Preparation method and application of extracellular polysaccharide riclin-based calcipotriol nanoemulsion

Abstract

The present invention discloses a riclin-based calcipotriol nanoemulsion, the raw materials of which include: calcipotriol, oil, and an aqueous solution containing an emulsifier; wherein the emulsifier is composed of an extracellular polysaccharide riclin and polyethylene glycol in a weight volume ratio of 12.5mg:0-0.02ml; the structural formula of the extracellular polysaccharide riclin is shown in formula (I). The present invention also discloses a method for preparing the above-mentioned riclin-based calcipotriol nanoemulsion, comprising the following steps: mixing an aqueous solution containing an emulsifier with oil dissolved with calcipotriol, and ultrasonically homogenizing to obtain the riclin-based calcipotriol nanoemulsion. The present invention also discloses the use of the above-mentioned riclin-based calcipotriol nanoemulsion in the preparation of a drug for treating psoriasis. The riclin-based calcipotriol nanoemulsion of the present invention has good stability, strong anti-psoriasis effect, rapid absorption, and low irritation to the body.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a preparation method and application of an extracellular polysaccharide riclin-based calcipotriol nanoemulsion. Background Art

[0002] Psoriasis is a common chronic immune-mediated inflammatory disease. Its main symptoms are abnormal proliferation of epidermal cells, papules of varying sizes, erythema, and silvery-white scales with clear boundaries. Psoriasis often occurs on the scalp, extensor side of the limbs, and back. Psoriasis can be divided into: psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis, and arthritic psoriasis.

[0003] The clinical pathology of psoriasis is mainly keratinocyte hyperplasia, angiogenesis and inflammatory response, so there is no cure or specific treatment. Psoriasis is associated with many diseases, such as psoriatic arthritis, inflammatory bowel disease, cardiovascular disease, diabetes, infection, malignant tumors, etc. Since psoriasis vulgaris is prone to recurrence and the incidence rate has been increasing year by year, it is difficult for patients to be cured, which is a major difficulty in the current medical community.

[0004] Due to the dangers of systemic medication, topical therapy is the cornerstone of psoriasis treatment. However, the penetration of conventional topical drug preparations into the thickened stratum corneum is not satisfactory. Clinical studies have shown that creams in the drug formulation of calcipotriol (CT) are more popular with patients and can effectively improve patient compliance. Although CT shows significant anti-psoriatic effects and higher compliance, CT often leads to a high incidence of skin irritation, limiting its long-term clinical use. In addition, the formation of scales and plaques and the thickening of the stratum corneum remain the main obstacles to the transdermal administration of CT. Therefore, further improving the transdermal permeability of CT and reducing skin irritation remain the key to optimizing local efficacy. Summary of the invention

[0005] Based on the technical problems existing in the background technology, the present invention proposes a preparation method and application of an extracellular polysaccharide riclin-based calcipotriol nanoemulsion. The riclin-based calcipotriol nanoemulsion of the present invention has good stability, strong anti-psoriasis effect, rapid absorption and low irritation to the body.

[0006] The present invention provides a riclin-based calcipotriol nanoemulsion, the raw materials of which include: calcipotriol, oil, and an aqueous solution containing an emulsifier; wherein the emulsifier is composed of an extracellular polysaccharide riclin and polyethylene glycol in a weight-to-volume ratio of 12.5 mg:0-0.02 ml; the structural formula of the extracellular polysaccharide riclin is shown in formula (I);

[0007]

[0008] R is -OCCH2CH2COOH or -H, and n is an integer greater than 0.

[0009] The molecular weight of the extracellular polysaccharide riclin is 2.0×10 6 -3.0×10 6 Da; the molecular weight of the emulsifier is preferably 2.5×10 6 Da.

[0010] The extracellular polysaccharide riclin is extracted from Agrobacterium ZCC3656. Its molecular chain contains a large number of hydroxyl and carboxyl groups and has good biocompatibility and biodegradability.

[0011] When R=H, the extraction method of extracellular polysaccharide riclin includes the following steps: taking Agrobacterium sp. ZCC3656, with a preservation number of CCTCC NO: M 2018797, and sequentially subjecting it to activation, seed culture, and fermentation culture; adding an ethanol aqueous solution to the fermentation liquid for alcohol precipitation, and drying the precipitate to obtain a crude extracellular polysaccharide riclin; taking an aqueous solution of the crude extracellular polysaccharide riclin, adding an inorganic base to react, and then taking the supernatant after the reaction, and performing alcohol precipitation with an ethanol aqueous solution, and taking the precipitate to obtain the extracellular polysaccharide riclin.

[0012] In the above-mentioned method for extracting the extracellular polysaccharide riclin, the fermentation culture temperature is 28-32°C, the fermentation culture time is ≥2 days; the volume ratio of the fermentation liquid to the ethanol aqueous solution is 1:2.5-5; the volume ratio of the supernatant after the reaction to the ethanol aqueous solution is 1:2.5-5; the volume fraction of ethanol in the ethanol aqueous solution is 90-98%; when an inorganic base is added for reaction, the reaction temperature is 100-110°C, the reaction time is 15-30min, the reaction pH is 8.8-9.2, and the reaction pressure is 2-5.8kPa.

[0013] The above-mentioned crude extracellular polysaccharide riclin contains a large amount of protein and other impurities. Adding an inorganic base for reaction can remove the protein and the succinyl group at the same time. This method can quickly remove the protein and shorten the purification time, and is suitable for industrial production.

[0014] When R=OCCH2CH2COOH, the extracellular polysaccharide riclin can be purchased from the market, or the crude extracellular polysaccharide riclin can be deproteinized and purified according to the method described in the literature (Yang Y, Sun X, Zhao Y, Ge W, Ding Z, Liu J, Wang L, Xu X, Zhang J. Anti-tumor activity and immunogenicity of a succinoglycan riclin. Carbohydr Polym. 2021 Mar 1; 255: 117370. doi: 10.1016 / j.carbpol.2020.117370. Epub 2020 Nov 12. PMID: 33436203).

[0015] Preferably, the concentration of the extracellular polysaccharide riclin in the aqueous solution is 10-15 mg / ml; more preferably, the concentration of the extracellular polysaccharide riclin in the aqueous solution is 12.5 mg / ml.

[0016] Preferably, the volume ratio of oil to aqueous solution is 5-7:3-5.

[0017] Preferably, the polyethylene glycol is polyethylene glycol 400.

[0018] Preferably, the oil is a pharmaceutically acceptable oil.

[0019] Preferably, the oil is at least one of sunflower oil, soybean oil, corn oil and olive oil.

[0020] Preferably, in the riclin-based calcipotriol nanoemulsion, the particle size of the droplets is ≤100 nm; preferably, the particle size of the droplets is ≤75 nm.

[0021] Preferably, in the riclin-based calcipotriol nanoemulsion, the final concentration of calcipotriol is 5-10 mg / ml; preferably, the final concentration of calcipotriol is 5 mg / ml.

[0022] The water mentioned above can be deionized water, purified water, sterile water, etc.

[0023] The invention selects extracellular polysaccharide riclin and polyethylene glycol as co-emulsifiers, wherein the extracellular polysaccharide riclin is the main emulsifier and the polyethylene glycol is the auxiliary emulsifier, and the two cooperate with each other in an appropriate ratio and are combined with water and oil in an appropriate ratio to form a riclin-based calcipotriol emulsion, and the water-insoluble calcipotriol is uniformly dispersed in the emulsion system. The droplet particle size in the emulsion is very small and is at the nanometer level, with the smallest being about 10 nm, almost reaching the molecular scale. The nanometer-level emulsion helps the drug calcipotriol to penetrate into the psoriasis stratum corneum, thereby improving the treatment efficiency, and can also reduce the inflammatory reaction of psoriasis and reduce the irritation of the drug calcipotriol to the skin. The riclin-based calcipotriol emulsion has good biocompatibility and superior anti-psoriatic reaction, meets clinical needs, and brings a safer and more effective preparation formula for the treatment of psoriasis.

[0024] The present invention also provides a method for preparing the riclin-based calcipotriol nanoemulsion, comprising the following steps: uniformly mixing an aqueous solution containing an emulsifier with oil in which calcipotriol is dissolved, and performing ultrasonic homogenization to obtain the riclin-based calcipotriol nanoemulsion.

[0025] Preferably, the mixing temperature is room temperature.

[0026] Preferably, the mixing method is stirring mixing, the stirring speed is 1000-3000rpm, and the stirring time is 20-40min.

[0027] Preferably, the temperature of ultrasonic homogenization is 0-10°C.

[0028] Preferably, the ultrasonic homogenization time is 4-10 min, and the ultrasonic amplitude is 10-30%.

[0029] The invention selects suitable preparation methods and process parameters to prepare riclin-based calcipotriol nanoemulsion with very small particle size, and enables the emulsion to have good emulsification stability.

[0030] The present invention also proposes the use of the riclin-based calcipotriol nanoemulsion in the preparation of a drug for treating psoriasis; preferably, the psoriasis is psoriasis vulgaris.

[0031] Beneficial effects:

[0032] 1. The present invention selects extracellular polysaccharide riclin and polyethylene glycol as co-emulsifiers, wherein the extracellular polysaccharide riclin is the main emulsifier and the polyethylene glycol is the auxiliary emulsifier. The two cooperate with each other in an appropriate ratio, and are combined with water and oil in an appropriate ratio. A suitable preparation process is used to prepare a riclin-based calcipotriol emulsion, which is insoluble in water and is innovatively uniformly dispersed in the emulsion system. The droplet size in the emulsion is very small and is at the nanometer level, with a minimum of about 10 nm, almost reaching the molecular scale. The nanometer-level emulsion helps the drug calcipotriol to penetrate into the psoriasis stratum corneum, thereby improving the treatment efficiency, and at the same time can reduce the inflammatory response of psoriasis and reduce the irritation of the drug calcipotriol to the skin; the riclin-based calcipotriol emulsion has good biocompatibility and superior anti-psoriatic response, meets clinical needs, and brings a safer and more effective formulation for the treatment of psoriasis;

[0033] 2. The droplets of the riclin-based calcipotriol emulsion of the present invention are of oil-in-water type and have a sustained release effect; the emulsification effect of the riclin-based calcipotriol emulsion of the present invention is long-lasting and stable; the extracellular polysaccharide riclin has good biocompatibility and has no adverse effect on the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the particle size result of riclin-based calcipotriol nanoemulsion at different storage times.

[0035] Figure 2 The graph shows the particle size results of riclin-based calcipotriol nanoemulsion at different temperatures.

[0036] Figure 3 : This is a graph showing the particle size and zeta potential of riclin-based calcipotriol nanoemulsion at different pH values, where a is the particle size and b is the zeta potential.

[0037] Figure 4 The drug release curves of riclin-based calcipotriol nanoemulsion and calcipotriol oil, wherein CRNE is riclin-based calcipotriol nanoemulsion and CT / Oil is calcipotriol oil.

[0038] Figure 5 The immune effect diagram of each group of psoriasis mice treated in Example 5, wherein a is a graph showing changes in mouse weight, b is a graph showing spleen size of mice, c is a graph showing spleen index of mice, control is a blank control group, Model is an imiquimod group, PC is a commercially available calcipotriol cream group, CT / Oil is a calcipotriol oil group, and CRNE is a riclin-based calcipotriol nanoemulsion group.

[0039] Figure 6The PASI scores of the mice with psoriasis treated in each group in Example 5 and the effect diagram of their skin tissues, wherein a is the PASI score, b is a photo of the mouse skin, c is a HE staining photo of the mouse skin tissue, d is the dermis thickness, e is the adventitia thickness, f is the ki67 cell nucleus basement membrane, g is the erythema score, control is the blank control group, Model is the imiquimod group, PC is the commercially available calcipotriol cream group, CT / Oil is the calcipotriol oil group, and CRNE is the riclin-based calcipotriol nanoemulsion group.

[0040] Figure 7 The figure is a skin irritation test score result diagram of each group in Example 6, wherein CK is saline, Capsaicin is capsaicin, PC is calcipotriol ointment, and CRNE is riclin-based calcipotriol nanoemulsion. DETAILED DESCRIPTION

[0041] The technical solutions of the present invention are described in detail below through specific embodiments. However, it should be clearly stated that these embodiments are for illustration only and are not to be construed as limiting the scope of the present invention.

[0042] The calcitriol (CT), sunflower oil, and polyethylene glycol 400 (PEG400) used below are all from Shanghai, China. Reagent Co., Ltd.; imiquimod cream was purchased from Sichuan Mingxin Pharmaceutical Co., Ltd. (Chengdu, China); commercially available calcipotriol cream was purchased from Shandong Likang Laboratory Co., Ltd. and LEO Pharmaceuticals Co., Ltd. of Ireland; petrolatum was provided by Shandong Likang Medical Technology Co., Ltd. (Shandong, China); BALB / c mice (male, 6-8 weeks old, 21±1g) were provided by the Experimental Animal Center of Yangzhou Medical College.

[0043] The water used below is deionized water.

[0044] Example 1

[0045] The method for extracting the extracellular polysaccharide riclin when R=H comprises the following steps:

[0046] Agrobacterium sp. ZCC3656, with a deposit number of CCTCC NO: M 2018797, was taken, its colony was activated, and then inoculated into a seed culture medium (LB liquid culture medium) for seed culture; the seed culture was then transferred to a fermentation broth (M9 culture medium, wherein the carbon source was 3% sucrose), and fermented at 30° C. for 2 days; the fermentation broth was taken, 3 times the volume of precooled 95% (v / v) ethanol aqueous solution was added for alcohol precipitation, the precipitate was collected by centrifugation (6000 g, 5 min), and the crude exopolysaccharide riclin was obtained by drying at 40° C.;

[0047] The crude extracellular polysaccharide riclin was prepared into an aqueous solution with a concentration of 10 mg / ml, adjusted to pH 9.0 with an aqueous NaOH solution, sterilized under high pressure at 105°C for 20 min, and then centrifuged (10000 g, 10 min) to obtain the supernatant, added with 3 times the volume of pre-cooled 95% (v / v) ethanol aqueous solution for alcohol precipitation, and centrifuged (6000 g, 10 min) to obtain the precipitate to obtain the extracellular polysaccharide riclin.

[0048] A method for preparing a riclin-based calcipotriol nanoemulsion comprises the following steps: taking an aqueous solution containing PEG400 and the extracellular polysaccharide riclin prepared in Example 1 and sunflower seed oil in which calcipotriol is dissolved, stirring at room temperature at 1500 rpm for 30 minutes to mix evenly, and then performing ultrasonic homogenization in an ice bath (temperature of 0-10° C.) using an ultrasonic processor with a voltage of 220 V, a frequency of 20 kHz, and a probe diameter of 6 mm to obtain the riclin-based calcipotriol nanoemulsion, wherein the concentration of the extracellular polysaccharide riclin in the aqueous solution is 12.5 mg / ml, and the final concentration of calcipotriol is 5 mg / ml.

[0049] The emulsion was prepared according to the preparation method of the riclin-based calcipotriol nanoemulsion in Example 1, and the ultrasonic homogenization time (denoted as X1), the ultrasonic homogenization amplitude (denoted as X2), the volume ratio of sunflower oil to the aqueous solution (denoted as X3), and the volume fraction of PEG400 in the aqueous solution (denoted as X4) were used as independent variables. The Design expert 11.0 response surface design (RSM) was used to screen and determine the optimal formula and process, and the effects of the four independent variables on the droplet size in the riclin-based calcipotriol nanoemulsion were investigated.

[0050] Using Design-Expert 11.0 software (Stat-Ease Inc., Minneapolis, USA), 29 experiments were generated based on BBD, and the four independent variables were operated at three levels. The specific level codes and independent variables are shown in Table 1. The results of the 29 experiments are shown in Table 2.

[0051] Table 1 Three level codes and independent variable information

[0052] Independent Variable Low level (-1) Medium level(0) High level (1) X1-ultrasonic homogenization time (min) 4 7 10 X2-ultrasonic homogenization amplitude (%) 10 20 30 X3-Volume ratio of sunflower oil to aqueous solution (v / v) 50:50 60:40 70:30 X4-volume fraction of PEG400 in aqueous solution (%) 0 1 2

[0053] Table 2 BBD matrix and its 29 groups of results

[0054]

[0055]

[0056] It can be seen from Table 2 that the droplet size of the riclin-based calcipotriol nanoemulsions obtained in 29 groups of experiments is all less than 100 nm, and the emulsions are at the nanometer level.

[0057] Design-Expert 11.0 software (Stat-Ease Inc., Minneapolis, USA) was used to perform variance analysis of the fitted cubic equation and calculate the model regression coefficient, and finally the optimal independent variable parameters were obtained. The optimal independent variable parameters were used to prepare the emulsion according to the preparation method of the riclin-based calcipotriol nanoemulsion in Example 1, and the particle size of the droplets in the emulsion was detected. The results are shown in Table 3.

[0058] Table 3 Results of optimal independent variable parameters and nanoemulsion droplet size

[0059] Serial number X1 X2 X3 X4 Measured value of droplet size (nm) Predicted droplet size (nm) 1 9.722 18.746 67.51:32.49 1.759 10.6564 10.193

[0060] It can be seen from Table 3 that when the ultrasonic homogenization time is 9.722 min, the ultrasonic homogenization amplitude is 18.746%, the volume ratio of sunflower oil to aqueous solution is 67.51:32.49, and the volume fraction of PEG400 in the aqueous solution is 1.759%, the minimum particle size of the prepared riclin-based calcipotriol nanoemulsion is 10.6564 nm.

[0061] Furthermore, through the analysis of variance of the cubic equation and the calculation of the regression coefficient of the model, it was determined that the independent variable X3 (the volume ratio of sunflower oil to aqueous solution) had the most significant effect on the droplet size in the nanoemulsion; while the independent variable X4 (ultrasonic homogenization amplitude) and the independent variable X4 (volume fraction of PEG400 in aqueous solution) had no significant effect.

[0062] Example 2

[0063] According to the method described in the literature (Yang Y, Sun X, Zhao Y, Ge W, Ding Z, Liu J, Wang L, Xu X, Zhang J. Anti-tumor activity and immunogenicity of a succinoglycan riclin. Carbohydr Polym. 2021 Mar 1; 255: 117370. doi: 10.1016 / j.carbpol.2020.117370. Epub 2020 Nov 12. PMID: 33436203), the crude extracellular polysaccharide riclin was deproteinized and purified to obtain the extracellular polysaccharide riclin when R = OCCH2CH2COOH.

[0064] The extracellular polysaccharide riclin prepared in Example 2 was selected, 4 optimal independent variable parameters were selected, and a nanoemulsion was prepared according to the preparation method of the riclin-based calcipotriol nanoemulsion in Example 1. The droplet size in the nanoemulsion was detected to be 10.6564 nm.

[0065] Example 3

[0066] The nanoemulsion was prepared according to the preparation method of the riclin-based calcipotriol nanoemulsion in Example 1, wherein the independent variable X1 was 9.722, the independent variable X2 was 18.746, the independent variable X3 was 67.51:32.49, and the independent variable X4 was 1.759.

[0067] The riclin-based calcipotriol nanoemulsion was stored in a glass bottle at 4°C for 30 days. The droplet size of the nanoemulsion was measured once a week using a DTS1060 cell (Zetasizer Nano ZS, Malvern Instruments, Ltd., Malvern, UK). Figure 1 shown. Figure 1 This is the particle size result of riclin-based calcipotriol nanoemulsion at different storage times.

[0068] Depend on Figure 1 It can be seen that the average droplet size of the riclin-based calcipotriol nanoemulsion remained unchanged during storage for 1 month.

[0069] The riclin-based calcipotriol nanoemulsion was stored at 0, 20, 40, 60, 80 and 100°C for 20 min, and then allowed to stand at room temperature for 24 h. The droplet size of the nanoemulsion was measured using a DTS1060 cell (Zetasizer Nano ZS, Malvern Instruments, Ltd., Malvern, UK). The results are as follows: Figure 2 shown. Figure 2 The graph shows the particle size results of riclin-based calcipotriol nanoemulsion at different temperatures.

[0070] Depend on Figure 2 It can be seen that when the temperature increases from 0 to 100°C, the droplet size of the riclin-based calcipotriol nanoemulsion is almost unaffected.

[0071] The pH of the riclin-based calcipotriol nanoemulsion was adjusted to 2, 4, 6, 8, 10 or 12 using 1 Mmol sodium hydroxide aqueous solution or 1 Mmol hydrochloric acid aqueous solution, and after standing at room temperature for 24 hours, the droplet size and zeta potential of the nanoemulsion were measured using DTS1060cell (Zetasizer Nano ZS, Malvern Instruments, Ltd., Malvern, UK). The results are as follows: Figure 3 shown. Figure 3 : This is a graph showing the particle size and zeta potential of riclin-based calcipotriol nanoemulsion at different pH values, where a is the particle size and b is the zeta potential.

[0072] Depend on Figure 3 It can be seen that under different pH conditions, the droplet size of the riclin-based calcipotriol nanoemulsion did not change significantly; when the pH value was 10, the minimum droplet size was 13 nm; when the pH value was 2.0-12.0, the zeta potential range of the riclin-based calcipotriol nanoemulsion was -8.63 mV to -36.1 mV.

[0073] Depend on Figure 1-3 It can be seen that the riclin-based calcipotriol nanoemulsion has good temperature resistance, pH resistance and time stability, and has the potential for local application against psoriasis.

[0074] Example 4

[0075] Detecting the encapsulation efficiency and in vitro release curve of calcipotriol in the riclin-based calcipotriol nanoemulsion prepared by the optimal independent variable parameters in Example 1 specifically comprises the following steps:

[0076] 10 mL of riclin-based calcipotriol nanoemulsion was placed in a dialysis bag (3500 Da), and then immersed in 100 mL of release medium (a mixed solution of pH = 5.8 phosphate buffered saline and methanol in a volume ratio of 7:3), and the experiment was carried out at 50 rpm and 37 ± 1 ° C; 6 mL of release medium was sampled at different time points (each time sampling, an equal amount of fresh release medium was added), and the calcipotriol content was detected and calculated by HPLC, and the cumulative drug release and encapsulation efficiency were calculated. The results are as follows Figure 4 As shown, and compared with calcipotriol oil, calcipotriol oil is a sunflower oil solution of calcipotriol, and the calcipotriol content is 5 mg / ml.

[0077] The results are as follows Figure 4 As shown, Figure 4The drug release curves of riclin-based calcipotriol nanoemulsion and calcipotriol oil, wherein CRNE is riclin-based calcipotriol nanoemulsion and CT / Oil is calcipotriol oil.

[0078] Depend on Figure 4 It can be seen that the riclin-based calcipotriol nanoemulsion can sustain release for more than 36 hours in vitro; compared with the calcipotriol oil, the release of calcipotriol in the riclin-based calcipotriol nanoemulsion is a very slow process, which shows that the riclin-based calcipotriol nanoemulsion is a very good calcipotriol sustained-release system, and its calcipotriol encapsulation rate reaches 91.77%.

[0079] Example 5

[0080] A psoriasis mouse model was established, and the riclin-based calcipotriol nanoemulsion prepared with the optimal independent variable parameters in Example 1 was used for treatment and its efficacy was observed. The specific steps are as follows:

[0081] 1. Establishment of psoriasis mouse model: BALB / c mice were shaved of their back hair to form an exposure area of ​​about 2 cm × 3 cm, and were housed in single cages; the mice were randomly divided into a blank control group, an imiquimod group, a commercially available calcipotriol cream group, a calcipotriol oil group (calcipotriol sunflower oil solution with a content of 5 mg / ml), and a riclin-based calcipotriol nanoemulsion group; the number of mice in each group was the same;

[0082] The blank control group mice were smeared with 62.5 mg of vaseline on their backs daily, and the mice in the imiquimod group, the commercial calcipotriol cream group, the calcipotriol oil group, and the riclin-based calcipotriol nanoemulsion group were smeared with 5% imiquimod cream 62.5 mg once a day in the morning for 6 consecutive days; in addition, starting from the second day, the commercial calcipotriol cream group, the calcipotriol oil group, and the riclin-based calcipotriol nanoemulsion group were smeared with commercial calcipotriol cream, calcipotriol oil, and riclin-based calcipotriol nanoemulsion once a day in the evening. The morphological and histological changes of the skin lesions of psoriasis mice were observed during the medication period; the mice were killed on the morning of the 7th day, and their back skin and spleen were preserved for further in-depth analysis.

[0083] 2. Psoriasis area and severity index (PASI) score of mice: Digital photography was used to record the lesions every day, and the scores of erythema, scales and thickness of the lesions were given to the mice according to the PASI scoring standard, with a score of 0-4. The three scores were added together to get the total score. The PAIS scoring standard is as follows: 0, none; 1, mild; 2, moderate; 3, severe; 4, extremely severe. The average score of each group of mice was taken and the trend line was drawn to observe the changes in the lesions of each group of mice.

[0084] 3. Pathological examination of mouse skin lesions: HE staining and Ki67 immunohistochemistry were used for observation. Four mice in each group were killed by cervical dislocation, and the corresponding skin lesion tissues were cut and fixed in 4% paraformaldehyde, dehydrated, paraffin-embedded, sliced, HE stained and Ki67 immunohistochemically processed, and then observed.

[0085] 4. Compare the body weight and final spleen size of each group of mice during treatment.

[0086] The results are as follows Figure 5-6 shown. Figure 5 The immune effect diagram of each group of psoriasis mice treated in Example 5, wherein a is a graph showing changes in mouse weight, b is a graph showing spleen size of mice, c is a graph showing spleen index of mice, control is a blank control group, Model is an imiquimod group, PC is a commercially available calcipotriol cream group, CT / Oil is a calcipotriol oil group, and CRNE is a riclin-based calcipotriol nanoemulsion group.

[0087] Depend on Figure 5 It can be seen that mice lost weight after imiquimod induction; riclin-based calcipotriol nanoemulsion significantly improved the weight loss and splenomegaly of mice induced by imiquimod;

[0088] After continuous use of IMQ for 6 days, the spleen index of the imiquimod group was (12.01±1.46), which was more than 2.6 times higher than that of the blank control group (4.61±0.24), indicating that the mouse psoriatic dermatitis model had been established; the spleen index of the riclin-based calcipotriol nanoemulsion group, the commercially available calcipotriol cream group, and the calcipotriol oil group decreased to varying degrees compared with the imiquimod group, and the spleen index of the riclin-based calcipotriol nanoemulsion group (7.94±0.45) was the smallest;

[0089] There was almost no difference in weight loss and spleen index between the calcipotriol oil group and the imiquimod group (P>0.05), indicating that riclin-based calcipotriol nanoemulsion can effectively reduce the immune response of imiquimod-induced psoriasis.

[0090] Figure 6 The PASI scores of the mice with psoriasis treated in each group in Example 5 and the effect diagram of their skin tissues, wherein a is the PASI score, b is a photo of the mouse skin, c is a HE staining photo of the mouse skin tissue, d is the dermis thickness, e is the adventitia thickness, f is the ki67 cell nucleus basement membrane, g is the erythema score, control is the blank control group, Model is the imiquimod group, PC is the commercially available calcipotriol cream group, CT / Oil is the calcipotriol oil group, and CRNE is the riclin-based calcipotriol nanoemulsion group.

[0091] Depend on Figure 6 It can be seen that compared with the blank control group, the scores of mice in the imiquimod group gradually increased for 6 consecutive days, indicating that imiquimod successfully induced psoriatic dermatitis in mice;

[0092] Typical psoriatic skin lesions developed during the evolution of psoriasis from day 1 to day 6 in the imiquimod, commercially available calcipotriol cream, calcipotriol oil, and riclin-based calcipotriol nanoemulsion groups; however, significant improvement in psoriasis lesions was observed on day 6 after the use of commercially available calcipotriol cream or riclin-based calcipotriol nanoemulsion; whereas psoriasis skin lesions in the calcipotriol oil group were as severe as those in the imiquimod group;

[0093] According to histological evaluation, the typical pathological changes of imiquimod-induced psoriasis skin lesions are epidermal thickening, mainly manifested by increased keratinocytes and severe acanthosis, which is consistent with the common phenotype of human psoriasis skin; histological H&E staining of the tissue showed that riclin-based calcipotriol nanoemulsion can effectively alleviate the symptoms of epidermal thickening in psoriasis mice; it is worth noting that the epidermal thickness of imiquimod-induced psoriasis-like lesions on the back increased significantly to 108.1±9.4μm, while the commercially available calcipotriol cream group and the riclin-based calcipotriol nanoemulsion group were inhibited to 62.3±8.9μm and 34.6±6.6μm, respectively; there was almost no difference between the calcipotriol oil group and the imiquimod group; this shows that the effect of riclin-based calcipotriol nanoemulsion in treating psoriasis is comparable to, or even better than, the commercially available calcipotriol cream.

[0094] Example 6

[0095] The effect of riclin-based calcipotriol nanoemulsion on skin sensitivity irritation was investigated. The specific steps are as follows:

[0096] The hair on the back of BALB / c mice was shaved to form an exposure area of ​​about 2 cm × 3 cm, and the mice were housed in single cages. The BALB / c mice were randomly divided into 4 groups, with the same number of mice in each group, namely saline treatment group, capsaicin treatment group, calcipotriol ointment treatment group, and riclin-based calcipotriol nanoemulsion treatment group. During the 4 consecutive days of treatment, 200 mg of the sample was evenly distributed on the back of the mice every day and left for 2 hours.

[0097] Skin irritation was evaluated using a dryness scale; the redness or erythema of the mouse skin was assessed based on visual observation, i.e., 0-no erythema; 1-light pink (mild erythema); 2-dark pink (moderate erythema); 3-light red (moderate to severe erythema); 4-extreme redness (severe erythema). The results are shown in Figure 7 shown.

[0098] Figure 7 The figure is a skin irritation test score result diagram of each group in Example 6, wherein CK is saline, Capsaicin is capsaicin, PC is calcipotriol ointment, and CRNE is riclin-based calcipotriol nanoemulsion.

[0099] Depend on Figure 7 It can be seen that: as expected, according to the Draize scale, capsaicin has the highest skin irritation score; calcipotriol cream causes mild skin erythema; while riclin-based calcipotriol nanoemulsion does not cause obvious changes in skin irritation; riclin-based calcipotriol nanoemulsion can significantly reduce the irritation of calcipotriol to the skin; this is consistent with the research results that nano drug delivery systems can reduce drug irritation. This also means that riclin-based calcipotriol nanoemulsion can have higher patient compliance due to lower skin irritation. Therefore, riclin-based calcipotriol nanoemulsion has a brighter commercial development potential.

[0100] In summary, the riclin-based calcipotriol nanoemulsion of the present invention has an attractive low skin irritation effect, can effectively delay the release of calcipotriol, alleviate the deficiency of poor drug permeability caused by psoriasis epidermal thickening, and enhance its therapeutic effect. An innovative formula is provided for the treatment of psoriasis vulgaris; the riclin-based calcipotriol nanoemulsion prepared by selecting the optimal parameters has a droplet size of up to 10.6564nm.

[0101] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A riclin-based calcipotriol nanoemulsion, characterized in that: The raw materials include: calcipotriol, oil, and an aqueous solution containing an emulsifier; wherein the emulsifier is composed of extracellular polysaccharide riclin and polyethylene glycol in a weight-to-volume ratio of 1.25 g:0-0.02 ml; the structural formula of the extracellular polysaccharide riclin is shown in formula (I); R is -OCCH2CH2COOH or -H, and n is an integer greater than 0; The molecular weight of the extracellular polysaccharide riclin is 2.0×10 6 -3.0×10 6 Da; The concentration of the exopolysaccharide riclin in the aqueous solution is 1.2-1.3 g / ml; The volume ratio of oil to water solution is 5-7:3-5; In the riclin-based calcipotriol nanoemulsion, the particle size of the droplets is ≤100 nm.

2. The riclin-based calcipotriol nanoemulsion according to claim 1, characterized in that The polyethylene glycol was polyethylene glycol 400.

3. The riclin-based calcipotriol nanoemulsion according to claim 1, characterized in that The oil is a pharmaceutically acceptable oil.

4. The riclin-based calcipotriol nanoemulsion according to claim 1, characterized in that The oil is at least one of sunflower oil, soybean oil, corn oil and olive oil.

5. The riclin-based calcipotriol nanoemulsion according to claim 1, characterized in that In the riclin-based calcipotriol nanoemulsion, the final concentration of calcipotriol is 0.005-0.01 mg / ml.

6. A method for preparing the riclin-based calcipotriol nanoemulsion according to any one of claims 1 to 5, characterized in that: The method comprises the following steps: uniformly mixing an aqueous solution containing an emulsifier with oil in which calcipotriol is dissolved, and ultrasonically homogenizing to obtain a riclin-based calcipotriol nanoemulsion.

7. The method for preparing the riclin-based calcipotriol nanoemulsion according to claim 6, characterized in that: The mixing temperature is room temperature.

8. The method for preparing the riclin-based calcipotriol nanoemulsion according to claim 6, characterized in that: The mixing method is stirring, the stirring speed is 1000-3000rpm, and the stirring time is 20-40min.

9. The method for preparing the riclin-based calcipotriol nanoemulsion according to claim 6, characterized in that: The temperature of ultrasonic homogenization is 0-10°C.

10. The method for preparing the riclin-based calcipotriol nanoemulsion according to claim 6, characterized in that: The ultrasonic homogenization time is 4-10 min, and the ultrasonic amplitude is 10-30%.

11. Use of the riclin-based calcipotriol nanoemulsion according to any one of claims 1 to 5 in the preparation of a drug for treating psoriasis.

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