An inverted dispersion nanohydrogel, its preparation method and application

Abstract

This invention discloses a Dian Dao San nano-hydrogel, its preparation method, and its application. The preparation method includes: preparing rhubarb ultrafine powder and nano-sulfur for later use; uniformly dispersing plant-derived prolysin or milk protein in an alkaline aqueous solution containing a solubilizer to obtain a dispersion of plant-derived prolysin or milk protein; adding rhubarb ultrafine powder and nano-sulfur to the dispersion, and adjusting the pH of the system to neutral to obtain the Dian Dao San nano-hydrogel. This invention combines ultrafine pulverization technology and gel technology to prepare Dian Dao San nano-hydrogel, effectively promoting the dissolution of the active ingredients of Dian Dao San, improving the efficacy of treating acne, and providing a moisturizing, skin-friendly, stable, safe, and effective product.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparation technology, and specifically relates to a Dian Dao San nanohydrogel, its preparation method and application. Background Technology

[0002] Diandao San comes from the "Medical Mirror". It is composed of two herbs, rhubarb and sulfur. It is a classic external compound formula in traditional Chinese medicine for treating acne, rosacea and seborrheic dermatitis. Rhubarb is cold in nature and bitter in taste, clearing heat and detoxifying, while sulfur is warm in nature and pungent in taste, killing parasites and relieving itching. The two herbs have opposite properties of cold and heat, hence the name Diandao San.

[0003] Currently, the clinical application of DDS (Diandao San) involves grinding rhubarb and sulfur into coarse powder, mixing equal amounts of the two powders, mixing with cold water to form a paste, and then applying it directly to the affected area (Observation on the efficacy of DDS in treating 30 cases of acne. Journal of Clinical Traditional Chinese Medicine. 2019, 31(08)). The actual efficacy of this drug still falls short of meeting people's needs and urgently needs improvement. Furthermore, it suffers from uneven application, poor skin affinity, and easy drying and peeling, resulting in a poor user experience and severely limiting the application and promotion of DDS. Summary of the Invention

[0004] To address the aforementioned issues, this invention combines ultrafine pulverization and gel technology to prepare DDS-NG nano-hydrogel, which effectively promotes the dissolution of DDS-NG active ingredients, enhances the efficacy of acne treatment, and provides a moisturizing and skin-friendly product with stable quality, safety, and effectiveness.

[0005] In one aspect, the present invention provides a method for preparing a didaosan nanohydrogel, the method comprising: preparing rhubarb ultrafine powder and nano sulfur for later use; uniformly dispersing plant-derived prolysin or milk protein in an alkaline aqueous solution containing a solubilizer to obtain a dispersion of plant-derived prolysin or milk protein; adding rhubarb ultrafine powder and nano sulfur to the dispersion, adjusting the pH of the system to neutral, thereby obtaining the didaosan nanohydrogel.

[0006] As a specific embodiment of the present invention, the fineness of the rhubarb ultrafine powder is 160~600 mesh, preferably 300 mesh; the size of the nano sulfur is 1~100 nm.

[0007] As a specific embodiment of the present invention, the plant-derived prolysin is either zein or wheat prolysin, preferably zein, and more preferably zein with a particle size of 50-80 nm.

[0008] In a specific embodiment of the present invention, the milk protein is casein.

[0009] In a specific embodiment of the present invention, the solubilizer is diethylene glycol monoethyl ether.

[0010] As a specific embodiment of the present invention, the pH of the alkaline aqueous solution is 10~12.8.

[0011] In a specific embodiment of the present invention, the mass ratio of rhubarb ultrafine powder to nano sulfur in the system is 1~3:1~3, the amount of plant-derived alcohol-soluble protein or milk protein added is 3~15%wt, and the amount of solubilizer added is 2~10%wt.

[0012] As a specific embodiment of the present invention, rhubarb ultrafine powder is first added to the dispersion, stirred at room temperature for 0.2~0.5 h, then nano sulfur is added, and then the pH of the system is adjusted to 7.4±0.2 with hydrochloric acid.

[0013] In a second aspect, the present invention provides an inverted dispersion nanohydrogel prepared by the aforementioned preparation method.

[0014] A third aspect of the present invention provides the use of the aforementioned inverted nanohydrogel in the preparation of a medicament for treating acne.

[0015] The beneficial effects of this invention are as follows:

[0016] The preparation method provided by this invention involves: 1) preparing rhubarb and sulfur into ultrafine powders, which, compared to their respective coarse powders, are more conducive to the dissolution and release of active ingredients; furthermore, an alkaline aqueous solution containing plant-derived prolysins or milk proteins is added. On the one hand, the slightly alkaline environment is conducive to the dissolution of active ingredients such as rhein, emodin, and aloe-emodin in rhubarb; on the other hand, the self-assembled nanoparticles of plant-derived prolysins or milk proteins have the effect of adsorbing free rhein, emodin, and aloe-emodin in the dispersion, which can further synergistically promote the release of these active ingredients. 1) It is dissolved from rhubarb powder; 2) As a preferred option, when diethylene glycol monoethyl ether is used as a solubilizer, this component can increase the solubility and stability of plant-derived alcohol-soluble proteins or milk proteins in alkaline aqueous solutions, and at the same time, it can act as a permeation enhancer to improve the affinity and transdermal permeability of the final prepared Dian Dao San nano-hydrogel with the skin; 3) For the first time, ultra-micro pulverization technology and gel technology are combined to change the dosage form of Dian Dao San, an ancient traditional powder, which is convenient to use and easy to carry, providing a new idea for the inheritance and innovation of this formula; 4) It is simple and easy to implement, and suitable for industrial production applications.

[0017] The inverted powder nano-hydrogel prepared by this invention has small particle size, uniform dispersion, high dissolution of medicinal ingredients, good therapeutic effect on acne, safety and non-irritation, and a delicate gel texture that is skin-friendly and moisturizing, does not easily dry out and fall off, and provides a good patient experience. Attached Figure Description

[0018] Figure 1 Particle size distribution diagrams of ordinary inverted powder (a) and inverted powder nanohydrogel (b);

[0019] Figure 2 FESEM images (×500) of inverted powder (a) and inverted powder nanohydrogel (b);

[0020] Figure 3 Standard curves for aloe-emodin, rhein, and emodin solutions;

[0021] Figure 4 Cumulative dissolution curves of (A) aloe-emodin, (B) rhein and (C) emodin in Dian Dao San and Dian Dao San nanohydrogel. ±S, n = 3);

[0022] Figure 5 Minimum inhibitory concentration (MIC) results of Dian Dao San solution (a) and Dian Dao San nanohydrogel (b) against Propionibacterium acnes (n = 3);

[0023] Figure 6 Rabbit skin irritation test (administration areas on normal and broken skin: 1-physiological saline, 2-pure water, 3-DDS suspension, 4-DDS-NG).

[0024] Figure 7 Comparison of skin lesions in acne model rats in different groups; (a) normal control group; (b) acne model group; (c) isotretinoin positive control group; (d) low-dose DDS-NG(L) nano-hydrogel group; (e) medium-dose DDS-NG(M) nano-hydrogel group; (f) high-dose DDS-NG(H) nano-hydrogel group;

[0025] Figure 8 : H&E sections of ear tissue from acne model rats in each group (×10); among which: (a) control group; (b) model group; (c) isotretinoin positive control group; (d) low-dose DDS-NG(L) nanohydrogel group; (e) medium-dose DDS-NG(M) nanohydrogel group; (f) high-dose DDS-NG(H) nanohydrogel group;

[0026] Figure 9 Levels of inflammatory factors (IL-1α, IL-6, and TNF-α) in rat ear tissue (n = 8); among which: compared with the control group, #### P <0.0001; compared with the model group, * P <0.05, ** P <0.01,*** P <0.001, **** P <0.0001;

[0027] Figure 10 Serum levels of inflammatory factors (IL-1α, IL-6, and TNF-α) in rats (n = 8); among which, compared with the control group, ### P <0.001, #### P <0.0001; compared with the model group, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001;

[0028] Figure 11 Atomic force microscopy image of a zein nanodispersion. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1

[0031] A method for preparing inverted dispersion nanohydrogels is as follows:

[0032] 1) After drying rhubarb slices in a 50 ℃ constant temperature drying oven for 2 hours, they are then pulverized using an ultra-micro pulverizer and passed through a 300-mesh sieve to obtain rhubarb ultra-micro powder for later use.

[0033] 2) Take the purchased nano sulfur and set it aside;

[0034] 3) Zeat protein was uniformly dispersed in an aqueous solution of sodium hydroxide at pH 12 containing diethylene glycol monoethyl ether to obtain a zeat protein nano-dispersion.

[0035] 4) First, add rhubarb ultrafine powder to the zein nano-dispersion, stir at room temperature for 0.2 h, then add nano sulfur. The mass ratio of rhubarb ultrafine powder to nano sulfur is 1:1. The amount of zein added is 3%wt, and the amount of diethylene glycol monoethyl ether added is 2%wt. Finally, quickly adjust the pH of the system to 6.8 with hydrochloric acid to obtain the inverted dispersion nanohydrogel DDS-NG.

[0036] Example 2

[0037] A method for preparing inverted dispersion nanohydrogels is as follows:

[0038] 1) After drying rhubarb slices in a 50 ℃ constant temperature drying oven for 2 hours, they are then pulverized using an ultra-micro pulverizer and passed through a 600-mesh sieve to obtain rhubarb ultra-micro powder for later use.

[0039] 2) Take the purchased nano sulfur and set it aside;

[0040] 3) Wheat gliadin was uniformly dispersed in an aqueous sodium hydroxide solution containing diethylene glycol monoethyl ether at pH 11 to obtain a wheat gliadin nano-dispersion.

[0041] 4) First, add rhubarb ultrafine powder to the wheat gliadin nano-dispersion, stir at room temperature for 0.5 h, then add nano sulfur. The mass ratio of rhubarb ultrafine powder to nano sulfur is 1:3. The amount of wheat gliadin added is 15%wt, and the amount of diethylene glycol monoethyl ether added is 10%wt. Finally, quickly adjust the pH to 7.2 with hydrochloric acid to obtain the inverted dispersion nano-hydrogel DDS-NG.

[0042] Example 3

[0043] A method for preparing inverted dispersion nanohydrogels is as follows:

[0044] 1) After drying rhubarb slices in a 50 ℃ constant temperature drying oven for 2 hours, they are then pulverized using an ultra-micro pulverizer and passed through a 160-mesh sieve to obtain rhubarb ultra-micro powder for later use.

[0045] 2) Take the purchased nano sulfur and set it aside;

[0046] 3) Casein was uniformly dispersed in an aqueous solution of sodium hydroxide at pH 12.8 containing diethylene glycol monoethyl ether to obtain a casein nano-dispersion.

[0047] 4) First, add rhubarb ultrafine powder to the casein nano-dispersion, stir at room temperature for 0.3 h, then add nano sulfur. The mass ratio of rhubarb ultrafine powder to nano sulfur is 3:1. The amount of casein added is 8%wt, and the amount of diethylene glycol monoethyl ether added is 6%wt. Finally, quickly adjust the pH to 7.2 with hydrochloric acid to obtain the inverted dispersion nanohydrogel DDS-NG.

[0048] Test case

[0049] 1. Particle size analysis

[0050] The particle size distribution of DDS and DDS-NG nanohydrogel from Example 1 was determined using a BT-9300S laser particle size analyzer. Each sample was measured three times, and the average value was taken. The results are shown in Table 1. The average particle size (D50) of the DDS nanohydrogel was significantly smaller than that of DDS. Figure 1 The figure shows the particle size distribution curves of the inverted dispersion and the inverted dispersion nanohydrogel. The inverted dispersion curve shows that the peak particle size distribution is after 100 μm, while the peak particle size distribution of the inverted dispersion nanohydrogel curve is between 10 and 100 μm, indicating a wider particle size distribution range. The D90 values ​​of the inverted dispersion and the inverted dispersion nanohydrogel are approximately 180.6 μm and 51.71 μm, respectively, which is basically consistent with the particle size distribution results in Table 1.

[0051] Table 1. Particle size distribution of DDS-NG and its nanohydrogel (DDS-NG) ± S, n = 3)

[0052]

[0053] 2. Morphological observation

[0054] The microstructure of the inverted powder and inverted powder nanohydrogel samples (Example 1) was observed using FESEM. First, the samples were adhered to a sample stage coated with conductive adhesive, followed by gold sputtering. The accelerating voltage was set to 5.0 kV. The morphological characteristics of the inverted powder and inverted powder nanohydrogel samples were observed under the electron microscope as follows: Figure 2 It can be seen that their particles are regular and irregular spherical and short rod-shaped. The particles in ordinary DDS are large and severely agglomerated, while DDS-NG is relatively uniformly dispersed and smaller in size at a magnification of 500x.

[0055] 3. Establishment of in vitro analytical methods for the effective components of Dian Dao San and Dian Dao San nano-hydrogel and determination of their content.

[0056] 3.1 Linear Range

[0057] Accurately weigh 1.67 mg, 1.32 mg, and 1.24 mg of aloe-emodin, rhein, and emodin reference standards, respectively, and place them in a 10 mL brown volumetric flask. Dissolve them by sonication with methanol, cool, and then add methanol to make up to volume to obtain the stock solution of the active ingredients of Diandao San (each 1 mL contains 167 μg of aloe-emodin, 132 μg of rhein, and 124 μg of emodin).

[0058] Chromatographic conditions: Column: 250 mm × 4.6 mm, 5 μm Agilent C18 column; Mobile phase: methanol-0.1% phosphoric acid (85:15, v:v); Detection time: 15 min; Flow rate: 1.0 mL / min; Column temperature: 30 ℃; Detection wavelength: 254 nm; Injection volume: 10 μL.

[0059] Preparation of reference solutions: Accurately measure an appropriate amount of standard stock solution and dilute it to a reference solution of aloe-emodin, rhein, and rhein inverted powder with concentrations of 33.4 µg / mL, 26.4 µg / mL, and 24.8 µg / mL.

[0060] Preparation of the test solution: Accurately weigh 1 g of inverted powder nanohydrogel, place it in a stoppered flat-bottomed flask, accurately add 25 mL of methanol, weigh the mass, heat under reflux for 1 h, cool, add methanol to the previously weighed mass, shake well, filter through a 0.45 µm filter membrane, and obtain the filtrate.

[0061] Accurately pipette 1, 2, 4, 6, 8, and 10 mL of the stock solution into separate 10 mL volumetric flasks, add methanol to the mark, mix well, and inject 10 μL of each flask under the chromatographic conditions described above. Plot the standard concentration as the x-axis (X) and the peak area as the y-axis to fit a standard curve.

[0062] The resulting equations are: aloe-emodin Y = 0.7843X - 0.0689 (R² = 0.9994), linear range 16.7–167 μg / mL; rhein Y = 0.5447X - 4.982 (R² = 0.9998), linear range 13.2–132 μg / mL; emodin Y = 0.5973X - 0.5104 (R² = 0.9996), linear range 12.4–124 μg / mL. The design point coordinates between 30 and 40 μg / mL were abnormal and therefore discarded. The results are shown below. Figure 3 .

[0063] 3.2 Determination of aloe-emodin, rhein, and emodin content

[0064] Accurately measure the sample solution of the inverted powder, filter it through a 0.22 μm microporous membrane, and collect the filtrate. The peak areas of aloe-emodin, rhein, and emodin in the extract were determined by HPLC, and the mass concentrations of aloe-emodin, rhein, and emodin in the sample solution were calculated. The results are shown in Table 2.

[0065] 3.3 Methodological Examination

[0066] 3.3.1 Precision

[0067] The same reference solution was tested 6 times under specified conditions. The RSDs of aloe-emodin, rhein, and emodin were calculated to be 0.08%, 0.06%, and 0.39%, respectively, indicating that the instrument has good precision.

[0068] 3.3.2 Recovery rate

[0069] The average recoveries of aloe-emodin, rhein, and emodin were 97.42%, 96.71%, and 96.28% under the specified chromatographic conditions, with RSDs of 1.71%, 1.70%, and 2.00%, respectively, indicating that the method meets the methodological requirements.

[0070] 3.3.3 Repeatability

[0071] The RSDs of aloe-emodin, rhein, and emodin were 1.59%, 1.37%, and 2.00% respectively, as determined under the specified chromatographic conditions, indicating that the method has good repeatability.

[0072] 3.3.4 Stability Test

[0073] The inverted powder nano-hydrogel test solution was prepared according to the above method. The RSDs of aloe-emodin, rhein, and emodin were 0.283%, 0.212%, and 0.18% at 0, 2, 4, 8, 12, and 24 h, respectively, under chromatographic conditions, indicating that the solution has good stability within 24 h.

[0074] 4. Determination of the in vitro dissolution rate of three indicator components in the inverted powder nanohydrogel

[0075] The dissolution rate of the active ingredient in the inverted powder nanogel of Example 1 was determined according to the small cup method, Method 3, General Chapter (0941), Part IV of the 2020 edition of the Chinese Pharmacopoeia. The experiment was repeated three times, and the cumulative dissolution rate (Yn′) was calculated. The cumulative dissolution formula is as follows:

[0076] Yi′ = Yi + (Y1 + Y2 + Y3 +......+ Y (i-1)) V2 / V1

[0077] Yi is the relative dissolution rate measured after the i-th calibration, Yi′ is the relative dissolution rate measured in the i-th actual measurement, V1 is the total volume of pure water, and V2 is the volume of pure water added after each sampling.

[0078] Cumulative dissolution curves were plotted using GraphPad Prism software version 6 (GraphPad software, Inc.). The results are shown below. Figure 4 .

[0079] It can be seen that the dissolution rates of the three active ingredients in the DDS-NG nanogel are higher than those in the DDS-NG nanogel.P <0.05). For example, at 0.3 h, the in vitro dissolution rates of aloe-emodin, rhein, and emodin in DDS-NG were 2.5 times, 1.3 times, and 3.0 times that of ordinary DDS (see [link to DDS]). Figure 4 ).

[0080] 5. Antibacterial test

[0081] 5.1 Drug susceptibility testing

[0082] Antimicrobial susceptibility testing was performed using the perforation method. First, take 1×10⁻⁶ of the test bacterial suspension. 8 Add 100 μL of the agar medium to the thioglycolate solid medium, spread it evenly with a spreader, and allow it to dry. Make four wells in the petri dish using a 200 μL pipette tip, making each well in one go. After making the wells, use a sterile needle to remove the medium from the agar wells. After each use, sterilize the needle with an alcohol lamp and seal the bottom with a flame to ensure the medium fully integrates with the dish and prevent leakage that could affect the results. Then, add 20 μL of the prepared invertase solution (passed through a 200-mesh sieve) and the invertase nano-hydrogel from Example 1 to the agar wells respectively. Cover the petri dish, place it in an anaerobic bag with an anaerobic gas generator, seal the bag, and incubate at 37 ℃ for 48 h. Results are shown below. Figure 5 .

[0083] 5.2 MIC determination of P. acnes by inverted nanogel

[0084] The MIC values ​​of the inverted powder nanohydrogel solution against *P. acnes* were determined using a 96-well microplate method with a two-fold dilution and plate coating technique. Sample solutions were diluted in 96-well plates at concentrations ranging from 200 to 1.5625 mg / mL, and then 100 µL of bacterial solution (1×10⁻⁶) was added to each well. 6 CFU / mL), final density 5×10 5CFU / well. Well 9 (P. acnes bacterial suspension + FT liquid medium) served as a negative control, and well 10 (FT liquid medium) served as a blank control. After culturing the 96-well microplates in an anaerobic culture bag at 37 ℃ for 48 h, 100 μL of suspension was absorbed from each well and inoculated into FT solid medium, and then incubated anaerobically at 37 ℃ for 48 h. The presence of sterile colonies was observed on a non-reflective, dark-colored surface. The drug concentration at which sterile colonies were formed was defined as the concentration that could completely inhibit the growth of P. acnes, i.e., the MIC value of P. acnes. The above experimental steps were repeated three times to obtain the final results. The results, as shown in Table 2, indicate that the diameters of the inhibition zones of Diandaosan and Diandaosan nanohydrogel were 18.77 ± 0.74 mm and 28.27 ± 1.16 mm, respectively. It is evident that the in vitro antibacterial effect of Diandaosan nanohydrogel is significantly better than that of Diandaosan, which is consistent with the high dissolution rate of the three active ingredients.

[0085] Table 2. Diameter of inhibition zone and MIC value of inverted powder and inverted powder nanogel against P. acnes (n = 3)

[0086]

[0087] 6. Skin irritation test:

[0088] Eight healthy male rabbits, weighing 2.0–2.5 kg, were purchased. This experiment used a self-control method with left and right sides as the same animal. Twenty-four hours before the experiment, the fur on both sides of the rabbits' backs was shaved, exposing approximately 40–60 cm of their backs. 2 Divide the exposed back into four equal sections. Prepare the broken skin: Disinfect the skin surface with 75% alcohol swabs. Using a sterile needle, draw a crisscross pattern on each of the four hairless skin sections, approximately 3 x 3 cm in size. 2 The degree of skin damage should be judged by slight bleeding, and the degree of damage on the four skin patches should be as consistent as possible. Figure 6 As shown.

[0089] The experimental groups were selected as Diandaosan and Diandaosan nano-hydrogel (Example 1), while the control groups were blank pure water and physiological saline. A rabbit autologous left-right side control method was used to investigate the skin irritation of Diandaosan ultrafine powder on rabbits. Exposed skin was divided into four zones (1, 2, 3, and 4), and the drug administration groups were as follows: Normal skin group: 1 (physiological saline), 2 (pure water), 3 (Diandaosan, 15%), 4 (Diandaosan nano-hydrogel, 15%); Damaged skin group: 1 (physiological saline), 2 (pure water), 3 (Diandaosan, 15%), 4 (Diandaosan nano-hydrogel, 15%), where 15% represents the drug content of Diandaosan and Diandaosan nano-hydrogel. After drug administration, medical gauze was used to cover the drug administration site on the rabbits with medical tape. Four hours after local application, the tested skin was washed with warm water to remove residual liquid. The results, based on the evaluation of skin irritation, showed that Diandaosan and Diandaosan nano-hydrogel had no significant irritation to either normal or damaged skin.

[0090] 7. Therapeutic effect of Dian Dao San nanohydrogel on acne model rats

[0091] Forty-eight healthy male rats were acclimatized for 7 days. Eight rats were randomly selected as a blank control group, and the remaining 40 rats were used to establish a right ear acne model using a modified Kligman method. First, 0.25–0.5 mL of 100% oleic acid was evenly applied into the opening of the inner ear canal of the rat's right ear. Then, 50 μL (6 × 10⁻⁶) was subcutaneously injected into the right auricle of the rat. 7 P. acnes bacterial suspension (CFU / mL). The left ear of rats was used for comparison without treatment. The above modeling was repeated for 4 weeks.

[0092] One day before drug administration, acne model rats were randomly divided into 5 experimental groups (n=8 per group): model group (equal volume of 0.9% sodium chloride solution), low-dose group of the inverted powder nanohydrogel from Example 1 (0.02 g / cm³), and acne model rats. 2 / d), Example 1: Medium-dose group of inverted powder nanohydrogel (0.04 g / cm³) 2 / d), Example 1: High-dose group of inverted powder nanohydrogel (0.08 g / cm³) 2 The rats were divided into a control group (5 g / kg / day) and a positive control group (0.025% isotretinoin cream, dose: 5 g / kg / day). Each group of rats was administered the corresponding drug once daily. Simultaneously, the control group and the model group were given an equal volume of 0.9% sodium chloride solution. The recovery status of the rats in each group was observed after 2 weeks. Figure 7 Physiological saline had no significant effect on improving the skin of the right ear in the model group. The low-dose group of the inverted powder nano-hydrogel (0.02 g / cm³) 2 / d) The symptoms in the right auricle of the rat gradually improved, the color became slightly red, the skin felt soft to the touch without any burning sensation. Medium dose group (0.04 g / cm³) 2 / d), high-dose group (0.08 g / cm³) 2 In the positive control group and the right ear auricle of rats, redness and swelling were relieved, skin temperature decreased, and the auricle became smooth and soft with tight pores and clearly visible capillaries. Compared with the model group, the low, medium, and high doses of the inverted powder nanohydrogel treatment groups all showed significant improvement in acne symptoms. The blank control group showed no significant change in acne symptoms.

[0093] After drug administration, a sample was taken from the right ear of each group of rats, and one rat was randomly selected for H&E staining and sectioning. The therapeutic effect after drug administration was then analyzed using histopathological tissue analysis. Figure 8 H&E staining results showed that the epidermis of the rat ear in the blank group was of moderate thickness, with hair follicles and dermis clearly visible, and inflammatory cells in the dermis were also clearly visible, with no other significant changes. Compared with the blank group, the model group rat ear epidermis was thickened, hair follicles were swollen, sebaceous glands in the dermis were hyperplastic, fibrous tissue was thickened, inflammatory necrosis formed in the dermal subcutaneous stroma, and inflammatory cell infiltration was visible. Compared with the model group, the positive drug group showed thinner fibrous tissue and a reduced number of infiltrated inflammatory cells. With the continuous increase of drug concentration, the epidermal thickness of the Dian Dao San nanohydrogel group gradually became more uniform, keratin, inflammatory cells and fibrous necrosis gradually decreased, inflammatory cells gradually disappeared, and the subcutaneous collagen fibers in the high-dose Dian Dao San nanohydrogel group were arranged in an orderly manner.

[0094] This invention investigated the effects of inverted powder nanohydrogel on changes in inflammatory factors in the ear tissue and serum of acne model rats. The results showed that the levels of inflammatory factors such as IL-1α, TNF-α, and IL-6 in the right ear tissue of the acne model group rats were significantly increased compared with those in the normal group. #### P <0.0001); Compared with the model group, the expression levels of inflammatory factors were significantly reduced to varying degrees after treatment with different doses of DDS-NG (<0.0001); * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001), such as Figure 9 As shown. Meanwhile, the serum levels of inflammatory factors such as IL-1α, TNF-α, and IL-6 in acne model rats were significantly increased compared to the normal group (as shown). ### P <0.001); Compared with the model group, the expression levels of inflammatory factors were significantly reduced to varying degrees after treatment with different doses of DDS-NG (<0.001); * P <0.05, ** P <0.01, ***P <0.001, **** P <0.0001), such as Figure 10 As shown in the figure, DDS-NG can significantly downregulate the inflammatory response in acne model rats.

[0095] 8. Clinical trial on young volunteers with acne:

[0096] Case selection: Eighteen patients with confirmed acne were selected. The left and right face comparison method was used to evaluate the clinical efficacy of the Diandaosan nanohydrogel (the left face was treated with the Diandaosan nanohydrogel of Example 1, while the right face was left untreated).

[0097] Medication selection: Diandao San nano hydrogel, for external use, twice a day. One course of treatment consists of 28 consecutive days of use, with a follow-up visit every 7 days. The observation period is one course of treatment.

[0098] Efficacy assessment: % reduction in lesions = (number of lesions before administration) (Number of lesions after drug administration) / Number of lesions before drug administration.

[0099] Table 3. Percentage reduction in total lesions on the left and untreated right cheeks treated with Dian Dao San nano-hydrogel.

[0100]

[0101] The percentage reduction in lesions was significantly higher on the left cheek than on the right cheek, with a significant reduction in pustules and inflammation. Patients also reported that the Dian Dao San nano-hydrogel was easy to use, had a delicate texture, and was skin-friendly and moisturizing. This indicates that the Dian Dao San nano-hydrogel for treating acne is safe and effective and can be promoted and used with confidence.

[0102] 9. Particle size of zein nanodispersion

[0103] The zein nanodispersion prepared in Example 1 was dropped onto a glass slide, dried, and then photographed using an atomic force microscope. The results showed that the diameter of the zein particles ranged from 50 to 80 nm. Figure 11 .

[0104] 10. Results Analysis

[0105] The average particle size of the inverted dispersion nanohydrogel (DDS-NG) prepared in this invention is 15.08 μm (see Table 1). Figure 1 , Figure 2 The active ingredients, aloe-emodin, rhein, and emodin, are evenly dispersed; their dissolution rates are significantly higher than those of ordinary DDS (Distilled Dispersant System). For example, at 0.3 h, the in vitro dissolution rates of aloe-emodin, rhein, and emodin in DDS-NG are 2.5 times, 1.3 times, and 3.0 times that of ordinary DDS (Distilled Dispersant System) (see [link to DDS-NG]). Figure 4 ).

[0106] DDS-NG showed significantly better in vitro antibacterial effects against Propionibacterium acnes than ordinary DDS, with an inhibition zone diameter increased by approximately 10 mm and the half-maximal inhibitory concentration (MIC) value reduced by half (see...). Figure 5 (Table 2) This is due to the high dissolution and release of the three active ingredients in the DDS-NG nanohydrogel, which exerts the drug effect.

[0107] Skin irritation test results showed that DDS-NG had no significant irritant effect on normal or damaged skin in rabbits (see...). Figure 6 ).

[0108] This invention uses a rat model of acne in the right ear to evaluate the efficacy of DDS-NG. The results show that DDS-NG can effectively alleviate acne symptoms. Figure 7 , Figure 8 ), and at the same time, it can significantly reduce the levels of inflammatory factors in the right ear tissue and serum of rats ( Figure 9 , Figure 10 The high-dose group showed the therapeutic effect of isotretinoin cream, a positive control drug, on rats with acne.

[0109] Meanwhile, the results of a self-controlled study of four acne patients who received DDS-NG treatment (Table 3) showed that the percentage reduction in acne lesions after administration was significantly higher than that on the untreated side. Patients also reported a significant reduction in pustules and inflammation. Furthermore, patients reported that the DDS-NG nano-hydrogel was convenient to use, had a delicate texture, and was skin-friendly and moisturizing. This indicates that the product of this invention is safe and effective in treating acne and can be promoted and used with confidence.

[0110] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for preparing an inverted dispersion nanohydrogel, characterized in that, The preparation method comprises the following steps: Preparation of ultra-fine powder of rhubarb and nano-sulfur is prepared; The plant-derived prolamine or milk protein is uniformly dispersed in an alkaline aqueous solution containing a solubilizer, i.e. diethylene glycol monoethyl ether, to obtain a dispersion of the plant-derived prolamine or milk protein; The ultra-fine powder of rhubarb and nano-sulfur are added to the dispersion, and the pH of the system is adjusted to neutral to obtain the nano-hydrogel of Danpousan.

2. The method for preparing the inverted dispersion nanohydrogel according to claim 1, characterized in that, The fineness of the ultra-fine powder of rhubarb is 160-600 mesh, and the size of the nano-sulfur is 1-100 nm.

3. The method of claim 2, wherein the inverted dispersion nanohydrogel is prepared by the steps of: The fineness of the ultra-fine powder of rhubarb is 300 mesh.

4. The method of claim 1, wherein the inverted dispersion nanohydrogel is prepared by the steps of: The plant-derived prolamine is any one of corn zein and wheat prolamine.

5. The method of claim 4, wherein the inverted dispersion nanohydrogel is prepared by the steps of: The plant-derived prolamine is corn zein.

6. The method of claim 5, wherein the inverted dispersion nanohydrogel is prepared by the steps of: The plant-derived prolamine is corn zein with a particle size of 50-80 nm.

7. The method for preparing the inverted dispersion nanohydrogel according to claim 1, characterized in that, The milk protein is casein.

8. The method of claim 1, wherein the inverted dispersion nanohydrogel is prepared by the steps of: The pH of the alkaline aqueous solution is 10-12.

8.

9. The method of claim 1, wherein the inverted dispersion nanohydrogel is prepared by the steps of: In the system, the mass ratio of the ultra-fine powder of rhubarb to nano-sulfur is 1-3:1-3, the addition amount of the plant-derived prolamine or milk protein is 3-15%wt, and the addition amount of the solubilizer is 2-10%wt.

10. The method for preparing the inverted dispersion nanohydrogel according to claim 1, characterized in that, The ultra-fine powder of rhubarb is first added to the dispersion, and then the nano-sulfur is added after stirring at room temperature for 0.2-0.5 h, and then the pH of the system is adjusted to 7.4±0.2 by using hydrochloric acid.

11. The nano-hydrogel of Danpousan prepared by the preparation method of any one of claims 1-10.

12. The use of the nano-hydrogel of Danpousan of claim 11 in the preparation of a medicament for treating acne.

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