaline metal compound to form a water-soluble salt. The arteannuic acid derivative provided by the invention solves the problem that arteannuic acid and dihydroarteannuic acid are insoluble in water, has the biological activity of whitening, anti-inflammatory and anti-tumor, and has good research and development prospects.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an artemisinic acid derivative and a preparation method and application thereof. Background Art

[0002] Human skin pigmentation is related to multiple factors such as pigment metabolism, inflammation, and skin barrier damage. It is a very complex process. Pigment metabolism disorders are cross-influenced by human internal factors and external environment, but all these influences will lead to common symptoms-inflammation, and inflammation will aggravate pigment metabolism disorders. Therefore, a single regulation is difficult to solve the pigmentation problem. Different degrees of pigmentation show different symptoms in clinical practice-freckles, chloasma, age spots and pigmentation caused by inflammation. Melanin can prevent human skin, hair and eyes from being damaged by ultraviolet rays, but excessive melanin can cause skin pigmentation to form spots and even melanoma. Tyrosinase is the key rate-limiting enzyme in melanin production. Inhibiting its activity can improve skin diseases related to excess melanin. Substances that inhibit tyrosinase include mercury compounds, hydroquinone, ascorbic acid and glutathione.

[0003] Among them, although mercury compounds have obvious whitening effects, they are highly toxic and easily deposited in the body, which may cause organ failure in the human body. Hydroquinone has serious skin irritation problems. Sulfhydryl compounds such as glutathione and cysteine ​​not only have pungent odors, but also have transdermal absorption problems. Ascorbic acid is easily oxidized in aqueous solution and cannot exert its effect continuously.

[0004] Artemisia annua contains abundant artemisinic acid, which is 8 to 10 times the content of artemisinin, and has pharmacological activities in many aspects such as antibacterial, antipyretic, and anti-tumor, but it is discarded during the extraction process of artemisinin, resulting in serious waste of resources. Studies have shown that artemisinic acid can reduce melanin by inhibiting the expression of the HMG CoA reductase gene, but artemisinic acid has poor water solubility and its activity intensity needs to be further improved.

[0005] Therefore, providing an active substance with excellent freckle-removing and whitening effects and high safety has good application prospects. Summary of the invention

[0006] To solve the above technical problems, the present invention provides an artemisinic acid derivative and its preparation method and application. The artemisinic acid derivative provided by the present invention solves the problem that artemisinic acid is poorly soluble in water, and has excellent whitening and anti-tumor biological activities, and has good research and development prospects.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides an artemisinic acid derivative, the artemisinic acid derivative comprising: a water-soluble organic salt formed by the reaction of artemisinic acid or dihydroartemisinic acid with a small molecule alkaline organic substance, or a water-soluble complex formed by the reaction of artemisinic acid or dihydroartemisinic acid with a small molecule peptide, or a water-soluble salt formed by the reaction of artemisinic acid or dihydroartemisinic acid with an alkaline metal compound;

[0009] The artemisinic acid derivative has the following structure:

[0010]

[0011] Wherein, R is selected from small molecule alkaline organic matter or small molecule peptide, and M is a metal.

[0012] The artemisinic acid derivatives provided by the present invention not only make up for the defect of poor water solubility of artemisinic acid, but also further enhance the biological activity, have excellent tyrosinase inhibition effect, and can inhibit the growth of mouse melanoma cells, have both whitening and anti-tumor activities, are non-toxic to normal cells, are highly safe, and have good research and development prospects.

[0013] Preferably, the small molecule alkaline organic matter includes alkaline amino acids or alkaloids.

[0014] Preferably, the basic amino acid includes lysine, arginine or histidine.

[0015] Preferably, the alkaloid comprises nicotinamide or ligustrazine.

[0016] Preferably, the small molecule peptide comprises glutathione.

[0017] Preferably, the alkaline metal compound comprises a sodium salt, a potassium salt, sodium hydroxide or potassium hydroxide.

[0018] Preferably, the sodium salt comprises any one of sodium carbonate or sodium bicarbonate or a combination of both.

[0019] Preferably, the potassium salt comprises any one of potassium carbonate or potassium bicarbonate or a combination of both.

[0020] Preferably, the artemisinic acid derivative is selected from any one of the following compounds:

[0021]

[0022] In a second aspect, the present invention provides a method for preparing the artemisinic acid derivative according to the first aspect, the preparation method comprising: reacting artemisinic acid or dihydroartemisinic acid with a modifier in water to obtain the artemisinic acid derivative; the modifier is a small molecule alkaline organic substance, a small molecule peptide or an alkaline metal compound.

[0023] Preferably, the molar ratio of artemisinic acid or dihydroartemisinic acid to the modifier is 1:(0.9-1.1), for example, it can be 1:0.92, 1:0.95, 1:0.98, 1:1, 1:1.02, 1:1.05, 1:1.08, etc.

[0024] Preferably, the amount of water is 5-20 times the total mass of artemisinic acid or dihydroartemisinic acid and the modifier, for example, 6, 8, 10, 12, 15, 18, etc.

[0025] Preferably, the reaction temperature is 20-30°C, for example, 22°C, 24°C, 25°C, 26°C, 28°C, etc.

[0026] Preferably, the reaction time is 30-60 s, for example, 35 s, 40 s, 45 s, 50 s, 55 s, etc.

[0027] Preferably, after the reaction is completed, a post-treatment step is further included, and the post-treatment includes filtration and drying.

[0028] Preferably, the drying comprises any one of freeze drying, spray drying or vacuum drying, or a combination of at least two thereof.

[0029] In a third aspect, the present invention provides a use of the artemisinic acid derivative according to the first aspect in a skin care product or medicine.

[0030] The artemisinic acid derivative provided by the present invention is used for preparing skin care products or medicines, and can give the skin care products and medicines excellent freckle-removing, whitening and anti-inflammatory effects.

[0031] Preferably, the cosmetics include essence, facial mask, microneedle, lotion, freeze-dried powder or facial cream.

[0032] Preferably, the mass percentage of the artemisinic acid derivative in the cosmetic is 1-40%, for example, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, etc.

[0033] Preferably, the dosage form of the drug includes tablets, capsules, granules, injections, sprays or films.

[0034] In a fourth aspect, the present invention provides an emulsion comprising one or a combination of at least two of the artemisinic acid derivatives according to the first aspect, a emollient, an emulsifier, a thickener, a stabilizer, a preservative and an antioxidant.

[0035] In a fifth aspect, the present invention provides a freeze-dried powder comprising one or a combination of at least two of the artemisinic acid derivatives according to the first aspect and a humectant.

[0036] In a sixth aspect, the present invention provides a tyrosinase inhibitor, wherein the tyrosinase inhibitor comprises any one or a combination of at least two of the artemisinic acid derivatives according to the first aspect.

[0037] Compared with the prior art, the present invention has at least the following beneficial effects:

[0038] The present invention provides an artemisinic acid derivative, which not only makes up for the defect of poor water solubility of artemisinic acid, but also further improves the biological activity, has excellent tyrosinase inhibition effect, can inhibit the growth of mouse melanoma cells, has whitening, anti-inflammatory and anti-tumor activities, is non-toxic to normal cells, has high safety, and has good research and development prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the result chart of HPLC of artemisinic acid.

[0040] Figure 2 This is the HPLC result chart of dihydroartemisinic acid.

[0041] Figure 3 This is the thin layer chromatography analysis chart in Example 1.

[0042] Figure 4 This is the thin layer chromatography analysis chart in Example 2.

[0043] Figure 5 This is the thin layer chromatography analysis chart in Example 3.

[0044] Figure 6 This is the thin layer chromatography analysis chart in Example 4.

[0045] Figure 7 This is the thin layer chromatography analysis chart in Example 5.

[0046] Figure 8 This is the thin layer chromatography analysis chart in Example 6.

[0047] Fig. 9 This is the thin layer chromatography analysis chart in Example 7.

[0048] Fig.10 This is a schematic diagram of the cytotoxicity experimental results of artemisinic acid and its derivatives.

[0049] Fig.11 This is a schematic diagram of the evaluation results of the whitening activity of artemisinic acid and its derivatives.

[0050] Fig.12 This is a picture of cell culture of artemisinic acid and its derivatives.

[0051] Fig.13 This is a graph showing the determination of the anti-inflammatory factor NO content.

[0052] Fig.14 This is a graph showing the determination of the anti-inflammatory factor IL-6 content.

[0053] Fig.15 This is a chart showing the determination of the content of the anti-inflammatory factor TNF-α. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0055] The reagents, materials and instruments used in the following examples can all be obtained through commercial means.

[0056] Experimental Example 1

[0057] Study on the Quality of Artemisinic Acid

[0058] Artemisinic acid (HPLC ≥ 98%) was dissolved in methanol and chromatographic column C18 (4.6nm*250nm, 5μm) was used at 35°C, UV203 nm, and flow rate of 1.0mL / min. Chromatographic conditions: phase A is acetonitrile; phase B is 0.1% phosphoric acid water; elution program: 0-20min, phase A: 37%, phase B: 63%; 20-25min, phase A: 37-90%, phase B: 63-10%. The results were processed and the purity of artemisinic acid was calculated based on the peak area. The analysis results are shown in Table 1. Figure 1 shown.

[0059] Table 1

[0060] Retention time / min Peak area Peak height Peak area ratio / % 8.016 15.96 1.35 0.34 8.752 8.75 0.61 0.19 9.72 4629.3 333.77 99.3 11.192 7.71 0.5 0.17

[0061] As shown in the table above, based on the peak area, it can be seen that the purity of artemisinic acid is 99.3%, which is greater than 98%.

[0062] Experimental Example 2

[0063] Study on the Quality of Dihydroartemisinic Acid

[0064] Dissolve dihydroartemisinic acid (HPLC ≥ 98%) in methanol and use a C18 (4.6nm*150nm, 5μm) chromatographic column at 35°C, UV203 nm, and a flow rate of 1.0mL / min. Chromatographic conditions: Phase A is acetonitrile; Phase B is 0.1% phosphoric acid water; elution program: 0-15min, Phase A: 56%, Phase B: 44%; 15-18min, Phase A: 56-95%, Phase B: 44-5%. Process the results and calculate the purity of dihydroartemisinic acid based on the peak area. The analysis results are shown in Table 2. Figure 2 shown.

[0065] Table 2

[0066] Retention time / min Peak area Peak height Peak area ratio / % 9.271 690.1396 45.631 98.07

[0067] As shown in the table above, based on the peak area, it can be seen that the purity of artemisinic acid is 98.07%, which is greater than 98%.

[0068] Example 1

[0069] Artemisinic acid nicotinamide (derivative 2) and its preparation method

[0070] The structural formula of artemisinin nicotinamide is as follows:

[0071]

[0072] The preparation method is as follows: add 2.44 g of nicotinamide to 100 mL of distilled water, stir until dissolved, add 4.68 g of artemisinic acid in portions under stirring, react completely under stirring (to form a clear solution), filter, and obtain a clear solution after the reaction of artemisinic acid and nicotinamide. Use a rotary evaporator to evaporate the solvent, and then vacuum dry at 60° C. to obtain a solid, which is ground into powder, namely artemisinic acid nicotinamide (4.3 g, yield 93%).

[0073] The results of thin layer chromatography analysis of derivative 2 are as follows Figure 3 As shown, 1 represents artemisinic acid, 2 represents derivative 2, and 3 represents nicotinamide; developing agent 1 is petroleum ether:ethyl acetate = 2:3, developing agent 2 is petroleum ether:acetone = 5:3, and developing agent 3 is petroleum ether:dichloromethane = 2:1, all of which are volume ratios.

[0074] The results showed that different developing solvent systems all showed that artemisinic acid and nicotinamide reacted to produce derivative 2.

[0075] Example 2

[0076] Artemisinic acid glutathione (derivative 3) and its preparation method

[0077] The structural formula of artemisinic acid glutathione is as follows:

[0078]

[0079] The preparation method is as follows: add 6.14g of glutathione to 100mL of distilled water, stir until dissolved, add 4.68g of artemisinic acid in portions under stirring, react completely under stirring (to form a clear solution), filter, and obtain a clear solution after the reaction of artemisinic acid and glutathione. Use a spray dryer, set the inlet temperature to 125°C, and the outlet temperature to 60°C, to obtain a white powder, namely artemisinic acid glutathione (4.2g, yield 89%).

[0080] Thin layer chromatography analysis of derivative 3 Figure 4 As shown, 1 represents artemisinic acid, 2 represents derivative 3, 3 represents glutathione, and the developing solvent system is petroleum ether: ethyl acetate: methanol: water = 1:4:1:0.2 (volume ratio).

[0081] Example 3

[0082] Artemisinic acid lysine (derivative 5) and its preparation method

[0083] The structural formula of artemisinic acid lysine is as follows:

[0084]

[0085] The preparation method is as follows: add 2.92g of lysine to 100mL of distilled water, stir until dissolved, add 4.68g of artemisinic acid in portions under stirring, react completely under stirring (to form a clear solution), filter, and obtain a clear solution after the reaction of dihydroartemisinic acid and lysine. Use a spray dryer, set the inlet temperature to 135°C, and the outlet temperature to 70°C, to obtain white powder dihydroartemisinic acid lysine (3.9g, yield 83%).

[0086] Thin layer chromatography analysis of derivative 5 Figure 5 As shown, 1 represents artemisinic acid, 2 represents derivative 5, 3 represents lysine, and the developing solvent system is ethyl acetate:methanol:water=1:4:1 (volume ratio).

[0087] Example 4

[0088] Dihydroartemisinic acid nicotinamide (derivative 6) and its preparation method

[0089] The structural formula of dihydroartemisinic acid nicotinamide is as follows:

[0090]

[0091] The preparation method is as follows: 2.44 g of nicotinamide is added to 100 mL of distilled water, stirred until dissolved, 4.73 g of dihydroartemisinic acid is added in portions under stirring, the reaction is completed under stirring (a clear solution is formed), and filtered to obtain a clear solution after the reaction of dihydroartemisinic acid and nicotinamide. The solvent is evaporated using a rotary evaporator, and then spray-dried to obtain a solid, which is ground into powder, namely dihydroartemisinic acid nicotinamide (4.1 g, yield 86%).

[0092] The results of TLC analysis of derivative 6 are shown in Figure 6 As shown, 1 represents dihydroartemisinic acid, 2 represents derivative 6, and 3 represents nicotinamide; the developing solvent system is petroleum ether:ethyl acetate=2:3 (volume ratio).

[0093] Example 5

[0094] Dihydroartemisinic acid glutathione (derivative 7) and its preparation method

[0095] The structural formula of dihydroartemisinic acid glutathione is as follows:

[0096]

[0097] The preparation method is as follows: add 6.14 g of glutathione to 100 mL of distilled water, stir until dissolved, add 4.73 g of dihydroartemisinic acid in portions under stirring, react completely under stirring (to form a clear solution), filter, and obtain a clear solution after the reaction of dihydroartemisinic acid and nicotinamide. Use a rotary evaporator to evaporate the solvent, then vacuum dry to obtain a solid, grind into powder, that is, dihydroartemisinic acid nicotinamide (4.5 g, yield 95%).

[0098] Thin layer chromatography analysis of derivative 7 Figure 7 As shown, 1 represents dihydroartemisinic acid, 2 represents derivative 7, 3 represents glutathione, and the developing solvent system is ethyl acetate:methanol:water=1:4:2 (volume ratio).

[0099] Example 6

[0100] Sodium artemisinate (derivative 1) and its preparation method

[0101] The structural formula of sodium artemisinate is as follows:

[0102]

[0103] In 100mL of distilled water, add 1.6g of sodium bicarbonate, stir until dissolved, add 4.68g of artemisinic acid in portions under stirring, react completely under stirring (to form a clear solution), filter, and obtain a sodium artemisinate solution. Use vacuum low-temperature drying method to dry, freeze at -40°C, vacuum dry, gradually heat to 55°C, and take a total of 16 hours to obtain white porous loose sodium artemisinate (4.5g, yield 96%).

[0104] The results of thin layer chromatography analysis of derivative 1 are as follows Figure 8 As shown, 1 represents artemisinic acid, 2 represents derivative 1; developing agent 1 is petroleum ether: dichloromethane = 2:3, developing agent 2 is petroleum ether: ethyl acetate = 1:1, and developing agent 3 is n-hexane: ethyl acetate = 2:3, all of which are volume ratios.

[0105] The results show that in different developing solvent systems, the two react to produce derivative 1. Since sodium bicarbonate is an inorganic substance, it will not show a peak in thin layer chromatography.

[0106] Example 7

[0107] Potassium dihydroartemisinic acid (derivative 4) and its preparation method

[0108] The structural formula of potassium dihydroartemisinate is as follows:

[0109]

[0110] In 100 mL of distilled water, add 2.00 g of potassium bicarbonate and stir until dissolved. Under stirring, add 4.73 g of dihydroartemisinic acid in portions. The reaction is complete under stirring (a clear solution is formed), and filtered to obtain a potassium dihydroartemisinic acid solution. The solvent is evaporated using a rotary evaporator, and then vacuum dried at 55° C. to obtain a white powder potassium dihydroartemisinic acid (4.6 g, yield 97%).

[0111] The results of thin layer chromatography analysis of derivative 4 are shown in Fig. 9 As shown, 1 is dihydroartemisinic acid, 2 is potassium dihydroartemisinic acid, and the developing solvent is petroleum ether:ethyl acetate = 2:3 (volume ratio).

[0112] Application Example 1

[0113] A liquid preparation (emulsion) and its preparation method

[0114] This application example provides an emulsion, the raw materials for its preparation include: 25g of artemisinic acid derivative, 10g of emollient, 5g of emulsifier, 1.5g of thickener, 0.05g of stabilizer, 0.15g of preservative, 0.01g of antioxidant and 78.28g of water. Among them, the emollient is glycerin, the emulsifier is glyceryl stearate, the thickener is xanthan gum, the stabilizer is EDTA-2 sodium, the preservative is phenoxyethanol, and the antioxidant is sodium pyrosulfite.

[0115] The preparation method of the emulsion comprises: mixing water, a softener, a stabilizer and a preservative uniformly, heating to 80° C., adding an emulsifier while stirring, homogenizing for 8 minutes, adding a thickener while stirring, then lowering the temperature to 35° C., then adding artemisinic acid derivative 2 and an antioxidant, and stirring uniformly to obtain the emulsion.

[0116] Application Example 2

[0117] A solid preparation (lyophilized powder) and its preparation method

[0118] This application example provides a freeze-dried powder, and its preparation raw materials include: 31g of artemisinic acid derivative, 20g of mannitol, 5g of trehalose, and 65g of deionized water.

[0119] The preparation method of the freeze-dried powder comprises: dissolving and mixing the above raw materials evenly, filtering through 0.45 μm and 0.22 μm filter elements respectively, adding the raw materials to a container, plugging and placing the container in a vacuum freeze dryer, pre-freezing at -40°C for 2.5 hours, evacuating to 0.19 mbar, heating and sublimation drying for 26 hours until the water is completely sublimated, and sealing the container under vacuum conditions; when in use, the powder dissolving liquid is sterile water or physiological saline.

[0120] Application Example 3

[0121] A whitening essence and preparation method thereof

[0122] This application example provides a whitening essence, in which the contents of the various components are: EDTA-2Na 0.25%, p-hydroxyacetophenone 0.6%, dipropylene glycol 5%, 1,2-hexanediol 0.4%, sodium hyaluronate 0.1%, panthenol 0.05%, derivatives 25%, and the balance is water.

[0123] The preparation method of the whitening essence comprises: mixing water, dipropylene glycol, EDTA-2Na, 1,2-hexanediol and sodium hyaluronate, heating to 85°C, and stirring at the temperature until the mixture is completely dissolved and uniformly dissolved to obtain an aqueous phase liquid; lowering the temperature to 65°C, adjusting the rotation speed to 220 r / min, and adding parahydroxypropiophenone; when the temperature is lowered to 45°C, adding panthenol and derivative 2, and mixing well to obtain a whitening essence.

[0124] Test Example 1

[0125] Sensitization safety assessment-ADRA test

[0126] Test samples: artemisinic acid, derivatives 1-7

[0127] Test method:

[0128] On the day of the experiment, the positive control and test solution were prepared, with a concentration of 1mmol / L. The positive control was phenylacetaldehyde with a purity of more than 90%. Solvent selection: acetonitrile, water, acetonitrile / water (V:V=1:1), isopropanol, acetone, acetone / acetonitrile (V:V=1:1), and other solvents that do not affect the stability of the peptide. If it still does not dissolve, try to dissolve it in 300μL DMSO and dilute it with 2700μL acetonitrile; or dissolve it in 1500μL DMSO and dilute it with 1500μL acetonitrile. The test substance and the derivative were mixed and reacted in the dark for 24±1h at a temperature of 25±1℃. HPLC determination was performed within 1h after the reaction, and all tests were completed within 30h. The injection bottle should be observed before and after the reaction and whether precipitation occurred should be recorded. When precipitation occurs before the reaction starts, the percentage of derivative consumption cannot be calculated, a positive result is available, and a negative result is uncertain; if precipitation occurs only after the reaction, use low-speed centrifugation at 100-400g to allow the precipitate to accumulate at the bottom of the injection bottle before injection.

[0129]

[0130] Judgment basis:

[0131] (1) When the test substance does not co-elute with either cysteine ​​derivatives or lysine derivatives, the 1:50 cysteine ​​derivative and 1:50 lysine derivative determination models shown in Table 3 are used for determination.

[0132] Table 3

[0133] Mean of NAC and NAL consumption percentages Prediction results 0% ≤ Mean of consumption percentage < 4.9% Negative 4.9%≤mean of consumption percentage≤100% Negative

[0134] (2) When the test substance co-eluted only with the lysine derivative, the 1:50 cysteine ​​derivative model shown in Table 4 was used for determination.

[0135] Table 4

[0136] Mean NAC consumption percentage ADRA prediction results 0% ≤ Mean of consumption percentage < 5.6% Negative 5.6%≤mean of consumption percentage≤100% Negative

[0137] The results of artemisinin acid sensitization are shown in Table 5:

[0138] Table 5

[0139] Sample name NAC consumption NAL consumption Consumption mean Prediction results Artemisinic acid 0.36％ 0％ 0.18％ Negative Dihydroartemisinic acid 0％ 0％ 0％ Negative Derivative 2 0％ 1.18％ 0.59％ Negative Derivative 3 1.08％ 0 0.54％ Negative Derivative 5 7.71％ 0.71％ 4.21％ Negative Derivative 6 0％ 0％ 0％ Negative Derivative 7 2.11％ 1.41％ 1.76％ Negative Derivative 1 4.41％ 0％ 2.2％ Negative Derivative 4 0％ 0％ 0％ Negative

[0140] The test results showed that the sensitization results of artemisinic acid, dihydroartemisinic acid and their derivatives were all negative, indicating that they have high safety.

[0141] Test Example 2

[0142] Determination of cytotoxicity of artemisinic acid and its derivatives by MTT assay

[0143] Test samples: artemisinic acid, derivatives 1-7

[0144] Test method:

[0145] B16F10 cells with good growth status were taken, digested and added with 10% FBS DMEM medium to make a cell suspension, and the cells were cultured at a concentration of 1×10 5 The plates were inoculated with 100 μL of DMSO / mL and cultured in a 37°C, 5% CO2 incubator for 24 h. Artemisinic acid and its derivatives (concentration 200 μM) were then added. A control group and a blank group were set up, with 3 replicates in each group. The plates were cultured for 24 h at 37°C, 5% CO2. 25 μL of MTT was then added for 2-4 h. The culture medium was washed off. DMSO 150 μL / well was added and shaken for 10 min. The absorbance was measured at 570 nm using an enzyme reader. The results are shown in Table 1. Fig.10 , as shown in Table 6.

[0146] Table 6

[0147] sample Cell survival rate / % Artemisinic acid (200 μM) 122.3 Dihydroartemisinic acid (200 μM) 142 Derivative 2 (200 μM) 142.7 Derivative 3 (200 μM) 163.7 Derivative 5 (200 μM) 204.7 Derivative 6 (200 μM) 178 Derivative 7 (200 μM) 166 Derivative 1 (200 μM) 154.7 Derivative 4 (200 μM) 144.3

[0148] Depend on Fig.10 The MTT test results in Table 6 show that the intervention with 6 concentrations of drugs promoted the growth and development of cells, indicating that artemisinic acid and its derivatives in the present invention have no toxic effects on B16F10 cells, thereby proving that the obtained product has low toxicity and its safety is greatly improved.

[0149] Test Example 3

[0150] Tyrosinase inhibitory effect assay

[0151] Test samples: artemisinic acid, derivatives 1-7

[0152] Test method 1:

[0153] L-tyrosine was used as the catalytic substrate of monophenolase. 200 μL of tyrosinase and 20 μL of inhibitors of different concentrations (0 mM, 0.625 mM, 1.25 mM, 2.5 mM, 5 mM, 10 mM) and PBS (blank group) were added to the 96-well plate in sequence. After 10 minutes of incubation at 37°C, 50 μL of L-tyrosine was added; after 5 minutes of reaction, the plate was placed in an ELISA reader and the absorbance was measured at 475 nm. The inhibitor concentration was plotted as the horizontal axis and the inhibition rate as the vertical axis to calculate the IC50. The positive control was α-arbutin, and the results are shown in Table 7.

[0154] Table 7

[0155] sample Inhibition rate / % α-Arbutin (10mM) 65 Derivative 2 (10 mM) 82 Derivative 3 (10 mM) 93 Derivative 5 (10 mM) 72 Derivative 6 (10 mM) 64 Derivative 7 (10 mM) 89 Artemisinic acid (10 mM) 56 Derivative 1 (10 mM) 65 Derivative 4 (10 mM) 51

[0156] From Table 7, it can be seen that derivatives 3 and 7 have excellent tyrosinase inhibitory effects, which are better than α-arbutin, and the formation of derivatives 3 and 7 further improves the water solubility of artemisinic acid and dihydroartemisinic acid, indicating that the combination of glutathione with artemisinic acid and dihydroartemisinic acid is synergistic. In addition, the inhibition rate of derivative 2 is also higher than that of artemisinic acid alone.

[0157] Test Method 2:

[0158] L-DOPA was used as the catalytic substrate of diphenolase. 50 μL L-DOPA and 20 μL inhibitors of different concentrations (0, 0.625 mM, 1.25 mM, 2.5 mM, 5 mM, 10 mM) and PBS (blank group) were added to the 96-well plate. Then 200 μL tyrosinase was added. After reacting at room temperature for 30 minutes, the plate was placed in an ELISA reader and the absorbance was measured at 475 nm. The inhibitor concentration was plotted as the horizontal axis and the inhibition rate as the vertical axis to calculate the IC50. The positive control was α-arbutin, and the results are shown in Table 8.

[0159] Table 8

[0160]

[0161]

[0162] From Table 8, it can be seen that derivatives 3 and 7 have excellent tyrosinase inhibitory effects, which are better than α-arbutin. After the formation of derivatives 3 and 7, the water solubility of artemisinic acid and dihydroartemisinic acid is further improved, indicating that glutathione is synergistic after combining with artemisinic acid and dihydroartemisinic acid. The inhibitory effect did not change after changing the substrate, indicating that the inhibitory effect of the derivatives is stable. This indirectly shows that artemisinic acid, dihydroartemisinic acid and their derivatives can play a whitening role by inhibiting tyrosinase.

[0163] Test Example 4

[0164] Evaluation of the whitening activity of artemisinic acid and its derivatives (cell experiment)

[0165] Test samples: artemisinic acid, derivatives 1-7

[0166] Test method:

[0167] B16F10 cells in the exponential growth phase were obtained, digested with trypsin, and inoculated into 6-well plates at a cell density of 7 × 10 4 / mL, 2mL / well. After overnight adhesion, the culture medium was replaced, and artemisinic acid and its derivatives were treated for 48 hours. Three replicates were made for each sample. The culture medium was discarded from the cultured cells and washed once with PBS. Place the cell culture dish on an ice plate, and 330μL of non-denaturing cell lysis solution (containing 1mM PMSF) was added to each dish. Lyse at 4°C for 20 minutes, and the cells were collected. Centrifuge at 13000r / min for 10 minutes, and the melanin precipitate is at the bottom of the centrifuge tube. Add 330μL NaOH (containing 10% DMSO), vortex for sufficient lysis, and place in an 80°C metal bath for lysis for 2 hours to fully dissolve the melanin precipitate. Vortex to mix, add 200μL of melanin dissolution solution to each well of the 96-well plate, set up 3 replicates, set up a blank well, and measure the OD value at 405nm. The test results are as follows Fig.11 , as shown in Table 9.

[0168]

[0169] Table 9

[0170] sample Relative melanin production / % Blank Group 100 α-Arbutin (100 μg / mL) 60.14 Derivative 2 (200 μM) 78.31 Derivative 3 (200 μM) 81.57 Derivative 5 (200 μM) 83.27 Derivative 6 (200 μM) 103.92 Derivative 7 (200 μM) 92.16 Artemisinic acid (200 μM) 77.10 Derivative 1 (200 μM) 83.26 Derivative 4 (200 μM) 99.34

[0171] Depend on Fig.11 As can be seen from Table 9, artemisinic acid and its derivatives can reduce the melanin content, while dihydroartemisinic acid and its derivatives do not reduce the melanin content. By comparing artemisinic acid and derivatives 1-3, it can be seen that the melanin inhibition effect of the artemisinic acid derivatives 2-3 provided by the present invention is better than that of artemisinic acid and artemisinic acid metal salts, and the above-mentioned derivatives have a lower effective concentration and higher safety. Furthermore, the whitening effect of the derivative 2 provided by the present invention at a concentration of 200μM is not significantly different from that of the positive control α-arbutin. It can be seen that the artemisinic acid derivatives provided by the present invention further enhance the whitening activity on the basis of improving safety.

[0172] Test Example 5

[0173] Evaluation of the whitening activity of artemisinic acid and its derivatives (animal experiment)

[0174] Test samples: artemisinic acid, dihydroartemisinic acid, derivatives 1-5

[0175] Test method:

[0176] The drug was applied to the brown skin of the guinea pigs and the model was established after UVB irradiation (UVB lamp with a wavelength of 310 nm and a total irradiation of 2000 mJ / cm 2After 24 hours of treatment, the back of the guinea pig was shaved and divided into two 2 cm × 2 cm hair removal areas. One area was used as the administration area, and 50 μL of 25 mg / mL artemisinic acid and its derivatives solution was applied with a pipette twice a day; the other area was used as the blank control area, and 50 μL of 1 / 15 M phosphate buffer (pH = 6.8) was applied each time. The hair was shaved with a razor before each administration. After 30 days of continuous administration, the skin tissue was taken, sliced, stained and analyzed for optical density. The optical density / melanocyte area value and optical density / slice area value in the skin slices stained with L-dopa or ammonia silver are shown in Table 10.

[0177] Table 10

[0178]

[0179]

[0180] As can be seen from Table 10, after continuous application of artemisinic acid and its derivative solutions on the skin of guinea pigs for 30 days, the optical density / melanocyte area value and the optical density / section area value in the skin sections stained with L-dopa or ammonia silver were significantly decreased compared with those of the control group, and the statistical data showed significant differences, which indicates that artemisinic acid and its derivatives can effectively inhibit the formation of melanin in the skin, and have a more obvious effect on skin whitening. In addition, derivatives 2-3 have better whitening activity than artemisinic acid or artemisinic acid metal salts, while dihydroartemisinic acid and its derivatives do not effectively inhibit melanin formation.

[0181] Test Example 6

[0182] Experimental study on the effects of artemisinic acid and its derivatives on the growth of mouse melanoma cells

[0183] Test samples: artemisinic acid, dihydroartemisinic acid, derivatives 1-7

[0184] Test method:

[0185] B16F10 cells in the exponential growth phase were obtained, digested with trypsin, and inoculated into 6-well plates at a cell density of 7 × 10 4 / mL, 2mL / well. After overnight adhesion, the culture medium was replaced, and artemisinic acid, dihydroartemisinic acid and its derivatives were treated for 48h. Three replicates were made for each sample. The cultured cells were observed and photographed. The culture medium was discarded and washed once with PBS. The cell culture dish was placed on an ice plate, and 330μL of non-denaturing cell lysis buffer (containing 1mM PMSF) was added to each dish. The cells were lysed at 4℃ for 20min and the cells were collected. Centrifuged at 13000r / min for 10min, and the supernatant in the centrifuge tube was the cell protein. The protein content was detected using the BCA protein content detection kit, 3 replicates were set, a blank well was set, and the OD value was measured at 562nm.

[0186] Test results such as Fig.12 , as shown in Table 11:

[0187] Table 11

[0188]

[0189]

[0190] like Fig.12 As shown, the culture medium of artemisinic acid and derivative 2 is pink in color, indicating that they consume less nutrients, which indirectly indicates that their cell number is reduced, indicating that they can inhibit the growth of mouse melanoma cells. This is also verified by the protein content determination data. Therefore, artemisinic acid and its derivatives have certain anti-tumor effects.

[0191] Test Example 7

[0192] Evaluation of anti-inflammatory activity of artemisinic acid and its derivatives

[0193] Test samples: artemisinic acid, derivatives 1-7

[0194] Test method:

[0195] RAW264.7 cells in logarithmic growth phase with good morphology were selected and inoculated into 24-well plates and incubated in an incubator for 24 hours. A blank control group, LPS-induced stimulation group, positive control group (dexamethasone, DEX) and sample group were set up and incubated in a 37°C, 5% CO2 incubator for 2 hours. Except for the blank control group, LPS was added to each well and incubated in an incubator for 24 hours. The cell supernatant was tested according to the instructions of the ELISA kit and the nitric oxide detection kit, and the contents of TNF-α, IL-6 and NO in the collected cell supernatant were detected respectively. The results are shown in Figure 2. Fig.13 , Fig.14 , Fig.15 , as shown in Table 12.

[0196] Table 12

[0197]

[0198]

[0199] Test results such as Fig.13 , Fig.14 , Fig.15 As shown in Table 12, artemisinic acid and its derivatives have almost no effect on NO and IL-6 targets, but have a high significance on TNF-α targets, and the effect is very good; and the derivatives have a better trend. This result also shows that artemisinic acid has a certain effect in relieving skin inflammation, and it is speculated that it can also assist whitening in the anti-inflammatory pathway.

[0200] Although dihydroartemisinic acid does not reduce melanin, it also has a certain anti-inflammatory effect.

[0201] Test Case 8

[0202] Solubility test

[0203] Test samples: artemisinic acid, dihydroartemisinic acid, derivatives 2-3, 5-7

[0204] The solubility of artemisinic acid, dihydroartemisinic acid, and derivatives 2-3 and 5-7 in water at 25°C was tested by gravimetric method. The test results are shown in Table 13.

[0205] Table 13

[0206] sample Solubility (g / 100g water) Derivative 2 6.5 Derivative 3 10.2 Derivative 5 7.2 Derivative 6 6.3 Derivative 7 9.6 Artemisinic acid ＜0.01 Dihydroartemisinic acid ＜0.01

[0207] It can be seen from the test results that the artemisinic acid derivatives provided by the present invention have high solubility in water, which solves the problem of poor water solubility of artemisinic acid or dihydroartemisinic acid.

[0208] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. An artemisinic acid derivative, characterized in that: The artemisinic acid derivatives include: water-soluble organic salts formed by the reaction of artemisinic acid or dihydroartemisinic acid with small molecular alkaline organic matter, or water-soluble complexes formed by the reaction of artemisinic acid or dihydroartemisinic acid with small molecular peptides, or water-soluble salts formed by the reaction of artemisinic acid or dihydroartemisinic acid with alkaline metal compounds; The artemisinic acid derivative has the following structure: Wherein, R is selected from small molecule alkaline organic matter or small molecule peptide, and M is a metal.

2. The artemisinic acid derivative according to claim 1, characterized in that The small molecule alkaline organic matter includes alkaline amino acids or alkaloids; Preferably, the basic amino acid comprises lysine, arginine or histidine; Preferably, the alkaloid comprises nicotinamide or ligustrazine; Preferably, the small molecule peptide comprises glutathione; Preferably, the alkaline metal compound comprises a sodium salt, a potassium salt, potassium hydroxide or sodium hydroxide.

3. The artemisinic acid derivative according to claim 1 or 2, characterized in that The artemisinic acid derivative is selected from any one of the following compounds:

4. A method for preparing an artemisinic acid derivative according to any one of claims 1 to 3, characterized in that: The preparation method comprises: reacting artemisinic acid or dihydroartemisinic acid with a modifier in water to obtain the artemisinic acid derivative; the modifier is a small molecule alkaline organic matter, a small molecule peptide or an alkaline metal compound.

5. The preparation method according to claim 4, characterized in that: The molar ratio of artemisinic acid or dihydroartemisinic acid to the modifier is 1:(0.9-1.1); Preferably, the amount of water used is 5-20 times the total mass of artemisinic acid or dihydroartemisinic acid and the modifier.

6. The preparation method according to claim 4 or 5, characterized in that: The reaction temperature is 20-30°C; Preferably, the reaction time is 30-60s; Preferably, after the reaction is completed, a post-treatment step is further included, and the post-treatment includes filtering and drying; Preferably, the drying comprises any one of freeze drying, spray drying or vacuum drying, or a combination of at least two of them.

7. Use of the artemisinic acid derivative according to any one of claims 1 to 3 in skin care products or medicines; Preferably, the cosmetics include essence, facial mask, microneedle, lotion, freeze-dried powder or facial cream; Preferably, the mass percentage of the artemisinic acid derivative in the cosmetic is 1-40%; Preferably, the dosage form of the drug includes tablets, capsules, granules, injections, sprays or films.

8. An emulsion, characterized in that The emulsion comprises one or a combination of at least two of the artemisinic acid derivatives according to any one of claims 1 to 3, a softener, an emulsifier, a thickener, a stabilizer, a preservative and an antioxidant.

9. A freeze-dried powder, characterized in that: The freeze-dried powder comprises one or a combination of at least two of the artemisinic acid derivatives according to any one of claims 1 to 3 and a moisturizer.

10. A tyrosinase inhibitor, characterized in that The tyrosinase inhibitor comprises any one or a combination of at least two of the artemisinic acid derivatives according to any one of claims 1 to 3.

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