TWI763071B - Microemulsion and its use, pharmaceutical composition and pharmaceutical use - Google Patents

Abstract

The present invention provides a microemulsion, comprising: an aqueous phase composition, including an aqueous solution and a moisturizing agent; an oil phase composition; and a surfactant composition; wherein, the water phase composition, the oil phase composition and the surfactant Based on the total weight of the composition, the content of the water phase composition is 25 to 39 weight percent, the content of the oil phase composition is 8 to 22 weight percent, and the content of the surfactant composition is 53 to 67 weight percent Percentage; The microemulsion of the present invention can be used as a carrier for enhancing the penetration of water-soluble active ingredients into the stratum corneum of the skin. The present invention further provides the use of a microemulsion, a pharmaceutical composition comprising the microemulsion, and the pharmaceutical use of the pharmaceutical composition for treating psoriasis.

Description

Microemulsion and its use, pharmaceutical composition and pharmaceutical use

The present invention provides a carrier, especially a microemulsion carrier. The present invention further provides the use of a microemulsion, a pharmaceutical composition comprising the microemulsion carrier and salvianolic acid B, and the use of the pharmaceutical composition for preparing a medicine.

Psoriasis, also known as psoriasis or psoriasis, is a common chronic dermatitis, a non-bacterial infectious skin disease, and occurs in the scalp, limbs, lower back, ears, finger joints, nails, navel, The groin and pubic area, and in severe cases, can spread all over the body. Histological features include hyperplasia of the epidermis, abnormal differentiation of keratinocytes, and vascular proliferation with vasodilation, resulting in abnormal hypertrophy, roughening, scaling, red plaques, papules, and spotting of the patient's skin.

Patients with psoriasis often have uncomfortable symptoms such as itchy, chapped and painful skin. Although there is no fatal risk, it is more troublesome. The appearance of the skin of patients with psoriasis will affect their normal social life, resulting in patients prone to embarrassment and loss of self-confidence and other depression problems. , and emotional stress is one of the factors that induce psoriasis, which makes psoriasis not only difficult to recover, but also more likely to recur again and again.

Current topical creams for the treatment of psoriasis include: steroids, vitamin A, vitamin D derivatives, coal tar, and salicylic acid. However, steroids are only suitable for short-term control; vitamin A and salicylic acid may cause skin irritation; long-term use of vitamin D derivatives may instead cause redness and itching of the skin; coal tar may dry the skin and make it prone to irritation odor, and sticking to other contact objects, such as furniture, etc., causing the problem of dirt, and it is inconvenient to use.

In fact, in addition to the above-mentioned psoriasis that cannot be cured and requires long-term treatment, other skin diseases, such as allergic skin diseases, may also recur frequently, and it is necessary to develop new active ingredient delivery systems to provide more active ingredients. The ingredients can be used in the daily care of problem skin and acute symptom treatment, so that patients have more choices to avoid further side effects or extra life troubles caused by long-term use of specific drugs.

In order to achieve the above purpose, the present invention provides a microemulsion, which comprises: an aqueous phase composition, including an aqueous solution and a moisturizing agent; an oil phase composition; and a surfactant composition; wherein, the water phase composition and the oil phase composition are composed of Based on the total weight of the composition and the surfactant composition, the content of the water phase composition is 25% to 39% by weight, the content of the oil phase composition is 8% to 22% by weight, and the surfactant The content of the composition is 53 to 67 weight percent.

The microemulsion described in the present invention is a carrier in a state of "micro-emulsion", that is, a delivery system of active ingredients that embeds the active ingredients into micro-emulsion droplets with sizes ranging from microns to nanometers. , and the micro-emulsion droplets of the active ingredient delivery system include water-phase composition, oil-phase composition and surfactant composition, through the above-mentioned specific ratios, they can form a thermodynamically stable (thermodynamically stable) etc. An isotropic mixture and usually clear.

For example, based on the total weight of the water phase composition, the oil phase composition and the surfactant composition, the content of the water phase composition is 25 weight percent, 26 weight percent, 27 weight percent, 28 weight percent , 29 weight percent, 30 weight percent, 31 weight percent, 32 weight percent, 33 weight percent, 34 weight percent, 35 weight percent, 36 weight percent, 37 weight percent, 38 weight percent, or 39 weight percent.

For example, based on the total weight of the water phase composition, the oil phase composition and the surfactant composition, the content of the oil phase composition is 8 weight percent, 9 weight percent, 10 weight percent, 11 weight percent , 12 weight percent, 13 weight percent, 14 weight percent, 15 weight percent, 16 weight percent, 17 weight percent, 18 weight percent, 19 weight percent, 20 weight percent, 21 weight percent, or 22 weight percent.

For example, based on the total weight of the water phase composition, the oil phase composition and the surfactant composition, the content of the surfactant composition is 53 weight percent, 54 weight percent, 55 weight percent, 56 weight percent percent, 57 weight percent, 58 weight percent, 59 weight percent, 60 weight percent, 61 weight percent, 62 weight percent, 63 weight percent, 64 weight percent, 65 weight percent, 66 weight percent, or 67 weight percent.

In addition, micro-emulsification is not the same as emulsification, because the emulsified product is turbid and will eventually undergo phase separation, lacking thermodynamic stability, and the process of emulsification is also more energy-intensive than micro-emulsification. Therefore, micro-emulsification and emulsification are different in three aspects: thermodynamic stability, product appearance and preparation method.

Adding a moisturizing agent to the water-phase composition of the present invention can moisturize the skin or soften the stratum corneum of the skin, and facilitate the penetration of the water-soluble active ingredient into the stratum corneum of the skin.

In one embodiment, the microemulsions of the present invention are intended for external use, ie, applied or applied to the skin or mucous membranes, eg, hair-bearing skin or skin within folds.

Preferably, the droplet size of the microemulsion is 500 nm to 900 nm, for example: 500 nm, 600 nm, 700 nm, 800 nm or 900 nm; more preferably 690 nm to 900 nm 700 nm.

Preferably, the polydispersity index of the size distribution of the microemulsion is 0.43 to 0.44; more preferably, it is 0.435.

Preferably, the interfacial potential of the microemulsion is -14 mV to -16 mV; more preferably, -14.5 mV to -15.45 mV.

Preferably, the viscosity of the microemulsion is 3000 cP to 3200 cP; more preferably 3100 cP to 3120 cP.

Preferably, the electrical conductivity of the microemulsion is 20 μS/cm to 30 μS/cm; more preferably 23.65 μS/cm to 24.65 μS/cm.

Preferably, the microemulsion is a bicontinuous microemulsion of oil-in-water and water-in-oil. The microemulsion of the present invention has both oil-in-water and water-in-oil dual phases, so not only helps to preserve the activity of the active ingredient, but also helps to improve the skin penetration rate of the active ingredient.

Preferably, the content of the water phase composition is 25 to 35 percent by weight, the content of the oil phase composition is 8 to 12 percent by weight, and the content of the surfactant composition is 53 to 53 percent by weight. 67 weight percent.

Preferably, the aqueous solution is a cell isotonic solution, and the moisturizing agent comprises sorbitol (sorbitol) and glycerol (glycerol), and based on the total weight of the cell isotonic solution, sorbitol and glycerol, the cell isotonic solution. The content is 31 to 39 percent by weight, the content of sorbitol is 45 to 55 percent by weight, and the content of glycerol is 12 to 18 percent by weight.

For example, based on the total weight of the cell isotonic solution, sorbitol and glycerol, the content of the cell isotonic solution is 31 weight percent, 33 weight percent, 35 weight percent, 37 weight percent or 39 weight percent; sorbitol The content is 45 weight percent, 47 weight percent, 49 weight percent, 51 weight percent, 53 weight percent or 55 weight percent; and the content of glycerin is 12 weight percent, 13 weight percent, 14 weight percent, 15 weight percent, 16 weight percent percent, 17 weight percent or 18 weight percent.

More preferably, the cell isotonic solution is PBS buffer.

In one embodiment, the PBS buffer comprises 0.01 molar phosphate buffer, 0.0027 molar potassium chloride and 0.137 molar sodium chloride, and has a pH of 7.4 at 25°C.

Preferably, the oil phase composition comprises silicone oil (silicone oil), squalene (squalene) and triglyceride (triglyceride), and the weight ratio of silicone oil, squalene and triglyceride is 8 to 12 ratios 0.8 to 1.2 vs 2.5 to 3.5.

For example, the weight ratio of silicone oil, squalene and triglyceride is 8, 9, 10, 11 or 12 to 0.8, 0.9, 1.0, 1.1 or 1.2 to 2.5, 2.7, 2.9, 3.1, 3.3 or 3.5.

More preferably, the viscosity of the silicone oil at 25°C is 180 mPa.s to 220 mPa.s, such as 180 mPa.s, 190 mPa.s, 200 mPa.s, 210 mPa.s or 220 mPa.s.

Preferably, the surfactant composition comprises polyoxyl hydrogenated castor oil, tween, polyethylene glycol (PEG) and 1,2-propanediol (1,2-PG), And the weight ratio of polyoxyethylene castor oil, Tween, polyethylene glycol and 1,2-propanediol is 8 to 12 to 0.8 to 1.2 to 0.8 to 1.2 to 0.8 to 1.2.

For example, the weight ratio of polyoxyethylene castor oil, Tween, polyethylene glycol and 1,2-propanediol is 8, 9, 10, 11 or 12 to 0.8, 0.9, 1.0, 1.1 or 1.2 to 0.8, 0.9, 1.0, 1.1 or 1.2 to 0.8, 0.9, 1.0, 1.1 or 1.2.

The above-mentioned polyoxyethylene castor oil is also called hydrogenated castor oil.

Preferably, the above-mentioned polyoxyethylene castor oil is polyoxyl 40 hydrogenated castor oil, also known as ^Kolliphor® RH 40, Cremophor RH 40 and macrogolglycerol hydroxystearate 40, polyethylene glycol (PEG)-40 castor oil, PEG -40 Hydrogenated Castor Oil and Macrogol 40 Hydrogenated Castor Oil).

Preferably, the above-mentioned Tween is Tween 80 (tween 80).

Preferably, the above polyethylene glycol is polyethylene glycol 400 (PEG 400).

According to the present invention, the above-mentioned microemulsion can enhance skin penetration and/or bioavailability of active ingredients.

In one embodiment, the microemulsion further carries an active ingredient, the active ingredient is water-soluble and has a molecular weight of 500 Daltons to 1000 Daltons.

For example, the molecular weight of the active ingredient is 500 Daltons, 550 Daltons, 600 Daltons, 650 Daltons, 700 Daltons, 750 Daltons, 800 Daltons, 850 Daltons, 900 Daltons, 950 Daltons or 1000 Daltons.

Preferably, the active ingredient is salvianolic acid B, which is water-soluble and has a molecular weight of 718.62.

More preferably, based on the total weight of the microemulsion, the safe concentration of the salvianolic acid B is 0.3 weight percent to 5 weight percent. The "safe concentration" refers to the concentration that can be legally used in the human body after weighing the benefits and risks of the active ingredient after use.

The present invention further provides a method for enhancing skin penetration and/or bioavailability of an active ingredient, wherein the active ingredient uses the above-mentioned microemulsion as a carrier.

The present invention further provides a method of moisturizing the skin or softening the stratum corneum of the skin comprising topical application of the above-mentioned microemulsion.

The present invention further provides a microemulsion pharmaceutical composition comprising the above microemulsion and a therapeutically effective concentration of salvianolic acid B.

Preferably, the therapeutically effective concentration is based on the total weight of the microemulsion pharmaceutical composition, and the concentration of salvianolic acid B is 0.3 weight percent to 5 weight percent.

The present invention further provides the use of the above-mentioned microemulsion pharmaceutical composition for preparing a drug for inhibiting the expression of cytokines or proliferating cell nuclear antigens by skin cells, wherein the cytokines are selected from the group consisting of: interleukin -17A (IL-17A), Interleukin-17F (IL-17F), Interleukin-22 (IL-22) and Interleukin-23 (IL-23).

The present invention further provides the use of the above-mentioned microemulsion pharmaceutical composition for preparing a medicine for treating, improving or alleviating skin symptoms caused by skin inflammation.

The present invention further provides the use of the above-mentioned microemulsion pharmaceutical composition for preparing a medicine for treating, improving or alleviating skin symptoms caused by autoimmunity.

The present invention further provides the use of the above-mentioned microemulsion pharmaceutical composition for preparing a medicine for treating, improving or alleviating psoriasis.

The present invention further provides the use of the above-mentioned microemulsion pharmaceutical composition for preparing a drug for improving or restoring skin barrier or skin moisture content, or improving abnormal skin moisture evapotranspiration, or improving abnormal proliferation of epidermal cells, wherein the skin is damaged skin or Inflamed skin.

The above therapeutically effective concentration refers to the concentration that can achieve the following effects: (1) the effect of inhibiting skin cells from expressing cytokines or proliferating cell nuclear antigen (proliferating cell nuclear antigen), wherein the cytokines are selected from the group consisting of: interleukin IL-17A (IL-17A), IL-17F (IL-17F), IL-22 (IL-22), and IL-23 (IL-23); (2) to treat, improve or slow skin symptoms caused by skin inflammation; (3) treatment, improvement or mitigation of skin symptoms caused by autoimmunity; (4) treatment, improvement or mitigation of psoriasis; or (5) improvement or restoration of skin barrier or skin moisture content, Or improve the abnormal evapotranspiration of skin water, or improve the effect of abnormal proliferation of epidermal cells, wherein the skin is damaged skin or inflamed skin.

To sum up, the microemulsion of the present invention can be used as a carrier, which can not only stably preserve the active ingredients, but also can be used to improve the skin penetration rate and/or bioavailability of the active ingredients, and at the same time moisturize the skin or soften the skin stratum corneum, so that the Water-soluble active ingredients that do not easily penetrate the stratum corneum of the skin can also be absorbed by the skin; more importantly, the microemulsion of the present invention has extremely low irritation and sensitization to damaged or inflamed skin. Therefore, the present invention provides users with damaged or inflamed skin with more diverse and safe options. Finally, the microemulsion of the present invention further comprises salvianolic acid B, which has the following effects: (1) inhibiting the protein expression of inflammatory cytokines and abnormal proliferation of epidermal cells; (2) treating, improving or slowing down the skin caused by skin inflammation (3) Treat, improve or slow down autoimmune-induced skin symptoms; (4) Treat, improve or slow down psoriasis; and (5) Improve or restore skin barrier or skin moisture content, or improve abnormal skin moisture evapotranspiration, A new topical medication option is available for patients with damaged or inflamed skin, helping to avoid other side effects from long-term use of certain medicines.

Several operation modes are provided below in order to illustrate the embodiments of the present invention; those skilled in the art can easily understand the advantages and effects achieved by the present invention through the content of this specification, and perform various operations without departing from the spirit of the present invention. Modifications and alterations to carry out or apply the teachings of the present invention.

Preparation Example 1: Three-phase Diagram Drawing

In order to find the region where the microemulsion can be formed, a "pseudo ternary phase diagram" was constructed by aqueous titration at room temperature in this experiment. Each component is respectively an aqueous phase composition, an oil phase composition and a surfactant composition; wherein, the aqueous phase composition comprises PBS buffer, sorbitol and glycerol, and is based on the total weight of PBS buffer, sorbitol and glycerol , the content of the PBS buffer is 35% by weight, the content of sorbitol is 50% by weight, and the content of glycerol is 15% by weight; the oil phase composition comprises silicone oil AR200 (silicon oil AR200), squalene and triglyceride ester, and the weight ratio of silicone oil AR200, squalene and triglyceride is 10:1:3; the surfactant composition comprises polyoxyethylene 40 hydrogenated castor oil (Cremophor RH 40), Tween 80 (tween 80) ), polyethylene glycol 400 (PEG 400) and 1,2-propylene glycol, and the weight ratio of polyoxyethylene 40 hydrogenated castor oil, Tween 80, polyethylene glycol 400 and 1,2-propylene glycol is 10:1: 1:1. All the above components were purchased from Merck KGaA, Germany.

First, the above-mentioned oil phase composition and surfactant composition are in a weight ratio of 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9 The groups of : 1 are respectively formulated into mixed solutions, and titrated with the aqueous phase composition until the mixture becomes turbid, record the content of the used aqueous phase composition, and draw the oil phase, surfactant and water phase to construct three phases. Phase diagram (as shown in Figure 1); wherein, the area on the right side of the black dotted line is the micro-emulsion area. Compared with other formulation ratios that do not form micro-emulsion on the left side, the formulation ratio in the micro-emulsion area can form a clear solution, and each component Not layered.

Preparation Example 2: Microemulsion

The microemulsion is prepared according to the proportions shown in point A to point E in FIG. 1 , and the formula ratio is shown in Table 1.

Table 1: Microemulsion formulations group Aqueous phase composition (wt%) Oil phase composition (wt%) Surfactant composition (wt%) A 30 10 60 B 20 20 60 C 10 30 60 D 10 40 50 E 10 50 40

The basis of the above weight percentage is the total weight of the water phase composition, the oil phase composition and the surfactant composition as 100 weight percent.

Preparation of experimental materials:

(1) Experimental animals:

The experimental animals in this experiment were male BALB/c mice aged 6-8 weeks, which were raised at the National Laboratory Animal Center in Tainan, and were controlled at a humidity of 40%, a temperature of 24±2°C and The animals were reared under a 12-hour light-dark cycle. All animal experiments in the present invention complied with recognized humane animal care standards and were conducted in accordance with the standard procedures approved and formulated by the Institutional Animal Care and Use Committee (IACUC) of Cathay General Hospital.

(2) Preparation of mouse skin:

Normal mice were sacrificed after anesthesia, and the full-thickness skin on the back of the mice was excised, first removed with a depilatory cream (Nair, Church & Dwight Co., Inc., Ewing, NJ, USA), and then surgically removed The subcutaneous tissue was washed with distilled water and used for the following experiments immediately.

Example 1: Skin irritation test

Four mice were used in the control group and the experimental group in this experiment. From day 0, imiquimod (IMQ) was applied for 6 consecutive days to induce psoriasis symptoms; For 5 days, the control group did not receive any treatment, while the experimental group applied the microemulsion of the above-mentioned groups A (Formulation A) to E (Formulation E) once a day, and sacrificed the mice on the 11th day. Immerse in 10% formalin aqueous solution for fixation, and then carry out embedding, sectioning and tissue staining to find the group with the least skin irritation. The experimental results are shown in Figure 2A to Figure 2F; among them, the black bottom of each group in the diagram Lines are scale and are 100 microns.

It can be seen from Figure 2A to Figure 2F that the experimental results obtained by applying the microemulsion of group A (Formulation A) are the most similar to the control group (Control), and the degree of skin thickening caused by the microemulsion of group A is the lowest, indicating that the microemulsion of group A is the lowest. Minimal irritation to skin.

Since the surfactant is irritating to the skin, it can be found from the experimental results that although the content of the surfactant composition of the group A microemulsion is higher than that of the group D microemulsion and the group E microemulsion, the microemulsion of group A is more sensitive to the skin. On the contrary, the irritation is lower, so the microemulsion of group A has the unexpected effect of low irritation.

Example 2: Stability test of microemulsion

The above-mentioned group A microemulsion was subjected to a centrifugal test to evaluate its physical stability, and a pressure test to evaluate its thermal stability; wherein, the centrifugal test was to centrifuge the group A microemulsion at 3500 rpm for 30 minutes; The microemulsion was first stored at 4°C for 48 hours, then stored at 45°C for 48 hours, and repeated 6 cycles; then, first stored at 25°C for 48 hours, and then stored at -21°C for 48 hours, And repeat 3 cycles. After the above test, the microemulsion of group A did not appear delamination, so the microemulsion of group A had good stability.

Experimental Example 3: Characterization of Microemulsion

(1) Measurement of droplet size (diameter) and zeta potential (interface potential)

The droplet size of group A microemulsion was measured at room temperature with a scattering angle of 90° using a nanoparticle size analyzer (SZ-100, Horiba Ltd., Kyoto, Japan), and the results were repeated in triplicate. The results are shown in Table 2.

(2) Conductivity measurement

Group A microemulsions were tested at room temperature using a conductivity meter (Eutech COND 6+, Eutech Instruments Ple Ltd, Thermo Fisher Scientific, Singapore) with triplicates and the results are shown in Table 2.

(3) Measurement of viscosity

Using a Visco-895 viscometer (Atago Co., Inc., Tokyo, Japan) with the needle number A3 RE-77106, the viscosity of group A microemulsion was measured at room temperature. The results are shown in Table 2.

Table 2: Physicochemical properties of group A microemulsion characteristic (unit) Group A Microemulsion Droplet size (nm) 696.2 ± 188.3 Size distribution (PI) 0.435 ± 0.004 Zeta potential (mV) (interface potential) -14.95 ± 0.64 Viscosity (cP) 3112.3 ± 5.8 Conductivity (μS/cm) 24.15 ± 0.07

PI in Table 2 is the polydispersity index.

It can be seen from Table 2 that the droplets of the microemulsion in group A are nano-scale, which can improve the skin penetration of the microemulsion; the zeta potential (interface potential) is an important indicator of suspension stability, and the higher the value of zeta potential, the higher the microemulsion. The higher the stability of the colloidal dispersion. In addition, when the conductivity of the microemulsion is 10.3 μS/cm to 52.5 μS/cm, it is defined as a bicontinuous microemulsion with oil-in-water and water-in-oil, because the conductivity of group A microemulsion The degree is about 24 μS/cm, indicating that the group A microemulsion is a bicontinuous phase microemulsion with oil-in-water and water-in-oil. It can be seen that the group A microemulsion has an oil-in-water microemulsion, which can improve the skin penetration rate of active ingredients, and has a water-in-oil microemulsion, which can reduce the irritation of the carrier to the skin, so it is especially suitable for damaged skin. or inflamed skin.

Experimental Example 4: Skin Penetration Experiment

The freshly prepared rat skin in the above experimental material preparation (2) was subjected to in vitro penetration experiments in a percutaneous absorption vertical diffusion tank (Silicon Technology, Taiwan). Among them, the experimental group used group A microemulsion as the vehicle; the control group used 75% ethanol as the vehicle of salvianolic acid B.

First, the skin samples were placed in diffusion cells (0.985 cm ² in area) and allowed to stand at 37±0.5°C for 30 minutes to achieve temperature equilibrium before the experiment, and then 200 μL containing 300 μg The microemulsion of group A of salvianolic acid B/mL was added to the sample cell (donor cells), and the PBS buffer with pH value of 7.4 was used as the receiver buffer. Ethanol was added to the receiving buffer to increase the solubility of salvianolic acid B to avoid the drug in the receiving buffer from affecting the experiment due to saturation; wherein, the weight ratio of ethanol and PBS buffer in the receiving buffer was 2 to 8 . The liquid in the receiver tank was stirred via a Teflon-coated stir bar at 600 rpm and equilibrated at 37 ± 0.5°C for 6 hours. The samples from the receiving tank were filtered with a 0.22 μm filter and analyzed by high-performance liquid chromatography (HPLC).

The control group was also tested and analyzed according to the above process, and the results are shown in Table 3.

Table 3: Skin penetration results of salvianolic acid B with different carriers parameter (unit) Experimental group (group A microemulsion) Control group (ethanol) J _s (ng/cm ² h) 574.44 ± 280.00* 52.66 ± 8.09 t _lag (hr) 1.78 ± 0.32 0.82 ± 0.14* Q (ng/cm ² ) 2411.23 ± 877.94* 305.14 ± 65.05 K _p ( x10 ^-3 cm/h) 1.91 ± 0.93* 0.18 ± 0.03

It can be seen from Table 3 that the skin flux (J _s ) and penetration coefficient (K _p ) of the experimental group are both more than 10 times that of the control group. In addition, the cumulative amount (Q) of the experimental group was about 8 times that of the control group. Although the control group used ethanol to achieve the steady state flux faster, in comparison, the group A microemulsion used in the experimental group was still more suitable as the carrier of the salvianolic acid B external medicine.

Experimental Example 5: Skin deposition test of active ingredients

Continuing from Experimental Example 4, it was divided into an experimental group and a control group, and the following experiments were carried out:

(1) Carry out a skin deposition test to quantify the deposition amount of salvianolic acid B on the skin: after the skin penetration test of Experimental Example 4 for 6 hours, wash with a mixed solution of ethanol and pure water (volume ratio 1:1) After the skin surface of the experimental group and the control group was removed five times with water to remove the excess drug on the surface, the skin was cut into small pieces, acetonitrile was added, and then homogenized to denature the protein. After completion, chloramphenicol was added as an internal standard to the resulting homogenate at a concentration of 0.1 mg/mL, and centrifuged at 13,200 rpm for 10 minutes at room temperature. Take 20 μL of the supernatant and carry out the following high-performance liquid chromatography analysis; when the high-performance liquid chromatography analysis, the concentration of salvianolic acid B is 0.1 μg/mL, and its extraction recovery rate from mouse skin is 91.5 ± 11.6 %.

(2) High performance liquid chromatography analysis: analyze with HPLC (Primaide 1110 pump, Primaide 1410 ultraviolet (UV) detector, Primaide 1210 autosampler, Hitachi, Tokyo, Japan), and use Mightysil RP- 18 column, 4.6 mm x 250 mm and particle size 5 μm (Kanto, Chemical Co. Inc., Tokyo, Japan). The mobile phase was methanol-ultrapure water (volume ratio was 60:40, pH was adjusted to 3 with orthophosphoric acid), and then filtered through a 0.22 μm Millipore filter membrane. The HPLC flow rate was set to 0.6 milliliters (mL)/min (min), the sample injection volume (ie, the injection volume) was 20 μL, and the salvianolic acid B and chloramphenicol (internal standard) were tested at a wavelength of 288 nm at room temperature. ) to check. Among them, the retention times of salvianolic acid B and chloramphenicol were 6.0 ± 0.1 minutes and 8.3 ± 0.1 minutes, respectively, and the limit of detection (LOD) and limit of quantitation (LOQ) of salvianolic acid B were Both were 0.1 μg/mL; and the accuracy and precision of salvianolic acid B at 0.1 μg/mL ranged from -2.6% to -13.2% and 0.8% to 5.1%, respectively. The results are as follows: shown in Figure 3.

As can be seen from Figure 3, the area under the curve (AUC _0-6 ) of the experimental group was 15.45 ± 1.84 μg/g tissue h; it was about 3 times that of the control group, which was 5.95 ± 4.22 μg/g tissue h; Compared with 75% ethanol, the microemulsion of group A used in group A is more suitable as the carrier of salvianolic acid B for external use.

Experimental Example 6: Skin barrier function test

(1) Induction of psoriasis: There are 4 experimental animals in each group, and according to the standard practice contained in the journal document: Methodological refinement of Aldara-induced psoriasiform dermatitis model in mice published in Scientific reports in 2019, the above 6-8 weeks old The backs of male BALB/c mice were shaved, and then 62.5 mg of ALDARA CREAM 5% (ALDARA CREAM 5%, 3M), imiquimod (IMQ), was applied daily for 6 consecutive days. The daily dose is 3.125 mg.

(2) Experimental treatment: During the experiment, the above-mentioned mice were kept in separate cages to prevent the interaction between mice from affecting the measurement of skin barrier efficacy; at the same time, each mouse toy was provided, such as: The wooden strips were randomly divided into the groups shown in Table 4 in order to meet the requirements of IACUC.

Table 4: Each group of skin barrier efficacy test group Experimental treatment result normal group No additional drug treatment was performed. Figure 4A, 6A control group Apply imiquimod (IMQ). Figure 4B, 6B Ethanol group Apply IMQ and 100 μL of 75% ethanol daily. Figure 4C, 6C Microemulsion group Apply IMQ and 100 μL of Group A microemulsion daily. Figure 4D, 6D Low Salvianolic Acid B Microemulsion Group Apply IMQ and 100 μL daily microemulsion of group A containing 100 μg/mL salvianolic acid B. Figure 4E, 6E High salvianolic acid B microemulsion group Apply IMQ and 100 μL daily microemulsion of group A containing 300 μg/mL salvianolic acid B. Figure 4F, 6F positive control group Apply IMQ and 60 mg daily of Scrub ^Shuang® Ointment. Figure 4G, 6G

The above-mentioned Esperson ^® Ointment contains 0.25% desoximetasone ⁽ DXM for short), and the drugmaker is Sanofi Co., Ltd.

Except for the normal group, the above-mentioned groups were subjected to their respective experimental treatments on the dorsal skin of the mice 3 to 4 hours after daily application of IMQ. The results are shown in Figure 4A to Figure 4G.

(3) Result evaluation: The MPA 2 system (Courage and Khazaka, Köln, Germany) equipped with Tewameter TM300, Corneometer CM825 and Mexmeter MX18 probes was used to measure in Table 4 on day 0, day 3 and day 6, respectively The transepidermal water loss (TEWL), skin hydration and skin erythema values of the dorsal surface of the mice in each group are shown in Figure 5A to Figure 5G.

It can be seen from Figure 5A to Figure 5C that the barrier measurements of the control group on day 0 were normal basal values: transdermal water loss was 7.39 ± 1.20 g/m ² /h; epidermal water was 58.25 ± 5.84 arbitrary units (arbitrary units, AU); the skin erythema generation value was 16.80 ± 1.74 AU.

It can be seen from Figure 5A and Figure 5C that, compared with the measured values of the control group on the 0th day, the transepidermal water loss and skin erythema generation values of the control group after applying imiquimod for 6 consecutive days were on the 6th day. At , both were significantly increased, 40.79 ± 8.05 g/m ² /h and 31.23 ± 4.17 AU, respectively; and it can be seen from Figure 5B that the epidermal moisture was also significantly reduced, only 3.69 ± 1.55 AU on the 6th day.

As can be seen from Figure 5A and Figure 5B, compared to the control group and the ethanol group, the microemulsion group had a relatively lower transepidermal water loss on day 3: 14.80 ± 3.47 g/m ² /h, and a significantly higher High epidermal moisture: 45.59 ± 6.85 AU; it can be seen that the microemulsion of group A used in the microemulsion group is indeed more suitable than the 75% ethanol used in the ethanol group as the carrier of salvianolic acid B external medicine, and has the effect of moisturizing the skin .

As can be seen from Figure 5D, the transdermal water loss of the control group on the 6th day was 40.79 ± 8.05 g/m ² /h, in contrast, the high salvianolic acid B microemulsion group and the positive control group were on the 6th day. The transdermal water loss is respectively 22.85 ± 6.69 g/m ² /h and 20.49 ± 5.69 g/m ² /h, it can be known that the high salvianolic acid B microemulsion group used in the A group containing high concentration of salvianolic acid B microemulsion The lotion, as well as the dehydrodicortisol (DXM) used in the positive control group, significantly restored the transdermal water loss.

As can be seen from Figure 5E, the epidermal moisture of the control group on the 3rd day was 22.29 ± 4.82 AU; in contrast, the high salvianolic acid B microemulsion group and the positive control group had significantly higher epidermal moisture on the 3rd day , and the measured values of the two groups were 52.29 ± 6.91 AU and 35.49 ± 5.56 AU, respectively. In addition, the epidermal moisture of the control group and the positive control group were 3.84 ± 1.60 AU and 6.10 ± 4.03 AU on the 6th day, respectively, and the high salvianolic acid B microemulsion group had significantly higher epidermal moisture on the 6th day: 13.94 ± 7.04 AU. It can be seen that the high salvianolic acid B microemulsion group has a better effect of improving epidermal moisture.

As can be seen from FIG. 5F , there was no significant difference between the control group, the high salvianolic acid B microemulsion group and the positive control group in the measurement value of skin erythema generation value.

To sum up, compared with the control group, both the high salvianolic acid B microemulsion group and the positive control group showed significant effects of improving transdermal water loss and epidermal water loss. It can be seen that the salvianolic acid B microemulsion of the present invention has the same performance as the market. It has the same or better efficacy as dehydrocortisol (DXM), and can be used to improve the damaged or inflamed skin barrier function due to psoriasis, and to improve or restore the skin barrier and skin moisture content, and to improve abnormal skin moisture evapotranspiration .

Experimental Example 7: Tissue staining

The mice in the normal group of Experimental Example 6 were sacrificed 48 hours after hair removal, and their full-thickness skin was taken as the negative control group for the analysis of inflammatory cytokines. The other part was subjected to protein extraction, and the obtained protein samples were stored at -80°C for later use; the rest of the groups were sacrificed after completing the above skin barrier efficacy test on the 6th day, and their full-thickness skins were taken for tissue staining and protein extraction.

For tissue staining, the skin samples were fixed in a 10% aqueous solution of formalin, routinely processed and embedded in paraffin, and then cut into 5-micron-thick sample sections and stained with hematoxylin and eosin. It was observed under an optical microscope (Olympus BX41, Tokyo, Japan), and the results were shown in Figure 6A to Figure 6G ; the black line at the bottom of each group in the figure is a scale bar and is 100 microns.

From Figure 4A to Figure 4G, it can be seen that, except for the normal group, the appearance and morphology of the mouse back of the other groups have scales, erythema and dry skin; and from the stained photos of the histopathology shown in Figure 6A to Figure 6G, it can be seen that, except for the normal group Acanthosis, parakeratosis, tortuous microvascular dilatation in papillary dermis, and inflammatory cell infiltration were found in mouse skin sections of other groups except for the other groups; however, the high salvianolic acid B microemulsion group and the positive control group showed There are relatively few serious clinical symptoms and pathological features. It can be seen that the high salvianolic acid B microemulsion group of the present invention is the same as the commercially available dehydrodicortisol (DXM), and can be used to improve damaged or inflamed skin with similar symptoms to psoriasis. Finally, because the improvement effect of severe clinical symptoms and pathological features in the low salvianolic acid B microemulsion group was not as good as that in the high salvianolic acid B microemulsion group, 300 μg/mL was the optimal concentration of salvianolic acid B. In other words, the salvianolic acid B concentration of 300 μg/mL has the dual advantages of being more effective and safe.

Experimental Example 8: Assessment of Inflammatory Cytokine Proteins

The protein samples described in Experiment 7 were subjected to the following experiments to analyze interleukin-17A (IL-17A), interleukin-17C (IL-17C), interleukin-17F (IL-17F), interleukin- 22 (IL-22), interleukin-23 (IL-23) and tumor necrosis factor alpha (tumor necrosis factor alpha, TNF-α) protein expression.

IL-17A, IL-17F, IL-22, IL-23, and TNF-alpha protein expression assays were performed using the LEGENDplex ^™ assay panel (mouse inflammation panel, BioLegend, San Diego, CA, USA); wherein, the protein samples and the labeled beads were water bathed together, and the respective cytokines were measured by a flow cytometer (Accuri C6, BD Biosciences, San Jose, CA, USA) according to the operation manual, and the results are shown in Figure 7A , as shown in Figure 7C to Figure 7F.

IL-17C protein expression analysis was performed using the mouse IL-17C detection kit (ABcam, Cambridge, MA, USA) according to the manual and on a microplate analyzer (BioTek, Winooski, VT, USA). The measurement was performed at a wavelength of 450 nm, and the results are shown in Figure 7B.

From Figure 7A, Figure 7C to Figure 7F, it can be seen that the expression of interleukin-17A, interleukin-17F, interleukin-22, interleukin-23 and tumor necrosis factor alpha protein in the control group was compared with that in the normal group. It can be seen that the expression of inflammatory cytokines is significantly increased after imiquimod is applied to the skin.

It can be seen from Figure 7A, Figure 7C, Figure 7D and Figure 7E that compared with the control group, the protein concentration of the high salvianolic acid B microemulsion group and the positive control group were significantly reduced, indicating that the high salvianolic acid B microemulsion of the present invention The group is the same as the commercially available deshydrocortisol, which can be used to inhibit the protein expression of interleukin-17A, interleukin-17F, interleukin-22 and interleukin-23.

It can be seen from Fig. 7B and Fig. 7F that the high salvianolic acid B microemulsion group of the present invention and the commercially available deshydrodicortisol do not inhibit the protein expression of interleukin-17C and tumor necrosis factor α.

To sum up, although the active ingredient and the carrier of the salvianolic acid B microemulsion of the present invention are different from those of the commercially available dehydrodicortisol, they can also be used to treat, improve or slow down the skin diseases of psoriasis symptoms.

Experimental Example 9: Evaluation of Proliferating Cell Nuclear Antigen (PCNA)

Epidermal protein was extracted from the epidermis prepared from full-thickness mouse skin, and the PCNA ELISA quantitative detection kit (Cell Biolabs Inc., San Diego, CA, USA) was used to detect the concentration of proliferating cell nuclear antigen. The results are shown in Figure 8. Show.

As can be seen from Figure 8, the protein expression of PCNA in the control group was significantly improved compared with the normal group; secondly, compared with the control group, PCNA in the high salvianolic acid B microemulsion group and the positive control group ( PCNA) protein concentrations were significantly reduced, indicating that the high salvianolic acid B microemulsion group of the present invention and the commercially available dehydrodicortisol can both be used to inhibit the protein expression of proliferating cell nuclear antigen, that is, inhibit the abnormal proliferation of keratinocytes. In other words, although the active ingredient and the carrier of the salvianolic acid B microemulsion of the present invention are different from those of the commercially available deshydrodicortisol, they can also be used to treat, improve or slow down the skin diseases of psoriasis symptoms.

To sum up, the high salvianolic acid B microemulsion group of the present invention can be used to improve damaged skin with similar symptoms to psoriasis, and can be used as a new treatment option for patients. Patients have more choices to avoid further side effects or additional life troubles caused by long-term use of certain drugs.

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Figure 1 is a three-phase diagram of a proportion of regions forming a microemulsion.

2A to 2F are the stained photos of mouse skin tissue sections in the skin irritation test.

Figure 3 shows the skin deposition amount of salvianolic acid B using different carriers.

Figures 4A to 4G are photographs of the backs of mice of each group in the skin barrier efficacy test.

Figures 5A to 5F are graphs of comparative analysis of transepidermal water loss, epidermal water and skin erythema generation values in the skin barrier efficacy test.

6A to 6G are the stained photos of mouse skin tissue sections of each group of mice in the skin barrier efficacy test.

Figures 7A to 7F are a comparison of the protein expression of IL-17A, IL-17C, IL-17F, IL-22, IL-23 and TNF-alpha in the skin barrier efficacy test diagram.

Figure 8 is a comparative analysis of the protein expression of PCNA in the skin barrier efficacy test.

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Claims (16)

A microemulsion comprising: an aqueous phase composition, including an aqueous solution and a moisturizing agent; an oil phase composition; and a surfactant composition; wherein, the water phase composition, the oil phase composition and the surfactant composition Based on the total weight of the material, the content of the water phase composition is 25% by weight to 39% by weight, the content of the oil phase composition is 8% by weight to 22% by weight, and the content of the surfactant composition is 53% by weight weight percent to 67 weight percent; and the aqueous solution is a cell isotonic solution, and the moisturizing agent comprises sorbitol (sorbitol) and glycerol (glycerol), and based on the total weight of the cell isotonic solution, sorbitol and glycerol, the The content of the cell isotonic solution is 31 to 39 percent by weight, the content of sorbitol is 45 to 55 percent by weight, and the content of glycerol is 12 to 18 percent by weight; the oil phase composition comprises silicone oil (silicone oil), squalene and triglyceride, and the weight ratio of silicone oil, squalene and triglyceride is 8 to 12 to 0.8 to 1.2 to 2.5 to 3.5; and the surfactant The composition contains polyoxyl hydrogenated castor oil, tween, polyethylene glycol (PEG) and 1,2-propanediol (1,2-PG), and polyoxyethylene castor oil, Tween The weight ratio of warm, polyethylene glycol and 1,2-propanediol is 8 to 12 to 0.8 to 1.2 to 0.8 to 1.2 to 0.8 to 1.2. The microemulsion of claim 1, wherein the microemulsion is a bicontinuous microemulsion of oil-in-water and water-in-oil. The microemulsion as claimed in claim 1, wherein the content of the water phase composition is 25 to 35 weight percent, the content of the oil phase composition is 8 to 12 weight percent, and the surfactant composition The content is 53 weight percent to 67 weight percent. The microemulsion according to claim 1, wherein the cell isotonic solution is PBS buffer; the viscosity of the silicone oil at 25°C is 180mPa.s to 220mPa.s; the polyoxyethylene castor oil is polyoxyethylene 40 hydrogenated polyoxyl 40 hydrogenated castor oil; the Tween is tween 80; or the polyethylene glycol is polyethylene glycol 400 (PEG 400). The microemulsion according to claim 1, further carrying an active ingredient, the active ingredient is water-soluble and has a molecular weight of 500 Daltons to 1000 Daltons. The microemulsion according to claim 5, wherein the active ingredient is salvianolic acid B. The microemulsion according to claim 6, wherein based on the total weight of the microemulsion, the safe concentration of the salvianolic acid B is 0.3 to 5 weight percent. A microemulsion pharmaceutical composition comprising the microemulsion according to any one of claims 1 to 4 and a therapeutically effective concentration of salvianolic acid B. The microemulsion pharmaceutical composition according to claim 8, wherein the therapeutically effective concentration is based on the total weight of the microemulsion pharmaceutical composition, and the concentration of the salvianolic acid B is 0.3 to 5 weight percent. A use of the microemulsion pharmaceutical composition according to claim 8 or 9 for the preparation of a drug for inhibiting the expression of cytokines or proliferating cell nuclear antigens in skin cells, wherein the cytokines are selected from the group consisting of : Interleukin-17A (IL-17A), Interleukin-17F (IL-17F), Interleukin-22 (IL-22) and Interleukin-23 (IL-23). A use of the microemulsion pharmaceutical composition as claimed in claim 8 or 9 for preparing a medicament for treating, improving or alleviating skin symptoms caused by skin inflammation. A use of the microemulsion pharmaceutical composition as claimed in claim 8 or 9 for preparing a medicament for treating, improving or alleviating skin symptoms caused by autoimmunity. A use of the microemulsion pharmaceutical composition as claimed in claim 8 or 9 for preparing a medicine for treating, improving or slowing down psoriasis. A use of the microemulsion pharmaceutical composition as claimed in claim 8 or 9 for preparing a drug for improving or restoring skin barrier or skin moisture content, or improving abnormal skin moisture evapotranspiration, or improving abnormal epidermal cell hyperplasia, wherein the skin is Damaged or inflamed skin. A method of enhancing skin penetration and/or bioavailability of an active ingredient, wherein the active ingredient uses a microemulsion as claimed in any one of claims 1 to 4 as a carrier. A method of moisturizing the skin or softening the stratum corneum of the skin comprising topical application of the microemulsion of any one of claims 1 to 4.

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