Clathrate compound containing arteannuic acid as well as preparation method and application of clathrate compound

Abstract

The invention relates to an artemisinic acid-containing clathrate compound as well as a preparation method and application thereof. The clathrate compound containing the artemisinic acid comprises the artemisinic acid and a cyclodextrin compound wrapping the artemisinic acid. The cyclodextrin compound comprises cyclodextrin and / or a cyclodextrin derivative. According to the invention, the arteannuic acid is wrapped by the cyclodextrin compound, so that the water solubility of the arteannuic acid is greatly improved, and the effects of the arteannuic acid in the aspects of inflammation resistance, melanogenesis inhibition and the like can be better exerted. Meanwhile, the cyclodextrin compound and the arteannuic acid have a remarkable synergistic effect in the aspects of resisting inflammation, inhibiting melanogenesis and the like, and the arteannuic acid is clathrated through the cyclodextrin compound, so that the water solubility of the arteannuic acid can be improved, and the self effect of the arteannuic acid can be better exerted; the synergistic effect of the cyclodextrin compound and arteannuic acid in the aspects of resisting inflammation, inhibiting melanogenesis and the like can also be exerted.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a clathrate containing artemisinic acid and a preparation method and application thereof. BACKGROUND

[0002] Artemisinic acid is one of sesquiterpenes in Artemisia annua L. plants, and is an important intermediate in the synthesis of artemisinin and its derivatives. Artemisinic acid has important medicinal and development values due to its suitable chemical conformation, and has multiple biological activities such as anti-malaria, anti-inflammatory and anti-tumor activities, and is particularly famous for its anti-malaria activity. The molecular formula of artemisinic acid is C 15 H 22 O2, and the molecular weight is 234.33, and the CAS number is 80286-58-4. Due to the special sesquiterpene structure, artemisinic acid is insoluble in water, but has a higher solubility in organic solvents such as ethanol, methanol and acetone.

[0003] CN101130088A discloses a supramolecular water-soluble lyophilized product of a poorly soluble drug and a preparation method thereof. The supramolecular water-soluble product is prepared by mixing a poorly soluble drug and a water-soluble cyclodextrin derivative at a weight ratio of 1:1-80 to form a stable supramolecular self-assembly water-soluble product. The low-temperature micronization technology can ensure that the physicochemical properties of the drug before and after crushing are consistent, and the safety of the preparation is improved due to the absence of organic solvents, surfactants and latent solvents.

[0004] CN101125127A discloses a lyophilized preparation of a drug artemisinin derivative and a preparation method thereof. The lyophilized preparation is composed of an artemisinin derivative and a water-soluble cyclodextrin derivative, and the artemisinin derivative contains artemisinin, dihydroartemisinin, artemether and artesunate.

[0005] However, since artemisinic acid is insoluble in water, the raw material is micronized, a solid dispersion is formed, and a proper amount of surfactant is added for solubilization in the prior art, so that artemisinic acid is further processed and applied. At present, there are few reports on improving the solubility of artemisinic acid by forming an artemisinic acid clathrate.

[0006] Therefore, it has become one of the technical problems to be solved to develop a clathrate containing artemisinic acid, improve the water solubility of artemisinic acid, and better exert the efficacy of artemisinic acid. SUMMARY

[0007] In view of the deficiencies in the prior art, the purpose of the present application is to provide a clathrate containing artemisinic acid and a preparation method and application thereof. The clathrate containing artemisinic acid improves the water solubility of artemisinic acid, and can better exert the effects of artemisinic acid in anti-inflammatory and melanin production inhibition.

[0008] To achieve the object of the present application, the present application adopts the following technical solutions:

[0009] In a first aspect, the present application provides a clathrate containing artemisinic acid, which comprises artemisinic acid and a cyclodextrin compound wrapping the artemisinic acid.

[0010] The cyclodextrin compound comprises cyclodextrin and / or cyclodextrin derivative.

[0011] The present application uses cyclodextrin compounds to wrap artemisinic acid, greatly improving the water solubility of artemisinic acid, and better exerting the effects of artemisinic acid in anti-inflammatory, melanin inhibition, etc.

[0012] Meanwhile, the present application creatively finds that cyclodextrin compounds and artemisinic acid have significant synergistic effects in anti-inflammatory, melanin inhibition, etc. By wrapping artemisinic acid with cyclodextrin compounds, not only the water solubility of artemisinic acid can be improved, but also the effects of artemisinic acid itself can be better exerted, and the synergistic effects of cyclodextrin compounds and artemisinic acid in anti-inflammatory, melanin inhibition, etc. can be realized.

[0013] Preferably, the cyclodextrin comprises β-cyclodextrin and / or γ-cyclodextrin.

[0014] Preferably, the cyclodextrin derivative comprises hydroxypropyl-β-cyclodextrin and / or sulfobutyl-β-cyclodextrin sodium.

[0015] Preferably, the average degree of substitution of the hydroxypropyl-β-cyclodextrin is 4.0-9.0, for example, it can be 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, etc.

[0016] Preferably, the average degree of substitution of the sulfobutyl-β-cyclodextrin sodium is 6.2-6.9, for example, it can be 6.2, 6.4, 6.6, 6.8, 6.9, etc.

[0017] Preferably, the cyclodextrin compound comprises hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium.

[0018] The hydroxypropyl-β-cyclodextrin is a β-cyclodextrin whose hydroxyl group is replaced by a hydroxypropyl group, and the sulfobutyl-β-cyclodextrin sodium is a β-cyclodextrin whose 2, 3, 6 hydroxyl groups are replaced by sulfobutyl sodium.

[0019] The present application creatively finds that hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium together as a clathrate agent to wrap artemisinic acid can significantly improve the solubility of artemisinic acid, and the two artemisinic acid derivatives have significant synergistic effects in improving the solubility of artemisinic acid.

[0020] In addition, hydroxypropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin sodium have significant synergies in anti-inflammatory and melanin inhibition, etc., and can improve the anti-inflammatory effect and melanin inhibition effect of cyclodextrin compounds. At the same time, the combination of hydroxypropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin sodium can better exert the synergies between cyclodextrin compounds and artemisinic acid.

[0021] Preferably, the molar ratio of hydroxypropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin sodium is (1-5):(1-5).

[0022] Specific point values in 1-5 can be selected as 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, etc.

[0023] Preferably, the molar ratio of artemisinic acid and cyclodextrin compounds is 1:(7-10), for example, it can be 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, etc.

[0024] In the present application, the molar ratio of artemisinic acid and cyclodextrin compounds is controlled at 1:(7-10), which can better improve the inclusion capacity of cyclodextrin compounds for artemisinic acid, improve the water solubility of artemisinic acid, and further improve the anti-inflammatory and melanin inhibition effects of cyclodextrin compounds and artemisinic acid.

[0025] The structure of the inclusion compound containing artemisinic acid according to the present application is shown in the following figure: Figure 1 As shown in the figure, hydroxypropyl and sulfobutyl sodium independently substitute the hydroxyl groups on C2, C3 and C6 of cyclodextrin to form hydroxypropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin sodium, respectively. The barrel-shaped cyclodextrin derivative (hydroxypropyl-beta-cyclodextrin and sulfobutyl-beta-cyclodextrin sodium) wraps artemisinic acid, and the molar ratio of artemisinic acid and cyclodextrin compounds is 1:(7-10).

[0026] In a second aspect, the present application provides a preparation method of the inclusion compound containing artemisinic acid according to the first aspect, and the preparation method comprises:

[0027] Mixing and stirring cyclodextrin compounds and artemisinic acid in a solvent to obtain a mixed solution; drying the mixed solution to obtain an inclusion compound containing artemisinic acid.

[0028] Preferably, the solvent includes any one or a combination of at least two of water, ethanol or acetone.

[0029] Preferably, the ratio of the total mass of the cyclodextrin compound and artemisinic acid to the volume of the solvent is 1: (10-80) g / mL, for example, it can be 1:10 g / mL, 1:20 g / mL, 1:30 g / mL, 1:40 g / mL, 1:50 g / mL, 1:60 g / mL, 1:70 g / mL, 1:80 g / mL, etc.

[0030] Preferably, the mixing and stirring process further includes adding a pH adjuster.

[0031] Preferably, the pH adjuster includes hydrochloric acid.

[0032] In the present application, during the preparation of the inclusion compound, the addition of hydrochloric acid to adjust the pH of the mixed solution can further improve the inclusion capacity of the cyclodextrin compound for artemisinic acid.

[0033] Preferably, the pH of the solution after adding the pH adjuster is 6.0-7.5, for example, it can be 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.5, etc.

[0034] Preferably, the temperature of the mixing and stirring is 10-60℃, and the time of the mixing and stirring is 2-24h.

[0035] Preferably, the rotation speed of the mixing and stirring is 300-5000 rpm.

[0036] Specific point values in 10-60℃ can be selected as 10℃, 20℃, 30℃, 40℃, 50℃, 60℃, etc., specific point values in 2-24h can be selected as 2h, 5h, 10h, 15h, 20h, 24h, etc., and specific point values in 300-5000 rpm can be selected as 300 rpm, 1000 rpm, 2000 rpm, 3000 rpm, 4000 rpm, 5000 rpm, etc.

[0037] Preferably, the drying method includes spray drying, freeze vacuum drying, or vacuum rotary thin film concentration drying.

[0038] In a third aspect, the present application provides a pharmaceutical preparation, which includes the inclusion compound containing artemisinic acid of the first aspect.

[0039] Preferably, the pharmaceutical preparation further includes an excipient.

[0040] Preferably, the excipient includes any one or a combination of at least two of a pharmaceutically acceptable carrier, a diluent, an excipient, a filler, a binder, a wetting agent, or a disintegrating agent.

[0041] Preferably, the dosage form of the pharmaceutical preparation comprises tablets, capsules, patches, oral liquids, gels, creams, soluble microneedles or injections.

[0042] In a fourth aspect, the present application provides a use of the clathrate containing artemisinic acid according to the first aspect or the pharmaceutical preparation according to the third aspect in the preparation of a product for inhibiting melanin production, a preparation for promoting apoptosis of melanoma cells or a preparation for inhibiting proliferation of melanoma cells.

[0043] In a fifth aspect, the present application provides a use of the clathrate containing artemisinic acid according to the first aspect or the pharmaceutical preparation according to the third aspect in the preparation of an anti-inflammatory product.

[0044] The present application creatively discovers that cyclodextrin compounds and artemisinic acid have significant synergies in anti-inflammatory, melanin production inhibition and the like, and the cyclodextrin compounds are used to clathrate artemisinic acid, which not only improves the water solubility of artemisinic acid, but also better plays the efficacy of artemisinic acid itself, and realizes the synergies of cyclodextrin compounds and artemisinic acid in anti-inflammatory, melanin production inhibition and the like.

[0045] The numerical range in the present application includes not only the point values listed above, but also any point values between the above numerical ranges which are not listed, and the present application does not list the specific point values included in the range for the sake of brevity and simplicity.

[0046] Compared with the prior art, the present application has the following beneficial effects:

[0047] The clathrate containing artemisinic acid developed by the present application uses cyclodextrin compounds to clathrate artemisinic acid, greatly improves the water solubility of artemisinic acid, and can better play the role of artemisinic acid in anti-inflammatory, melanin production inhibition and the like.

[0048] Meanwhile, cyclodextrin compounds and artemisinic acid have significant synergies in anti-inflammatory, melanin production inhibition and the like, and the cyclodextrin compounds are used to clathrate artemisinic acid, which not only improves the water solubility of artemisinic acid, but also better plays the efficacy of artemisinic acid itself, and realizes the synergies of cyclodextrin compounds and artemisinic acid in anti-inflammatory, melanin production inhibition and the like.

[0049] Further, hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium are used as clathrates to clathrate artemisinic acid, which can significantly improve the solubility of artemisinic acid, and the two cyclodextrin derivatives have significant synergies in improving the solubility of artemisinic acid. Meanwhile, the two cyclodextrin derivatives have significant synergies in anti-inflammatory and melanin production inhibition and the like.

[0050] Further, the molar ratio of artemisinic acid and cyclodextrin compounds is controlled at 1:7-10, which can better improve the inclusion capacity of cyclodextrin compounds for artemisinic acid, and further improve the anti-inflammatory and melanin generation inhibiting effects of cyclodextrin compounds and artemisinic acid.

[0051] Further, in the preparation process of the inclusion compound containing artemisinic acid, hydrochloric acid is added to adjust the pH of the mixed solution, which can further improve the inclusion capacity of cyclodextrin compounds for artemisinic acid. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 is a structural diagram of the inclusion compound containing artemisinic acid involved in the present application. DETAILED DESCRIPTION

[0053] In order to further illustrate the technical means adopted by the present application and its effects, the technical solutions of the present application will be further described below in combination with the preferred embodiments of the present application, but the present application is not limited in the scope of the embodiments.

[0054] In the following specific embodiments, the sources of various reagents are shown in Table 1:

[0055] Table 1

[0056]

[0057]

[0058] Example 1

[0059] The present embodiment provides an inclusion compound containing artemisinic acid, and the preparation method thereof comprises:

[0060] (1) The cyclodextrin compounds (the molar ratio of hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium is 1:1) are mixed with 40% (v / v) acetone aqueous solution at a mass volume ratio of 1:35 g / mL, then artemisinic acid (the molar ratio of artemisinic acid and cyclodextrin compounds is controlled at 1:8) is added, hydrochloric acid is added to make the pH value of the solution 7.0, and the solution is stirred at 1000 rpm at 30°C for 12 h to make the artemisinic acid completely dissolved, to obtain a mixed solution.

[0061] (2) The mixed solution is spray dried, the inlet temperature is 70°C, the outlet temperature is 30°C, the inlet air temperature is 180°C, the outlet air temperature is 150°C, the inlet air speed is 3 m / s, and the outlet air speed is 8 m / s, and the inclusion compound containing artemisinic acid is obtained after drying.

[0062] Example 2

[0063] The present embodiment provides an inclusion compound containing artemisinic acid, and the preparation method thereof comprises:

[0064] (1) The cyclodextrin compound (molar ratio of hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium is 1:5) is mixed with water in a mass volume ratio of 1:20 g / mL, then artemisinic acid (control the molar ratio of artemisinic acid and cyclodextrin compound is 1:7) is added, hydrochloric acid is added to make the pH value of the solution 6.0, and the solution is stirred at 2000 rpm for 16 h at 20°C to make the artemisinic acid completely dissolved to obtain a mixed solution.

[0065] (2) The mixed solution is freeze-dried, the pre-freezing time is 4 h, the temperature is -45°C, the vacuum degree is 1013 mbar; the sublimation drying time is 10 h, the temperature is -16-5°C, the vacuum degree is 0.2 mbar; the resolution drying time is 4 h, the temperature is 16-40°C, the vacuum degree is 0.2-1013 mbar, and the inclusion compound containing artemisinic acid is obtained after drying.

[0066] Example 3

[0067] This example provides an inclusion compound containing artemisinic acid, and the preparation method comprises:

[0068] (1) The cyclodextrin compound (molar ratio of hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium is 5:1) is mixed with 50% (v / v) ethanol aqueous solution in a mass volume ratio of 1:60 g / mL, then artemisinic acid (control the molar ratio of artemisinic acid and cyclodextrin compound is 1:10) is added, hydrochloric acid is added to make the pH value of the solution 7.5, and the solution is stirred at 5000 rpm for 8 h at 40°C to make the artemisinic acid completely dissolved to obtain a mixed solution.

[0069] (2) The mixed solution is spray-dried, the inlet temperature is 70°C, the outlet temperature is 30°C, the inlet air temperature is 180°C, the outlet air temperature is 150°C, the inlet air speed is 3 m / s, and the outlet air speed is 8 m / s, and the inclusion compound containing artemisinic acid is obtained after drying.

[0070] Example 4

[0071] This example provides an inclusion compound containing artemisinic acid, which is only different from example 1 in that the total amount of substance of the cyclodextrin compound and the molar ratio of artemisinic acid and the cyclodextrin compound are controlled unchanged, and the cyclodextrin compound is replaced by single sulfobutyl-β-cyclodextrin sodium; the remaining steps are consistent with example 1.

[0072] Example 5

[0073] The present example provides a clathrate containing artemisinic acid, which differs from Example 1 only in that the total amount of substance of the cyclodextrin compound and the molar ratio of artemisinic acid and cyclodextrin compound are controlled, and the cyclodextrin compound is replaced with a single hydroxypropyl-β-cyclodextrin; the remaining steps are consistent with Example 1.

[0074] Example 6

[0075] The present example provides a clathrate containing artemisinic acid, which differs from Example 1 only in that the total amount of substance of the cyclodextrin compound and the molar ratio of artemisinic acid and cyclodextrin compound are controlled, and the cyclodextrin compound is replaced with a single β-cyclodextrin; the remaining steps are consistent with Example 1.

[0076] Example 7

[0077] The present example 1 provides a clathrate containing artemisinic acid, which differs from Example 1 only in that the molar ratio of artemisinic acid and cyclodextrin compound is adjusted to 1:12, and the remaining steps are consistent with Example 1.

[0078] Example 8

[0079] The present example 1 provides a clathrate containing artemisinic acid, which differs from Example 1 only in that the molar ratio of artemisinic acid and cyclodextrin compound is adjusted to 1:6, and the remaining steps are consistent with Example 1.

[0080] Example 9

[0081] The present example 1 provides a clathrate containing artemisinic acid, which differs from Example 1 only in that no hydrochloric acid is added during preparation, and the remaining steps are consistent with Example 1.

[0082] Comparative Example 1

[0083] The present comparative example provides artemisinic acid without clathration, which differs from Example 1 in that no cyclodextrin compound is added in the preparation method, i.e. after mixing artemisinic acid with a 40% (v / v) aqueous acetone solution, hydrochloric acid is added, stirring and drying. The specific parameters in the preparation process are consistent with Example 1.

[0084] Comparative Example 2

[0085] The present comparative example provides a cyclodextrin mixture, which differs from Example 1 in that no artemisinic acid is added in the preparation method, i.e. after mixing the cyclodextrin compound with a 40% (v / v) aqueous acetone solution, hydrochloric acid is added to make the solution pH value 7.0, stirring and drying. The specific parameters in the preparation process are consistent with Example 1.

[0086] Comparative Example 3

[0087] The comparative example provides a cyclodextrin mixture, which is only different from Comparative Example 2 in that the total amount of substance of cyclodextrin compounds is kept unchanged, and the cyclodextrin compounds are replaced by a single sulfobutyl-β-cyclodextrin sodium, and the remaining steps are consistent with Comparative Example 2.

[0088] Comparative Example 4

[0089] The comparative example provides a cyclodextrin mixture, which is only different from Comparative Example 2 in that the total amount of substance of cyclodextrin compounds is kept unchanged, and the cyclodextrin compounds are replaced by a single sulfobutyl-β-cyclodextrin sodium, and the remaining steps are consistent with Comparative Example 2.

[0090] Test Example 1

[0091] This test example tests the solubility of artemisinic acid in different solvents.

[0092] Referring to the method for solubility test in the General Rules of Chinese Pharmacopoeia 2020 Edition Part I, the inclusion compound containing artemisinic acid prepared in each example and artemisinic acid not subjected to inclusion in Comparative Example 1 are dissolved in water, 50% (v / v) ethanol aqueous solution, and 50% (v / v) methanol aqueous solution, respectively. The solubility of each sample to be tested in different solvents is shown in Table 2.

[0093] Table 2

[0094]

[0095] From the data in Table 2, it can be seen that by wrapping artemisinic acid with cyclodextrin or cyclodextrin derivatives to form an inclusion compound, the solubility of artemisinic acid in different solvents can be greatly improved.

[0096] From the comparison of the data of Example 1 and Examples 4-5, it can be seen that the co-use of hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium as inclusion agents for inclusion of artemisinic acid can significantly improve the solubility of artemisinic acid, and the two artemisinic acid derivatives have a significant synergistic effect in improving the solubility of artemisinic acid.

[0097] From the comparison of the data of Example 1 and Example 6, it can be seen that compared with artemisinic acid, the artemisinic acid derivative composed of hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium can further improve the inclusion capacity for artemisinic acid and improve the solubility of artemisinic acid in different solvents.

[0098] From the comparison of the data of Example 1 and Examples 7-8, it can be seen that by controlling the molar ratio of artemisinic acid and cyclodextrin compounds to be 1:(7-10), the inclusion capacity of cyclodextrin compounds for artemisinic acid can be better improved, and the solubility of artemisinic acid can be improved.

[0099] From the comparison of the data of Example 1 and Example 9, it can be seen that in the preparation of the inclusion compound, the pH value of the mixed solution is adjusted to 6.0-7.5 by adding hydrochloric acid, which can further improve the inclusion capacity of dextrin compounds for artemisinic acid and improve the solubility of artemisinic acid.

[0100] Test Example 2

[0101] In this test example, the inhibitory ability of different samples on melanin production is tested.

[0102] (1) Test sample: samples prepared in each example and comparative example.

[0103] (2) Test method:

[0104] (2.1) Resuscitation and culture of MNT-1 cells:

[0105] In a 100 mm cell culture dish, 10 mL of MNT-1 cell special medium was added, and a frozen tube containing MNT-1 cells was taken out from a -80℃ refrigerator and quickly placed in a preheated 37℃ water bath for continuous shaking to quickly melt the cell freezing solution within 1 min. The melted cell suspension was added to the above cell culture dish and blown evenly, and the cells were cultured at 37℃ in a 5% CO2 environment. After the cells adhered, fresh MNT-1 special medium was used for continuous culture, and when the cells grew to about 80%, they were subcultured. The cells used in all experiments were kept within 3-20 passages.

[0106] (2.2) CCK-8 method for determining melanocyte activity:

[0107] MNT-1 cells in good condition in the exponential growth phase were trypsinized for 40 s, and a cell counting plate was used for counting. The cells were inoculated in a 96-well plate at a density of 1×10 5 μL per well, and the cells were cultured at 37℃ in a 5% CO2 environment for 24 h. Then fresh medium containing 200 μM of each test sample or medium without test sample (control group) was used for culture for 24 h. 10 μL of CCK-8 solution was added to each well, incubated at 37℃ for 1 h, shaken on a shaker for 10 min, and the OD value of each well was determined at 450 nm wavelength. The relative cell viability of MNT-1 cells treated with each test sample at a concentration of 200 μM was more than 99%, indicating that 200 μM was a safe concentration, and further melanin content determination could be carried out.

[0108] (2.3) NaOH lysis method for determining melanin content:

[0109] MNT-1 cells were inoculated in a 6-well plate at a density of 1×10 5The cells were cultured in 96-well plates at a density of 5×103cells / mL, 2 mL / well. After overnight adhesion, the medium was replaced, and the cells were cultured for another 24 h, and then the medium was replaced with a medium containing 200 μM of each sample to be tested for 48 h (2.5 mL / well), and each sample was tested in triplicate. After 48 h of culture, the medium was removed, and the cells were washed once with PBS (added slowly to prevent the cells from being blown up), and the cell culture dish was placed on an ice plate, 330 μL of non-denaturing cell lysis solution (containing 1% PMSF) was added to each dish, and the cells were lysed at 4°C for 20 min. The cells were collected by centrifugation at 13,000 r / min for 10 min at 4°C. The black pigment precipitate was at the bottom of the centrifuge tube. 330 μL of NaOH (containing 10% DMSO) was added and vortexed to facilitate complete lysis. The mixture was placed in a metal bath at 80°C for 2 h to completely dissolve the black pigment precipitate. The mixture was vortexed and then centrifuged at 1,000 r / min for 5 min. 200 μL of black pigment lysis solution was added to each well of a 96-well plate, and three replicates were set up. A blank group (200 μL of PBS solution) and a control group (cells without drug) were also set up. The OD value was measured at 405 nm.

[0110] The relative cell viability was calculated as follows: relative cell viability = OD value of the sample group / OD value of the control group x 100%.

[0111] The relative melanin content was calculated as follows: relative melanin content = (OD value of the sample group - OD value of the blank group) / (OD value of the control group - OD value of the blank group) x 100%.

[0112] The results of the relative cell viability and the relative melanin content of each test group are shown in Table 3.

[0113] Table 3

[0114]

[0115]

[0116] As can be seen from the data of Example 1 and Comparative Examples 1 and 2, both artemisinic acid and cyclodextrin compounds can reduce the amount of melanin produced to some extent. In addition, artemisinic acid and cyclodextrin compounds have a significant synergistic effect in reducing the amount of melanin produced and inhibiting melanin production.

[0117] As can be seen from the data of Comparative Example 2 and Comparative Examples 3 and 4, hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium have a significant synergistic effect in inhibiting melanin production.

[0118] As can be seen from the data of Example 1 and Examples 4 and 5, hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium together as inclusion agents to include artemisinic acid, the two compounds cooperate with each other, have a synergistic effect, and can better play the synergistic effect of artemisinic acid and cyclodextrin compounds in inhibiting melanin production. As can be seen from the data of Example 1 and Comparative Examples 1 and 2, both artemisinic acid and cyclodextrin compounds can reduce the amount of melanin produced to some extent. In addition, artemisinic acid and cyclodextrin compounds have a significant synergistic effect in reducing the amount of melanin produced and inhibiting melanin production.

[0119] From the comparison of the data of Example 1 and Example 6, it can be seen that compared with artemisinic acid, the artemisinic acid derivative composed of hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium can better reduce the relative viability and melanin production of MNT-1 cells.

[0120] From the comparison of the data of Example 1 and Example 7-8, it can be seen that by controlling the molar ratio of artemisinic acid and cyclodextrin compounds at 1:(7-10), the inclusion capacity of cyclodextrin compounds for artemisinic acid can be better improved, and the synergistic effect of cyclodextrin compounds and artemisinic acid on inhibiting melanin production can be improved.

[0121] From the comparison of the data of Example 1 and Example 9, it can be seen that in the preparation process of the inclusion compound, the addition of hydrochloric acid to adjust the pH of the mixed solution can further improve the inclusion capacity of cyclodextrin compounds for artemisinic acid, and the synergistic effect of cyclodextrin compounds and artemisinic acid on inhibiting melanin production can be improved.

[0122] Test Example 3

[0123] In this test example, the inhibitory ability of different samples on inflammatory factors is tested.

[0124] (1) Test sample: samples prepared in each example and comparative example.

[0125] (2) Test method:

[0126] (2.1.1) MTT method for detecting cell activity:

[0127] RAW264.7 cells in good shape and in logarithmic growth phase were selected and inoculated in a 96-well plate, and incubated in an incubator for 24 h. Complete culture medium containing each test sample and complete culture medium without test sample (blank group) were used to incubate in an incubator for 24 h, respectively. The original supernatant was removed, MTT test solution was added to each well, and incubated in an incubator for 4 h. The absorbance at 570 nm was measured by an enzyme marker instrument, and the cell survival rate was calculated:

[0128] Cell survival rate = OD value of test sample group / OD value of blank group x 100%.

[0129] The test results of the survival rate of RAW264.7 cells in each test group are shown in Table 4. The survival rate of RAW264.7 cells treated with each test sample at a concentration of 5 μM is more than 99%, indicating that 5 μM is a safe concentration, and further anti-inflammatory activity test can be carried out.

[0130] (2.2.2) Anti-inflammatory activity test:

[0131] (2.2.2.1) Test grouping and intervention method:

[0132] ①Blank group: the logarithmic growth period, good morphology RAW264.7 cells were inoculated in 24-well plates in the incubator for 24h;

[0133] ②LPS-induced stimulation group: the logarithmic growth period, good morphology RAW264.7 cells were inoculated in 24-well plates, and LPS was added (the amount added was 10 μL / well) for induction and stimulation, and incubated in the incubator for 24h;

[0134] ③Positive control group (dexamethasone) group: the logarithmic growth period, good morphology RAW264.7 cells were inoculated in 24-well plates, and dexamethasone (final concentration 5 μM) was added, and LPS (the amount added was 10 μL / well) was added for induction and stimulation after two hours, and incubated in the incubator for 24h;

[0135] ④Sample group: the logarithmic growth period, good morphology RAW264.7 cells were inoculated in 24-well plates, and 5 μM of each example and comparative example of the sample to be tested was added to each well, and LPS (the amount added was 10 μL / well) was added for induction and stimulation after two hours, and incubated in the incubator for 24h.

[0136] The cell supernatant was detected according to the steps of the ELISA kit and the nitric oxide detection kit instructions, and the contents of TNF-α, IL-6 and NO in the collected cell supernatant were detected.

[0137] The content test results of TNF-α, IL-6 and NO in each test group are shown in Table 4.

[0138] Table 4

[0139]

[0140]

[0141] From the data comparison of Example 1 and Comparative Example 1, Comparative Example 2, it can be seen that artemisinic acid and cyclodextrin compounds can reduce the expression level of verification factors to a certain extent; in addition, artemisinic acid and cyclodextrin compounds show significant synergistic effect in reducing the expression level of inflammatory factors.

[0142] From the data comparison of Comparative Example 2 and Comparative Example 3-4, it can be seen that hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium have significant synergistic effect in anti-inflammatory.

[0143] From the data comparison of Example 1 and Example 4-5, it can be seen that hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium together as inclusion agent for artemisinic acid, they cooperate with each other, synergistically, and can better play the synergistic effect of artemisinic acid and cyclodextrin compounds in anti-inflammatory.

[0144] From the comparison of the data of Example 1 and Example 6, it can be seen that compared with artemisinic acid, the artemisinic acid derivative composed of hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium has better anti-inflammatory effect.

[0145] From the comparison of the data of Example 1 and Example 7-8, it can be seen that by controlling the molar ratio of artemisinic acid and cyclodextrin compounds at 1:(7-10), the inclusion capacity of cyclodextrin compounds for artemisinic acid can be better improved, and thus the anti-inflammatory effect of cyclodextrin compounds and artemisinic acid can be improved.

[0146] From the comparison of the data of Example 1 and Example 9, it can be seen that in the preparation process of the inclusion compound, by adding hydrochloric acid to adjust the pH of the mixed solution, the inclusion capacity of cyclodextrin compounds for artemisinic acid can be further improved, and thus the anti-inflammatory effect of cyclodextrin compounds and artemisinic acid can be improved.

[0147] The applicant declares that the technical solutions of the present application are illustrated by the above examples, but the present application is not limited to the above examples, that is, it does not mean that the present application must rely on the above examples to be implemented. It should be understood by those skilled in the art that any improvement of the present application, equivalent replacement of each raw material of the product of the present application, addition of auxiliary ingredients, selection of specific modes, etc. all fall within the protection scope and disclosure scope of the present application.

[0148] The preferred embodiments of the present application are described in detail above, but the present application is not limited to the specific details in the above embodiments, and within the technical concept scope of the present application, various simple modifications of the technical solutions of the present application can be made, and these simple modifications all belong to the protection scope of the present application.

[0149] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined by any suitable means without contradiction, and in order to avoid unnecessary repetition, the present application will not further describe various possible combination manners.

Claims

1. An inclusion compound containing artemisinic acid, characterized in that: The inclusion compound containing artemisinic acid comprises artemisinic acid and a cyclodextrin compound encapsulating artemisinic acid; The cyclodextrin compounds include cyclodextrin and / or cyclodextrin derivatives.

2. The inclusion compound containing artemisinic acid according to claim 1, characterized in that The cyclodextrin includes β-cyclodextrin and / or γ-cyclodextrin; Preferably, the cyclodextrin derivative comprises hydroxypropyl-β-cyclodextrin and / or sulfobutyl-β-cyclodextrin sodium; Preferably, the cyclodextrin compounds include hydroxypropyl-β-cyclodextrin and sulfobutyl-β-cyclodextrin sodium.

3. The inclusion compound containing artemisinic acid according to claim 2, characterized in that: The molar ratio of the hydroxypropyl-β-cyclodextrin and the sulfobutyl-β-cyclodextrin sodium is (1-5):(1-5).

4. The inclusion compound according to any one of claims 1 to 3, characterized in that The molar ratio of artemisinic acid to cyclodextrin compounds is 1:(7-10).

5. A method for preparing an inclusion compound containing artemisinic acid according to any one of claims 1 to 4, characterized in that: The preparation method comprises: The cyclodextrin compound and artemisinic acid are mixed and stirred in a solvent to obtain a mixed solution; the mixed solution is dried to obtain an inclusion compound containing artemisinic acid.

6. The preparation method according to claim 5, characterized in that The solvent includes any one of water, ethanol or acetone or a combination of at least two; Preferably, the ratio of the total mass of the cyclodextrin compound and artemisinic acid to the volume of the solvent is 1:(10-80) g / mL; Preferably, the mixing and stirring process further includes adding a pH regulator; Preferably, the pH adjuster comprises hydrochloric acid; Preferably, the pH value of the solution after adding the pH regulator is 6.0-7.

5.

7. The preparation method according to claim 5 or 6, characterized in that: The mixing temperature is 10-60°C, and the mixing time is 2-24h; Preferably, the mixing and stirring speed is 300-5000 rpm; Preferably, the drying method includes spray drying, freeze vacuum drying or vacuum rotary film concentration drying.

8. A pharmaceutical preparation, characterized in that The pharmaceutical preparation comprises the inclusion compound containing artemisinic acid according to any one of claims 1 to 4; Preferably, the pharmaceutical preparation further comprises excipients; Preferably, the excipients include any one or a combination of at least two of a pharmaceutically acceptable carrier, diluent, excipient, filler, binder, wetting agent or disintegrant; Preferably, the dosage form of the pharmaceutical preparation includes tablets, capsules, patches, oral liquids, gels, creams, soluble microneedles or injections.

9. Use of the inclusion compound comprising artemisinic acid according to any one of claims 1 to 4 or the pharmaceutical preparation according to claim 8 in the preparation of a product for inhibiting melanogenesis, a preparation for promoting apoptosis of melanoma cells, or a preparation for inhibiting proliferation of melanoma cells.

10. Use of the inclusion compound comprising artemisinic acid according to any one of claims 1 to 4 or the pharmaceutical preparation according to claim 8 in the preparation of an anti-inflammatory product.

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