Matrine and oleanolic acid ionic liquid as well as preparation method and application thereof

Abstract

The invention discloses matrine-oleanolic acid ionic liquid and a preparation method and application thereof.The preparation method comprises the steps that matrine and oleanolic acid are dissolved in an organic solvent, heating and stirring are conducted, then rotary evaporation is conducted, and the matrine-oleanolic acid ionic liquid is obtained; wherein the mass ratio of the sum of the matrine and the oleanolic acid to the organic solvent is 1: (5-30), and the molar ratio of the matrine to the oleanolic acid is (3-10): 1. The ionic liquid disclosed by the invention not only can effectively improve the water solubility of oleanolic acid, but also can effectively inhibit the activity of 5 alpha-reductase and prevent the 5 alpha-reductase from catalyzing testosterone to be converted into DHT, so that the problems of rapid entry into a retrogression period, miniaturization, hair loss and the like caused by the fact that DHT acts on hair follicles after being combined with an androgen receptor are prevented. Meanwhile, expression of IL-1beta and TNF-alpha inflammatory factors in skin hair follicles can be inhibited, and the purposes of preventing alopecia and promoting hair growth are achieved. The effects of controlling oil, resisting inflammation, relieving, removing dandruff, relieving itching and the like are also achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of ionic liquids, and in particular relates to a matrine-oleanolic acid ionic liquid and a preparation method and application thereof. Background Art

[0002] With rising living standards, demand for scalp care and anti-hair loss cosmetics is increasing. While a wide variety of scalp care and anti-hair loss products are currently available on the market, their effectiveness varies widely, and some products have side effects. Therefore, developing safe, effective cosmetics that provide both scalp care and anti-hair loss benefits is crucial.

[0003] Both matrine and oleanolic acid are natural products with a wide range of biological activities. Matrine has strong antibacterial and anti-inflammatory effects, can effectively inhibit bacteria and fungi on the scalp, reduce inflammatory reactions around hair follicles, and improve the hair loss microenvironment. On the other hand, matrine can inhibit the activity of type II 5α-reductase, reduce the conversion of testosterone to dihydrotestosterone (DHT), and thus alleviate androgenic alopecia. Oleanolic acid (oleanolic aCid) is a pentacyclic triterpenoid compound. Similar to matrine, oleanolic acid has a certain inhibitory effect on 5α-reductase. Secondly, oleanolic acid has excellent anti-inflammatory and antioxidant properties, which can help reduce scalp redness and irritation and protect the scalp from the damaging effects of free radicals. However, the physicochemical properties of oleanolic acid show that oleanolic acid is insoluble in water at room temperature or under heating conditions. Its molecular structure is highly hydrophobic. The extremely poor water solubility of oleanolic acid limits its practical application in cosmetics. Summary of the Invention

[0004] The purpose of this section is to summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0005] As one aspect of the present invention, the present invention provides a method for preparing a matrine-oleanolic acid ionic liquid, which comprises dissolving matrine and oleanolic acid in an organic solvent, heating and stirring, and then rotary evaporating to obtain the matrine-oleanolic acid ionic liquid; wherein the mass ratio of the sum of the mass of matrine and oleanolic acid to the mass of the organic solvent is 1:5 to 30, and the molar ratio of matrine to oleanolic acid is 3 to 10:1.

[0006] In the preparation method of the matrine-oleanolic acid ionic liquid of the present invention, the organic solvent includes ethanol, methanol, ether or acetone.

[0007] As the preparation method of the matrine-oleanolic acid ionic liquid of the present invention, wherein the heating and stirring is performed at a heating temperature of 30 to 60°C.

[0008] As the preparation method of the matrine-oleanolic acid ionic liquid of the present invention, wherein, the heating and stirring, the heating temperature is 40-50° C.; the stirring speed is 50-800 rpm; and the stirring time is 30-300 min.

[0009] In the method for preparing the matrine-oleanolic acid ionic liquid of the present invention, the temperature of the rotary evaporation is 40-60°C.

[0010] In the preparation method of the matrine-oleanolic acid ionic liquid of the present invention, the mass ratio of the sum of the mass of matrine and oleanolic acid to the mass of the organic solvent is 1:10-20.

[0011] The present invention also provides application of the matrine-oleanolic acid ionic liquid prepared by the preparation method of the matrine-oleanolic acid ionic liquid in the preparation of cosmetics.

[0012] Wherein, the cosmetics include anti-hair loss products.

[0013] The matrine-oleanolic acid ionic liquid has the effects of removing dandruff, relieving itching, controlling oil and anti-inflammatory, and the cosmetics include cosmetics with the effects of removing dandruff, relieving itching, controlling oil and anti-inflammatory.

[0014] Beneficial effects of the present invention: The present invention provides a matrine and oleanolic acid ionic liquid. This ionic liquid not only effectively increases the water solubility of oleanolic acid but also effectively inhibits 5α-reductase activity, preventing the catalytic conversion of testosterone to DHT by 5α-reductase. This prevents DHT from binding to androgen receptors and acting on hair follicles, leading to rapid catagen, miniaturization, and hair loss. Furthermore, the present invention inhibits the expression of inflammatory factors such as IL-1β and TNF-α in hair follicles, preventing hair loss and promoting hair growth. It also offers benefits such as oil control, anti-inflammatory, soothing, and anti-dandruff and anti-itching properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments, wherein:

[0016] Figure 1 This is a physical picture of the matrine-oleanolic acid ionic liquid prepared in Example 2.

[0017] Figure 2 This is a physical picture of the matrine-oleanolic acid mixture prepared in Comparative Example 1.

[0018] Figure 3 These are infrared spectra of the matrine-oleanolic acid ionic liquid prepared in Example 2 and the matrine-oleanolic acid mixture prepared in Comparative Example 1.

[0019] Figure 4 The figure shows the effects of different examples on the proliferation of hair papilla cells.

[0020] Figure 5 This is the effect of samples of different concentrations on the proliferation of dermal papilla cells in Example 2.

[0021] Figure 6 This is the effect of samples of different concentrations on VEGF in Example 2.

[0022] Figure 7 This is the effect of samples of different concentrations on IL-1α in Example 2.

[0023] Figure 8 This is the effect of samples of different concentrations on IL-6 in Example 2.

[0024] Figure 9 This is the effect of samples of different concentrations on lipid secretion in Example 2.

[0025] Figure 10 This is the effect of samples of different concentrations on IL-6 in Comparative Example 1.

[0026] Figure 11 This is the effect of samples of different concentrations on VEGF in Comparative Example 1.

[0027] Figure 12 This is the effect of samples of different concentrations on IL-6 in Comparative Example 1.

[0028] Figure 13 This is the effect of samples of different concentrations on lipid secretion in Comparative Example 1. DETAILED DESCRIPTION

[0029] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0030] Preparation method of matrine-oleanolic acid ionic liquid: matrine powder and oleanolic acid powder are dissolved in ethanol, methanol, ether or acetone solvent, matrine and oleanolic acid are added in a molar ratio of 3 to 10:1, and the material-liquid ratio (mass ratio) is 1:5 to 30, and stirred at 30 to 60° C. and 50 to 800 rpm for 30 to 300 minutes, and finally rotary evaporated at 40 to 60° C. to obtain matrine-oleanolic acid ionic liquid.

[0031] Example 1:

[0032] Matrine powder and oleanolic acid powder were dissolved in ethanol, matrine and oleanolic acid were added in a molar ratio of 3:1, the material-liquid ratio was 1:10, and the mixture was stirred at 40°C and 300 rpm for 60 minutes. Finally, the ethanol was removed by rotary evaporation at 50°C to obtain a light yellow viscous matrine-oleanolic acid ionic liquid.

[0033] Example 2:

[0034] Matrine powder and oleanolic acid powder were dissolved in ethanol, matrine and oleanolic acid were added in a molar ratio of 5:1, the material-liquid ratio was 1:10, and the mixture was stirred at 40°C and 300 rpm for 60 minutes. Finally, the ethanol was removed by rotary evaporation at 50°C to obtain a light yellow viscous matrine-oleanolic acid ionic liquid.

[0035] Example 3:

[0036] Matrine powder and oleanolic acid powder were dissolved in ethanol, matrine and oleanolic acid were added in a molar ratio of 10:1, the material-liquid ratio was 1:10, stirred at 40°C and 300 rpm for 60 minutes, and finally rotary evaporated at 50°C to obtain a light yellow viscous matrine-oleanolic acid ionic liquid.

[0037] Comparative Example 1:

[0038] Matrine powder and oleanolic acid powder were dissolved in ethanol, matrine and oleanolic acid were added in a molar ratio of 2:1, the material-liquid ratio was 1:10, stirred at 40°C and 300 rpm for 60 minutes, and finally rotary evaporated at 50°C to obtain a white solid powder.

[0039] The experiment found that when the molar ratio of matrine to oleanolic acid was not within the range of 3 to 10:1, no ionic liquid could be formed. In Comparative Example 1, when the molar ratio of matrine to oleanolic acid was 2:1, no ionic liquid could be formed, and a white solid powder was finally obtained.

[0040] Experimental analysis:

[0041] Structural analysis: The structure of the substance is analyzed by Fourier total reflectance infrared spectroscopy (FTIR);

[0042] Oleanolic acid solubility in ionic liquids: 0.5 g of matrine-oleanolic acid ionic liquid prepared in Example 1, 2, or 3 was dissolved in a predetermined amount of deionized water at room temperature. After 24 hours of immersion, a clear, transparent solution was obtained. The minimum amount of deionized water required was used to calculate the solubility: oleanolic acid solubility / (g / mL) = 0.5 g * oleanolic acid mass fraction / minimum amount of deionized water required. The results of the oleanolic acid solubility test are shown in Table 1.

[0043] Table 1 Solubility of oleanolic acid

[0044] Sample source Solubility of oleanolic acid in ionic liquids (g / mL) Example 1 0.007 Example 2 0.011 Example 3 0.018

[0045] Dermal papilla cell proliferation, vascular endothelial growth factor and inflammatory factor detection experiments:

[0046] Cell culture and passage: Take out the cryopreserved tube of dermal papilla cells stored in liquid nitrogen, shake in a 37℃ water bath, and add the cryopreserved liquid to the DMEM high glucose medium (containing 10% fetal bovine serum and 1×10^ 5 U / L penicillin, 100mg / L streptomycin) in a T25 cell culture flask, shake it with the cross method, and place the culture flask in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. After observing the cells for 4 hours, change the medium and continue to culture. When the cell density exceeds 80%, passaging the cells: aspirate the cell culture medium, gently rinse twice with PBS, add trypsin containing 0.25% EDTA, digest in the incubator for 3 minutes, and terminate the cell digestion with fresh DMEM complete medium. Centrifuge the cell suspension at 1500rpm for 5 minutes. Add fresh DMEM complete medium to blow the cell pellet, and the number of passaging flasks is determined according to the cell number, status, and experimental requirements. Place the culture flask in a cell culture incubator for culture.

[0047] Cell proliferation assay:

[0048] Digest the cells in logarithmic growth phase and count them on a hemocytometer. Inoculate 1×10 cells per well. 4 Cells were plated in a 96-well cell culture plate. After 24 hours of culture, the supernatant was discarded. Different concentrations of each sample solution dissolved in culture medium were added to the sample wells. A blank control group was used without drug addition (the physical mixing group consisted of matrine powder and oleanolic acid powder mixed at a molar ratio of 3:1 and added to the culture medium to dilute to a concentration of 10 μg / mL. Oleanolic acid was insoluble in water and formed into a suspended particle). Multiple replicates were set up for each well. After 24 hours of culture, the supernatant was discarded and the cells were washed with PBS. 100 μL of freshly prepared MTT solution at 0.5 mg / mL (dissolved in DMEM culture medium) was added to each well. After incubation for 4 hours, the supernatant was discarded, and 100 μL of DMSO was added to each well to dissolve the crystals. The cells were shaken at 37°C for 5 minutes. The absorbance was read at 490 nm on a microplate reader. The cell proliferation was calculated using the formula:

[0049]

[0050] Where: A0 represents the absorbance value of the solvent group at 490 nm;

[0051] A1 represents the absorbance value of the sample group at 490 nm;

[0052] A2 represents the absorbance value of the blank control group at 490 nm.

[0053] Detection of vascular endothelial growth factor (VEGF) in dermal papilla cells: After 24 hours of culture, the cells adhered to the plate and the complete medium was aspirated. The plate bottom was rinsed twice with PBS. 2 mL of freshly prepared drug was added to each well. Blank controls and samples of varying concentrations were set up and cultured for another 48 hours. The VEGF concentration in the culture medium was determined according to the ELISA kit instructions.

[0054] Inflammatory factor detection: Dermal papilla cell suspension was prepared at a rate of 1×10 6 The cells were seeded at a density of 100 cells / well in a 12-well plate. After culturing for 24 h, the cells were incubated with a solution containing drug (dihydrotestosterone (DHT), matrine-oleanolic acid ionic liquid or comparative example 1 for 24 h. The cell supernatant was collected and the secretion of inflammatory factors by dermal papilla cells was detected according to the operating instructions of the ELISA kit.

[0055] Experiment on inhibiting excessive sebum secretion induced by androgen:

[0056] Cell culture and passage: Take out the sebaceous cell SZ95 cryopreservation tube stored in liquid nitrogen, shake it in a 37℃ water bath, and add it to the DMEM high glucose medium (containing 10% fetal bovine serum and 1×10 5 U / L penicillin, 100mg / L streptomycin) in a T25 cell culture flask, shake it with the cross method, and place the culture flask in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. After observing the cells for 4 hours, change the medium and continue to culture. When the cell density exceeds 80%, passaging the cells: aspirate the cell culture medium, gently rinse twice with PBS, add trypsin containing 0.25% EDTA, digest in the incubator for 3 minutes, and terminate the cell digestion with fresh DMEM complete medium. Centrifuge the cell suspension at 1500rpm for 5 minutes. Add fresh DMEM complete medium to blow the cell pellet, and the number of passaging flasks is determined according to the cell number, status, and experimental requirements. Place the culture flask in a cell culture incubator for culture.

[0057] Nile red staining: SZ95 cells in the logarithmic growth phase were digested and counted, and the cell suspension was adjusted to 1×10 4 / mL, transferred to each sample well of a 96-well plate with a black bottom and transparent type in an amount of 100 μL per well. The culture medium in the well plate was aspirated the next day and rinsed once with PBS. A dihydrotestosterone (DHT) group was added as a model group, a matrine-oleanolic acid ionic liquid group was added as an experimental group, and a non-drug group was used as a blank control group. After incubation for 48 hours, the DMEM complete medium was discarded, and 10 μg / mL Nile red was added after being carefully rinsed twice with PBS, and allowed to stand for 15 minutes in a dark environment at 37°C. The released fluorescence was detected with a multifunctional microplate reader with a fluorescence function at an excitation wavelength of 485 nm and an absorption wavelength of 565 nm, with three replicates per group. The effects of dihydrotestosterone (DHT) and the samples prepared in Example 2 and Comparative Example 1 on the lipid synthesis rate of SZ95 cells were calculated according to the following formula.

[0058]

[0059] Volunteer evaluation:

[0060] A scalp care solution was prepared using the matrine-oleanolic acid supramolecule prepared in Example 2 for use in volunteer evaluation experiments. The formula of the scalp care solution is shown in Table 2.

[0061] Table 2 Scalp care solution formula

[0062] Raw material name weight% Function Deionized water to 100 solvent Matrine-oleanolic acid ionic liquid 3 Active ingredients D-Panthenol 0.5 moisturizer Hydrolyzed sodium hyaluronate 0.01 moisturizer Butanediol 3 solvent 1,2-Hexanediol 2 preservative Phenoxyethanol 0.5 preservative

[0063] Scalp care solution's anti-hair loss effects:

[0064] Methods: Sixty volunteers aged 18-59 years (30 women, 30 men) with seborrheic alopecia were recruited. Male hair loss was graded as Hamilton III-IV, while female hair loss was graded as Ludwig 1-2. Each participant used one bottle of purified water scalp care solution (placebo) daily for one month. The efficacy of the scalp care solution in removing oil and preventing hair loss was assessed using self-evaluation. Fifty-eight volunteers indicated that the placebo solution had no substantial effect. These 58 participants continued the study, each using one bottle of the scalp care solution (with the formula shown in Table 2) daily for three months. The efficacy of the scalp care solution in removing oil and preventing hair loss was assessed using self-evaluation.

[0065] The anti-dandruff and anti-itching effects of scalp care solution:

[0066] Methods: 60 volunteers (30 women and 30 men) aged 18-59 years with severe dandruff and scalp itching were recruited. Each participant used one bottle of purified water scalp treatment solution (placebo) daily for one month. The anti-dandruff and anti-itching efficacy was assessed using self-evaluation. All participants reported no effect, so the study was continued, with each participant using one bottle daily for four weeks. The soothing (itching) and anti-dandruff efficacy were assessed using self-evaluation.

[0067] Table 3 Anti-hair loss analysis

[0068] Effect Effective number of people % invalid 17 29.3 Improved 20 34.5 Significant improvement 21 36.2 total 58 100

[0069] Table 4 Analysis of oil removal efficacy

[0070] Effect Effective number of people % invalid 8 13.8 Improved 18 31.0 Significant improvement 32 55.2 total 58 100

[0071] Table 5 Analysis of anti-dandruff and antipruritic efficacy

[0072] Effect Effective number of people % invalid 5 8.3 Improved 27 45.0 Significant improvement 28 46.7 total 60 100

[0073] In summary, the ionic liquid of the present invention not only increases the solubility of oleanolic acid, but also enables matrine and oleanolic acid to work synergistically to achieve the effects of anti-hair loss, oil control, anti-inflammatory and soothing, and anti-dandruff and antipruritic.

[0074] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a matrine-oleanolic acid ionic liquid, characterized in that: The method comprises dissolving matrine and oleanolic acid in an organic solvent, heating and stirring, and then rotary evaporating to obtain a matrine-oleanolic acid ionic liquid; wherein the mass ratio of the sum of the mass of matrine and oleanolic acid to the mass of the organic solvent is 1:5-30, and the molar ratio of matrine to oleanolic acid is 3-10:

1.

2. The method for preparing the matrine-oleanolic acid ionic liquid according to claim 1, wherein: The organic solvent includes ethanol, methanol, ether or acetone.

3. The method for preparing the matrine-oleanolic acid ionic liquid according to claim 1 or 2, wherein: The heating and stirring process has a heating temperature of 30-60°C.

4. The method for preparing the matrine-oleanolic acid ionic liquid according to claim 1 or 2, wherein: The heating and stirring process has a heating temperature of 40 to 50° C., a stirring speed of 50 to 800 rpm, and a stirring time of 30 to 300 min.

5. The method for preparing the matrine-oleanolic acid ionic liquid according to claim 1 or 2, characterized in that: The temperature of the rotary evaporation is 40-60°C.

6. The method for preparing the matrine-oleanolic acid ionic liquid according to claim 1 or 2, characterized in that: The mass ratio of the sum of the mass of matrine and oleanolic acid to the mass of the organic solvent is 1:10-20.

7. The matrine-oleanolic acid ionic liquid prepared by the preparation method of matrine-oleanolic acid ionic liquid according to claim 1.

8. Use of the matrine-oleanolic acid ionic liquid prepared by the preparation method of the matrine-oleanolic acid ionic liquid according to claim 1 in the preparation of cosmetics.

9. The use according to claim 8, characterized in that: The cosmetics include anti-hair loss products.

10. The use according to claim 8, characterized in that: The matrine-oleanolic acid ionic liquid has the effects of removing dandruff and relieving itching, controlling oil and anti-inflammatory, and the cosmetics include cosmetics with the effects of removing dandruff and relieving itching, controlling oil and anti-inflammatory.

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