Artemisinic acid derivative, and preparation method therefor and use thereof

Abstract

The present invention relates to an artemisinic acid derivative, and a preparation method therefor and the use thereof. The artemisinic acid derivative comprises: a water-soluble organic salt formed by means of reacting artemisinic acid or dihydroartemisinic acid with a small-molecule basic organic substance, or, a water-soluble complex formed by means of reacting artemisinic acid or dihydroartemisinic acid with a small-molecule peptide, or, a water-soluble salt formed by means of reacting artemisinic acid or dihydroartemisinic acid with a basic metal compound. The artemisinic acid derivative provided by the present invention solves the problem that artemisinic acid and dihydroartemisinic acid are poorly soluble in water, has whitening, anti-inflammatory and anti-tumor biological activities, and thus has good research and development prospects.

Description

Artemisinic acid derivative and preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an artemisinic acid derivative and preparation method and application thereof. BACKGROUND

[0002] Human skin pigmentation is related to many factors such as pigment metabolism, inflammation, and skin barrier damage, and is a very complex process. Pigment metabolism disorder is affected by internal and external environment of human body, but all these influences will cause a common symptom-inflammation, and inflammation will exacerbate pigment metabolism disorder. Therefore, single regulation is difficult to solve the problem of pigmentation. Different degrees of pigment deposition show different symptoms in clinical practice-freckles, chloasma, age spots and pigment deposition caused by inflammation. Melanin can prevent human skin, hair and eyes from being damaged by ultraviolet rays, but excessive melanin can cause skin pigment deposition to form chloasma and even melanoma. Tyrosinase is a key rate-limiting enzyme for melanin production, and inhibition of its activity can improve skin diseases related to excessive melanin. Substances with inhibitory effect on tyrosinase include mercury compounds, hydroquinone, ascorbic acid and glutathione.

[0003] Among them, although the mercury compound has obvious whitening effect, it has high toxicity and is easy to deposit in the body, which may cause internal organ failure of the human body, hydroquinone has a serious problem of skin irritation, and mercapto compounds such as glutathione and cysteine not only have irritating odor, but also have transdermal absorption problem, and ascorbic acid is easy to oxidize in aqueous solution state and cannot continuously play a role.

[0004] Artemisinic acid is rich in artemisia, and its content is 8-10 times that of artemisinin. It has pharmacological activities in many aspects such as antibacterial, antipyretic and antitumor, but it is discarded in the extraction process of artemisinin, causing serious waste of resources. Studies have shown that artemisinic acid can reduce melanin by inhibiting the expression of HMGCoA reductase gene, but artemisinic acid has poor water solubility, and the activity intensity needs to be further improved.

[0005] Therefore, it has good application prospect to provide an active substance with excellent freckle-removing and whitening effect and high safety.

[0006] SUMMARY

[0007] To solve the above technical problems, the present application provides an artemisinic acid derivative and a preparation method and application thereof. The artemisinic acid derivative provided by the present application solves the problem of poor water solubility of artemisinic acid, and has excellent whitening and antitumor biological activity, and has good research and development prospect.

[0008] To achieve this purpose, the following technical solutions are adopted in the present application:

[0009] In a first aspect, the present application provides a derivative of artemisinic acid, which comprises: a water-soluble organic salt formed by reacting artemisinic acid or dihydroartemisinic acid with a small-molecule basic organic compound, or a water-soluble complex formed by reacting artemisinic acid or dihydroartemisinic acid with a small-molecule peptide, or a water-soluble salt formed by reacting artemisinic acid or dihydroartemisinic acid with a basic metal compound.

[0010] The derivative of artemisinic acid has the following structure:

[0011] wherein R is selected from a small-molecule basic organic compound or a small-molecule peptide, and M is a metal.

[0012] The derivative of artemisinic acid provided by the present application not only makes up for the poor water solubility of artemisinic acid, but also further improves the biological activity, has excellent tyrosinase inhibitory effect, can inhibit the growth of mouse melanoma cells, has both whitening and anti-tumor activity, is non-toxic to normal cells, has high safety, and has good research and development prospects.

[0013] Preferably, the small-molecule basic organic compound comprises a basic amino acid or a biogenic alkaloid.

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

[0015] Preferably, the biogenic alkaloid comprises nicotinamide or tetramethylpyrazine.

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

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

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

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

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

[0021] In a second aspect, the present application provides a preparation method of the derivative of artemisinic acid according to the first aspect, which comprises: reacting artemisinic acid or dihydroartemisinic acid with a modifier in water to obtain the derivative of artemisinic acid; and the modifier is a small-molecule basic organic compound, a small-molecule peptide or a basic metal compound.

[0022] Preferably, the molar ratio of the artemisinic acid or dihydroartemisinic acid to the modifier is 1:(0.9-1.1), which can be 1:0.92, 1:0.95, 1:0.98, 1:1, 1:1.02, 1:1.05, 1:1.08, etc.

[0023] Preferably, the amount of water is 5-20 times the total mass of the artemisinic acid or dihydroartemisinic acid and the modifier, which can be 6, 8, 10, 12, 15, 18, etc.

[0024] Preferably, the temperature of the reaction is 20-30℃, which can be 22℃, 24℃, 25℃, 26℃, 28℃, etc.

[0025] Preferably, the time of the reaction is 30-60s, which can be 35s, 40s, 45s, 50s, 55s, etc.

[0026] Preferably, after the reaction is completed, a step of post-treatment is further included, and the post-treatment includes filtration and drying.

[0027] Preferably, the drying includes any one or a combination of at least two of freeze-drying, spray-drying or vacuum drying.

[0028] In a third aspect, the present application provides an application of the artemisinic acid derivative according to the first aspect in a skin care product or a medicine.

[0029] The artemisinic acid derivative provided by the present application is used for preparing a skin care product or a medicine, which can endow the skin care product or the medicine with excellent depigmentation and whitening effects and anti-inflammatory effects.

[0030] Preferably, the cosmetic product includes an essence, a mask, a microneedle, an emulsion, a freeze-dried powder or a cream.

[0031] Preferably, the mass percentage of the artemisinic acid derivative in the cosmetic product is 1-40%, which can be 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, etc.

[0032] Preferably, the dosage form of the medicine includes a tablet, a capsule, a granule, an injection, a spray or a film.

[0033] In a fourth aspect, the present application provides an emulsion, which includes one or a combination of at least two of the artemisinic acid derivatives according to the first aspect, an emollient, an emulsifier, a thickening agent, a stabilizer, a preservative and an antioxidant.

[0034] In a fifth aspect, the present application provides a freeze-dried powder, which includes one or a combination of at least two of the artemisinic acid derivatives according to the first aspect and a humectant.

[0035] In a sixth aspect, the present application provides a tyrosinase inhibitor, which comprises any one of the artemisinic acid derivatives according to the first aspect or a combination of at least two thereof.

[0036] Compared with the prior art, the present application has at least the following beneficial effects:

[0037] The artemisinic acid derivative provided by the present application 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 the activities of whitening, anti-inflammation and anti-tumor, is non-toxic to normal cells, has high safety, and has good research and development prospects. BRIEF DESCRIPTION OF DRAWINGS

[0038] Fig. 1 is an HPLC result diagram of artemisinic acid.

[0039] Fig. 2 is an HPLC result diagram of dihydroartemisinic acid.

[0040] Fig. 3 is a thin layer chromatography analysis diagram in Example 1.

[0041] Fig. 4 is a thin layer chromatography analysis diagram in Example 2.

[0042] Fig. 5 is a thin layer chromatography analysis diagram in Example 3.

[0043] Fig. 6 is a thin layer chromatography analysis diagram in Example 4.

[0044] Fig. 7 is a thin layer chromatography analysis diagram in Example 5.

[0045] Fig. 8 is a thin layer chromatography analysis diagram in Example 6.

[0046] Fig. 9 is a thin layer chromatography analysis diagram in Example 7.

[0047] Fig. 10 is a schematic diagram of the cytotoxicity experiment results of artemisinic acid and its derivatives.

[0048] Fig. 11 is a schematic diagram of the whitening activity evaluation results of artemisinic acid and its derivatives.

[0049] Fig. 12 is a cell culture picture of artemisinic acid and its derivatives.

[0050] Fig. 13 is an anti-inflammatory factor NO content determination diagram.

[0051] Fig. 14 is an anti-inflammatory factor IL-6 content determination diagram.

[0052] Fig. 15 is an anti-inflammatory factor TNF-α content determination diagram. DETAILED DESCRIPTION

[0053] The technical solutions of the present application are further illustrated below in combination with the drawings and through specific embodiments. However, the following examples are merely simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0054] The reagents, materials and instruments used in the following examples can be obtained through commercial means.

[0055] Experimental Example 1

[0056] Quality research of artemisinic acid

[0057] Artemisinic acid (HPLC≥98%) was dissolved in methanol, and a C18 (4.6 nm*250 nm, 5 μm) chromatographic column was used, the temperature was 35°C, UV 203 nm, and the flow rate was 1.0 mL / min. The chromatographic conditions were as follows: the A phase was acetonitrile; the B phase was 0.1% phosphoric acid water; the elution program was as follows: 0-20 min, A phase: 37%, B phase: 63%; 20-25 min, A phase: 37-90%, B phase: 63-10%. The results were processed, the purity of artemisinic acid was calculated according to the peak area, and the analysis results are shown in Table 1 and Figure 1.

[0058] Table 1

[0059] As shown in the above table, according to the peak area, the purity of artemisinic acid is 99.3%, which is greater than 98%.

[0060] Experimental Example 2

[0061] Quality research of dihydroartemisinic acid

[0062] Dihydroartemisinic acid (HPLC≥98%) was dissolved in methanol, and a C18 (4.6 nm*150 nm, 5 μm) chromatographic column was used, the temperature was 35°C, UV 203 nm, and the flow rate was 1.0 mL / min. The chromatographic conditions were as follows: the A phase was acetonitrile; the B phase was 0.1% phosphoric acid water; the elution program was as follows: 0-15 min, A phase: 56%, B phase: 44%; 15-18 min, A phase: 56-95%, B phase: 44-5%. The results were processed, the purity of dihydroartemisinic acid was calculated according to the peak area, and the analysis results are shown in Table 2 and Figure 2.

[0063] Table 2

[0064] As shown in the above table, according to the peak area, the purity of artemisinic acid is 98.07%, which is greater than 98%.

[0065] Example 1

[0066] Artemisinic acid nicotinamide (derivative 2) and a preparation method thereof

[0067] The structural formula of artemisinic acid nicotinamide is as follows:

[0068] The preparation method is as follows: 2.44 g of nicotinamide is added to 100 mL of distilled water, stirred to dissolve, and 4.68 g of artemisinic acid is added in portions under stirring until the reaction is complete (a clear solution is formed). Filtration is performed to obtain a clear solution after the reaction of artemisinic acid and nicotinamide. The solvent is evaporated using a rotary evaporator, and then vacuum drying is performed at 60°C to obtain a solid, which is ground into a powder, i.e. artemisinic acid nicotinamide (4.3 g, a yield of 93%).

[0069] The thin layer chromatography analysis result of derivative 2 is shown in FIG. 3, wherein 1 represents artemisinic acid, 2 represents derivative 2, and 3 represents nicotinamide; the developing agent 1 is petroleum ether: ethyl acetate = 2:3, the developing agent 2 is petroleum ether: acetone = 5:3, and the developing agent 3 is petroleum ether: dichloromethane = 2:1, all of which are volume ratios.

[0070] The results show that different developing agent systems all show that artemisinic acid and nicotinamide react to generate derivative 2.

[0071] Example 2

[0072] Artemisinic acid glutathione (derivative 3) and a preparation method thereof

[0073] The structural formula of artemisinic acid glutathione is as follows:

[0074] The preparation method is as follows: 6.14 g of glutathione is added to 100 mL of distilled water, stirred to dissolve, and 4.68 g of artemisinic acid is added in portions under stirring until the reaction is complete (a clear solution is formed). Filtration is performed to obtain a clear solution after the reaction of artemisinic acid and glutathione. A spray dryer is used, the inlet temperature is set to 125°C, and the air outlet temperature is 60°C, so that a white powder is obtained, i.e. artemisinic acid glutathione (4.2 g, a yield of 89%).

[0075] The thin layer chromatography analysis of derivative 3 is shown in FIG. 4, wherein 1 represents artemisinic acid, 2 represents derivative 3, and 3 represents glutathione, and the developing agent system is petroleum ether: ethyl acetate: methanol: water = 1:4:1:0.2 (volume ratio).

[0076] Example 3

[0077] Artemisinic acid lysine (derivative 5) and a preparation method thereof

[0078] The structural formula of artemisinic acid lysine is as follows:

[0079] The preparation method is as follows: 2.92 g of lysine is added into 100 mL of distilled water, stirred until dissolved, 4.68 g of artemisinic acid is added in portions under stirring, and the reaction is completed (a clear solution is formed) under stirring. Filtration is performed to obtain a clear solution after lysine reacts with artemisinic acid. A spray dryer is used, the inlet temperature is set to 135°C, and the outlet air temperature is set to 70°C, to obtain white powder lysine artemisinic acid (3.9 g, a yield of 83%).

[0080] Thin layer chromatography analysis of derivative 5 is shown in Figure 5, wherein 1 represents artemisinic acid, 2 represents derivative 5, and 3 represents lysine; the developing system is ethyl acetate:methanol:water = 1:4:1 (volume ratio).

[0081] Example 4

[0082] Nicotinamide artemisinic acid (derivative 6) and a preparation method thereof

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

[0084] The preparation method is as follows: 2.44 g of nicotinamide is added into 100 mL of distilled water, stirred until dissolved, 4.73 g of artemisinic acid is added in portions under stirring, and the reaction is completed (a clear solution is formed) under stirring. Filtration is performed to obtain a clear solution after nicotinamide reacts with artemisinic acid. A rotary evaporator is used to evaporate the solvent, and then spray drying is performed to obtain a solid, which is ground into powder, to obtain nicotinamide artemisinic acid (4.1 g, a yield of 86%).

[0085] Thin layer chromatography analysis results of derivative 6 are shown in Figure 6, wherein 1 represents artemisinic acid, 2 represents derivative 6, and 3 represents nicotinamide; the developing system is petroleum ether:ethyl acetate = 2:3 (volume ratio).

[0086] Example 5

[0087] Glutathione artemisinic acid (derivative 7) and a preparation method thereof

[0088] The structural formula of glutathione artemisinic acid is as follows:

[0089] The preparation method is as follows: 6.14 g of glutathione is added into 100 mL of distilled water, stirred until dissolved, 4.73 g of artemisinic acid is added in portions under stirring, and the reaction is completed (a clear solution is formed) under stirring. Filtration is performed to obtain a clear solution after lysine reacts with artemisinic acid. A rotary evaporator is used to evaporate the solvent, and then vacuum pressure drying is performed to obtain a solid, which is ground into powder, to obtain nicotinamide artemisinic acid (4.5 g, a yield of 95%).

[0090] The thin layer chromatography analysis of derivative 7 is shown in Figure 7, wherein 1 represents dihydroartemisinic acid, 2 represents derivative 7, and 3 represents glutathione, and the developing agent system is ethyl acetate:methanol:water = 1:4:2 (volume ratio).

[0091] Example 6

[0092] Sodium artemisinicate (derivative 1) and its preparation method

[0093] The structural formula of sodium artemisinicate is as follows:

[0094] In 100 mL of distilled water, 1.6 g of sodium bicarbonate was added and stirred until dissolved. Under stirring, 4.68 g of artemisinic acid was added in portions and the reaction was completed (a clear solution was formed) under stirring. Filtration was performed to obtain a sodium artemisinicate solution. Vacuum low-temperature drying was performed, i.e., freezing at -40°C, vacuum drying, and gradually increasing the temperature to 55°C, for a total of 16 h, to obtain white porous loose sodium artemisinicate (4.5 g, yield 96%).

[0095] The thin layer chromatography analysis results of derivative 1 are shown in Figure 8, wherein 1 represents artemisinic acid, and 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.

[0096] It can be seen from the results that derivative 1 is generated by the reaction of the two in different developing agent systems. Sodium bicarbonate is an inorganic substance and does not show a peak in thin layer chromatography.

[0097] Example 7

[0098] Potassium dihydroartemisinicate (derivative 4) and its preparation method

[0099] The structural formula of potassium dihydroartemisinicate is as follows:

[0100] In 100 mL of distilled water, 2.00 g of potassium bicarbonate was added and stirred until dissolved. Under stirring, 4.73 g of dihydroartemisinic acid was added in portions and the reaction was completed (a clear solution was formed) under stirring. Filtration was performed to obtain a potassium dihydroartemisinicate solution. The solvent was evaporated using a rotary evaporator, and then vacuum drying was performed at 55°C to obtain white powder potassium dihydroartemisinicate (4.6 g, yield 97%).

[0101] The thin layer chromatography analysis results of derivative 4 are shown in Figure 9, wherein 1 represents dihydroartemisinic acid, and 2 represents potassium dihydroartemisinicate, and the developing agent is petroleum ether:ethyl acetate = 2:3 (volume ratio).

[0102] Application Example 1

[0103] A liquid preparation (emulsion) and a method for preparing the same

[0104] The application example provides an emulsion, and raw materials for preparing the emulsion include: artemisinic acid derivative 25g, emollient 10g, emulsifier 5g, thickening agent 1.5g, stabilizer 0.05g, preservative 0.15g, antioxidant 0.01g and water 78.28g. The emollient is glycerol, the emulsifier is glycerol stearate, the thickening agent is xanthan gum, the stabilizer is EDTA-2 sodium, the preservative is phenoxyethanol, and the antioxidant is sodium pyrosulfite.

[0105] The preparation method of the emulsion comprises the following steps: uniformly mixing water, an emollient, a stabilizer and a preservative, heating to 80 DEG C, then stirring to add an emulsifier, homogenizing for 8 min, then stirring to add a thickening agent, then reducing the temperature to 35 DEG C, then adding artemisinic acid derivative 2 and an antioxidant, and uniformly stirring to obtain the emulsion.

[0106] Application example 2

[0107] A solid preparation (lyophilized powder) and a preparation method thereof

[0108] The application example provides a lyophilized powder, and raw materials for preparing the lyophilized powder include: artemisinic acid derivative 31g, mannitol 20g, trehalose 5g and deionized water 65g.

[0109] The preparation method of the lyophilized powder comprises the following steps: uniformly dissolving and mixing the raw materials, filtering through filter cores with a diameter of 0.45 mu m and 0.22 mu m respectively, then adding the filter cores into a container, sealing the container, then placing the container into a vacuum freeze dryer, pre-freezing at-40 DEG C for 2.5h, vacuumizing to 0.19mbar, heating and sublimating to dry for 26h until water is completely sublimated, and sealing the container under vacuum condition; and when the lyophilized powder is used, a powder dissolving solution is sterile water or normal saline.

[0110] Application example 3

[0111] A whitening essence and a preparation method thereof

[0112] The application example provides a whitening essence, and the content of each component is as follows: EDTA-2Na 0.25%, p-hydroxyphenylacetone 0.6%, dipropylene glycol 5%, 1,2-hexanediol 0.4%, sodium hyaluronate 0.1%, panthenol 0.05%, derivative 2 25%, and the balance is water.

[0113] The preparation method of the whitening essence comprises the following steps: mixing water, dipropylene glycol, EDTA-2Na, 1,2-hexanediol and sodium hyaluronate, heating to 85 DEG C, and uniformly stirring until the water phase liquid is completely dissolved; reducing the temperature to 65 DEG C, adjusting the rotating speed to 220r / min, and then adding p-hydroxyphenylacetone; when the temperature is reduced to 45 DEG C, adding panthenol and derivative 2, and uniformly mixing to obtain the whitening essence.

[0114] Test Example 1

[0115] Sensitization safety evaluation-ADRA experiment

[0116] Test samples: artemisinic acid, derivatives 1-7

[0117] Test method:

[0118] The positive control and test substance solution were prepared on the day of the test, with a concentration of 1 mmol / L, and the positive control was phenylacetaldehyde with a purity of more than 90%. The solvents selected were 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 is still not dissolved, it can be sequentially dissolved in 300 μL DMSO and diluted with 2700 μL acetonitrile; or dissolved in 1500 μL DMSO and diluted with 1500 μL acetonitrile. The test substance and derivative were mixed in the dark for 24 ± 1 h at a temperature of 25 ± 1°C, and HPLC determination was performed within 1 h after the end of the reaction, and all tests were completed within 30 h. The sample bottle needs to be observed and recorded before and after the reaction to see if there is any precipitation or other conditions. If precipitation occurs before the reaction starts, the percentage of derivative consumption cannot be calculated, and the positive result can be used, while the negative result is uncertain; if precipitation occurs only after the reaction, use low-speed centrifugation of 100-400 g to make the precipitate accumulate at the bottom of the sample bottle, and then sample.

[0119] Judgment basis:

[0120] (1) When the test substance does not co-elute with cysteine derivatives and lysine derivatives, use the 1:50 cysteine derivative and 1:50 lysine derivative determination model as shown in Table 3 to determine.

[0121] Table 3

[0122] (2) When the test substance only co-elutes with lysine derivatives, use the 1:50 cysteine derivative model as shown in Table 4 to determine.

[0123] Table 4

[0124] The sensitization results of artemisinic acid are shown in Table 5:

[0125] Table 5

[0126] The test results show that the sensitization results of artemisinic acid, dihydroartemisinic acid and their derivatives are negative, indicating that they have high safety.

[0127] Test Example 2

[0128] MTT assay for the cytotoxicity of artemisinin and its derivatives

[0129] Test samples: Artemisinin, derivatives 1-7

[0130] Test method:

[0131] Take B16F10 cells in good growth condition, digest them, and add 10% FBS to DMEM medium to prepare a cell suspension. Then, arrange the cells at a concentration of 1×10⁻⁶. 5 Artemisinin and its derivatives (200 μM) were inoculated into 96-well plates at 37°C and 5% CO2 for 24 h. Then, artemisinin and its derivatives were added, with a control group and a blank group, each with 3 replicates. The plates were incubated at 37°C and 5% CO2 for another 24 h. Then, 25 μL of MTT was added and the plates were incubated for another 2-4 h. The medium was washed off, and 150 μL of DMSO was added to each well. The plates were shaken for 10 min, and the absorbance was measured at 570 nm using a microplate reader. The results are shown in Figure 10 and Table 6.

[0132] Table 6

[0133] As shown in Figure 10 and Table 6, the MTT assay results indicate that the use of six different drug concentrations promoted cell growth and development, demonstrating that artemisinin and its derivatives in this invention have no toxic effects on B16F10 cells. This proves that the obtained product has low toxicity and its safety has been greatly improved.

[0134] Test Example 3

[0135] Tyrosinase inhibition effect determination

[0136] Test samples: Artemisinin, derivatives 1-7

[0137] Test Method 1:

[0138] Using L-tyrosine as the substrate for monophenolase, 200 μL of tyrosinase and 20 μL of different concentrations of inhibitors (0 mM, 0.625 mM, 1.25 mM, 2.5 mM, 5 mM, 10 mM) and PBS (for the blank control) were added sequentially to 96-well plates. After incubation at 37°C for 10 min, 50 μL of L-tyrosine was added; after reacting for 5 min, the plates were placed in a microplate reader, and the absorbance was measured at 475 nm. The IC50 was calculated by plotting the inhibitor concentration on the x-axis and the inhibition rate on the y-axis. α-Arbutin was used as the positive control. The results are shown in Table 7.

[0139] Table 7

[0140] As can be seen from Table 7, the derivatives 3 and 7 have excellent tyrosinase inhibiting effect, which is superior to that of a- arbutin, and the water solubility of artemisinic acid and dihydroartemisinic acid is further improved after forming the derivatives 3 and 7, indicating that the combination of glutathione and artemisinic acid and dihydroartemisinic acid is synergistic. In addition, the inhibition rate of the derivative 2 is also higher than that of artemisinic acid alone.

[0141] Test method 2:

[0142] L-DOPA was used as the catalytic substrate of diphenolase, and 50 μL of L-DOPA and 20 μL of different concentrations of inhibitors (0, 0.625 mM, 1.25 mM, 2.5 mM, 5 mM, 10 mM) and PBS (blank group added) were sequentially added to a 96-well plate. Then 200 μL of tyrosinase was added, and after 30 min of reaction at room temperature, it was placed in a microplate reader, and the absorbance value was measured at 475 nm. The inhibitor concentration was plotted as the abscissa, and the inhibition rate was plotted as the ordinate to calculate the IC50. The positive control was a- arbutin, and the results are shown in Table 8.

[0143] Table 8

[0144] As can be seen from Table 8, the derivatives 3 and 7 have excellent tyrosinase inhibiting effect, which is superior to that of a- arbutin, and the water solubility of artemisinic acid and dihydroartemisinic acid is further improved after forming the derivatives 3 and 7, indicating that the combination of glutathione and artemisinic acid and dihydroartemisinic acid is synergistic. After changing the action substrate, the inhibitory effect does not change, indicating that the inhibitory effect of the derivative is stable. It is indirectly indicated that artemisinic acid and dihydroartemisinic acid and their derivatives can play a whitening role by inhibiting tyrosinase.

[0145] Test example 4

[0146] Evaluation of whitening activity of artemisinic acid and its derivatives (cell experiment)

[0147] Test sample: artemisinic acid, derivatives 1-7

[0148] Test method:

[0149] B16F10 cells in exponential growth phase were trypsinized, and the cells were inoculated in a 6-well plate at a cell density of 7 x 10 4The concentration of the sample was 200 μg / mL, 2 mL / well. After overnight adhesion, the medium was replaced, and the artemisinic acid and its derivatives were treated for 48 h, with 3 replicate wells for each sample. The cultured cells were discarded, and the medium was washed once with PBS. The cell culture dish was placed on an ice plate, 330 μL of non-denatured cell lysate (containing 1 mM PMSF) was added to each dish, and the cells were lysed at 4°C for 20 min. The cells were collected. Centrifugation was performed at 13,000 r / min for 10 min, and the melanin precipitate was at the bottom of the centrifuge tube. 330 μL of NaOH (containing 10% DMSO) was added, vortexed to facilitate complete lysis, and placed in a 80°C metal bath for 2 h to completely dissolve the melanin precipitate. Vortex mixing was performed, 200 μL of melanin lysate was added to each well of a 96-well plate, 3 replicate wells were set, and a blank well was set. The OD value was measured at 405 nm, and the test results are shown in FIG. 11 and Table 9.

[0150] Table 9

[0151] As can be seen from FIG. 11 and Table 9, artemisinic acid and its derivatives can reduce the content of melanin, and dihydroartemisinic acid and its derivatives do not reduce the content of melanin. By comparing artemisinic acid and derivatives 1-3, it can be seen that the melanin inhibiting effect of artemisinic acid derivatives 2-3 provided by the present application is better than that of artemisinic acid and artemisinic acid metal salt, and the effective concentration of the above-mentioned derivatives is reduced, and the safety is higher. Further, the derivative 2 provided by the present application has no significant difference in whitening effect with the positive control a-arbutin at a concentration of 200 μM. Therefore, the artemisinic acid derivatives provided by the present application further improve the whitening activity on the basis of improving safety.

[0152] Test Example 5

[0153] Evaluation of the whitening activity of artemisinic acid and its derivatives (animal experiment)

[0154] Test sample: artemisinic acid, dihydroartemisinic acid, derivatives 1-5

[0155] Test method:

[0156] The guinea pigs with brown skin were selected for administration, and were modeled by UVB ultraviolet irradiation (UVB ultraviolet lamp tube irradiation wavelength was 310 nm, and the cumulative irradiation total amount was 2000 mJ / cm 2After the above, the guinea pig's back was shaved and divided into two 2cm x 2cm depilated areas, one of which was used as the drug administration area, and 50μL of 25mg / mL artemisinic acid and its derivatives solution was applied to the area each time, twice a day; the other was used as the blank control area, and 50μL of 1 / 15M phosphate buffer (pH = 6.8) was applied to the area each time. The skin was shaved with a razor before each administration, and after 30 days of continuous administration, the skin tissue was taken, sectioned, stained, and subjected to optical density analysis. The results of the optical density / melanocyte area value and the optical density / slice area value in the L-dopa-stained or silver-ammonia-stained skin sections are shown in Table 10.

[0157] Table 10

[0158] As can be seen from Table 10, after 30 days of continuous application of artemisinic acid and its derivatives solution to the guinea pig skin, the optical density / melanocyte area value and the optical density / slice area value in the L-dopa-stained or silver-ammonia-stained skin sections were significantly lower than those of the control group, and the statistical data had significant differences, which indicated that artemisinic acid and its derivatives could effectively inhibit the generation of melanin in the skin, had obvious skin whitening effects, and derivatives 2-3 had better whitening activity than artemisinic acid or artemisinic acid metal salt, while dihydroartemisinic acid and its derivatives did not effectively inhibit the generation of melanin.

[0159] Test Example 6

[0160] Experiment of artemisinic acid and its derivatives on the growth of mouse melanoma cells

[0161] Test samples: artemisinic acid, dihydroartemisinic acid, and derivatives 1-7

[0162] Test method:

[0163] B16F10 cells in the exponential growth phase were trypsinized, and the cells were inoculated in a 6-well plate at a cell density of 7x10 4 After overnight adhesion, the culture medium was replaced, and artemisinic acid, dihydroartemisinic acid, and their derivatives were treated for 48h, with 3 replicate wells for each sample. The cultured cells were observed and photographed. The culture medium was discarded, and the cells were washed once with PBS. The cell culture dish was placed on an ice plate, 330μL of non-denaturing cell lysis solution (containing 1mM PMSF) was added to each dish, and the cells were lysed at 4°C for 20min. The cells were collected. Centrifugation was performed at 13000r / min for 10min, and the supernatant in the centrifuge tube was the cell protein. The protein content was detected using a BCA protein content detection kit, 3 replicate wells were set, a blank well was set, and the OD value was measured at 562nm.

[0164] The test results are shown in Figure 12 and Table 11.

[0165] Table 11

[0166] As shown in Figure 12, the artemisinic acid and derivative 2 culture medium color presents pink, which indicates that it consumes less nutrients, indirectly indicates that the number of cells is reduced, which indicates that it can inhibit the growth of mouse melanoma cells, and the protein content determination data also verifies this, so artemisinic acid and its derivatives have certain anti-tumor effect.

[0167] Test Example 7

[0168] Evaluation of Anti-inflammatory Activity of Artemisinic Acid and Its Derivatives

[0169] Test sample: artemisinic acid, derivatives 1-7

[0170] Test method:

[0171] Logarithmic growth phase, well-shaped RAW264.7 cells were selected and inoculated in a 24-well plate in an incubator for 24 h. Set up a blank control group, an LPS-induced stimulation group, a positive control group (dexamethasone, DEX) and a sample group, incubate in a 37°C, 5% CO2 incubator for 2 h, add LPS to each well except the blank control group, and incubate in the incubator for 24 h. The cell supernatant was detected according to the ELISA kit and nitric oxide detection kit instructions. The contents of TNF-α, IL-6 and NO in the collected cell supernatant were detected, and the results are shown in Figures 13, 14, 15 and Table 12.

[0172] Table 12

[0173] The test results are shown in Figures 13, 14, 15 and Table 12. The anti-inflammatory target points of artemisinic acid and its derivatives have little effect on NO and IL-6 target points, but have high significance and good effect on TNF-α target point; and the derivatives have better trend. This result also shows that artemisinic acid has certain effect on relieving skin inflammation, and it is speculated that it can also assist whitening in the anti-inflammatory pathway.

[0174] Dihydroartemisinic acid and its derivatives will not reduce melanin, but they also have certain anti-inflammatory effect.

[0175] Test Example 8

[0176] Solubility test

[0177] Test sample: artemisinic acid, dihydroartemisinic acid, derivatives 2-3, 5-7

[0178] The solubility of artemisinic acid, dihydroartemisinic acid, derivatives 2-3, 5-7 in water at 25°C was tested by weight method, and the test results are shown in Table 13.

[0179] Table 13

[0180] From the test results, the artemisinic acid derivative provided by the present application has higher solubility in water, which solves the problem of poor water solubility of artemisinic acid or dihydroartemisinic acid.

[0181] The applicant states that the above description is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto. It should be understood by those skilled in the art that any changes or replacements within the technical scope disclosed by the present application can be easily thought of by any person skilled in the art, and all of them fall within the protection scope and disclosure scope of the present application.

Claims

1. An artemisinic acid derivative, characterized in that, The artemisinic acid derivative comprises: a water-soluble organic salt formed by reacting artemisinic acid or dihydroartemisinic acid with a small-molecule basic organic substance, or a water-soluble complex formed by reacting artemisinic acid or dihydroartemisinic acid with a small-molecule peptide, or a water-soluble salt formed by reacting artemisinic acid or dihydroartemisinic acid with a basic metal compound; The artemisinic acid derivative has the following structure: wherein R is selected from a small-molecule basic organic substance or a small-molecule peptide, and M is a metal.

2. The artemisinic acid derivative according to claim 1, characterized in that, The small-molecule basic organic substance comprises a basic amino acid or a biogenic alkaloid. Preferably, the basic amino acid comprises lysine, arginine, or histidine. Preferably, the biogenic alkaloid comprises nicotinamide or tetramethylpyrazine. Preferably, the small-molecule peptide comprises glutathione. Preferably, the basic 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 process for the preparation of a derivative of artesunate 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; and the modifier is a small-molecule basic organic substance, a small-molecule peptide, or a basic 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 is 5-20 times the total mass of artemisinic acid or dihydroartemisinic acid and the modifier.

6. The production method according to claim 4 or 5, characterized by, The reaction temperature is 20-30°C. Preferably, the reaction time is 30-60 s. Preferably, the reaction is followed by a post-treatment step, and the post-treatment comprises filtration and drying. Preferably, the drying comprises any one or a combination of at least two of freeze-drying, spray-drying, and vacuum drying.

7. Use of the artemisinic acid derivative of any one of claims 1-3 in a cosmetic product or a pharmaceutical product. Preferably, the cosmetic product comprises an essence, a mask, a microneedle, a lotion, a lyophilized powder, or a cream. Preferably, the mass percentage of the artemisinic acid derivative in the cosmetic product is 1-40%. Preferably, the dosage form of the pharmaceutical product comprises a tablet, a capsule, a granule, an injection, a spray, or a film.

8. An emulsion characterized in that, The lotion comprises one or a combination of at least two of the artemisinic acid derivatives of any one of claims 1-3, an emollient, an emulsifier, a thickening agent, a stabilizer, a preservative, and an antioxidant.

9. A lyophilized powder characterized in that, The lyophilized powder comprises one or a combination of at least two of the artemisinic acid derivatives of any one of claims 1-3 and a humectant.

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

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