Traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis and preparation method thereof

Abstract

The application provides a traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis and a preparation method, and belongs to the technical field of biological medicine. The traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis comprises Wenqingyin microemulsion and a gel framework, the Wenqingyin microemulsion is loaded on the gel framework, and the gel framework is carbomer-940. The Wenqingyin microemulsion comprises 2.55% of oleic acid, 12.73% of castor oil polyoxyethylene ether, 12.73% of 1,2-pentanediol and 72% of an aqueous phase. The microemulsion gel comprises 3.5% of CP940, 10% of glycerol and 1% of triethanolamine. The traditional Chinese medicine compound for treating atopic dermatitis has remarkable effects and small side effects. The microemulsion gel has the advantages of convenient administration and drug withdrawal, avoidance of liver first-pass effect and reduction of gastrointestinal tract stimulation by being applied to the skin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis and a preparation method thereof. BACKGROUND

[0002] Atopic dermatitis (AD) is also known as atopic eczema, which is a chronic recurrent skin disease characterized by dryness, itching and inflammation. According to statistics, the global prevalence of AD in children is as high as 20%, and the prevalence in adults is 10%. The number of AD patients is still increasing year by year. At present, the western medicine mainly uses corticosteroids, antibiotics, dupilumab and other immunosuppressive agents to treat AD. However, due to the easy recurrence of moderate and severe AD, continuous treatment is needed, but long-term use of the above drugs will cause various side effects, such as skin atrophy, high blood pressure and diarrhea, etc., and also produce drug resistance and dependence. Therefore, it is very important to find safe and effective AD treatment drugs with less side effects.

[0003] Wenqing Yin is from Wanbing Huichun written by Gong Tingxian in Ming Dynasty, which can invigorate the blood, tonify blood, clear heat and resolve toxicity. Modern pharmacological studies have shown that it has the effects of anti-ulcer, anti-inflammatory analgesic, antipyretic sedative, hemostatic and immune regulation, and is often used in the treatment of various skin diseases such as facial hormone-dependent dermatitis, chronic eczema, chronic urticaria, psoriasis, acne and AD. Transdermal drug delivery system refers to a drug delivery system that absorbs into the human blood circulation through the skin at a certain controlled rate to produce systemic or local therapeutic effect, which can overcome the shortcomings of traditional oral administration and injection administration, has the advantages of convenient administration and drug withdrawal, avoids liver first-pass elimination, and can maintain blood drug concentration for a long time. At present, the form of Wenqing Yin for treating skin diseases is mainly oral administration, so it is of great significance to develop an external preparation of Wenqing Yin for treating AD.

[0004] Microemulsion (ME) is a colloidal dispersion system with particle size of 10-100 nm, which is dispersed in another liquid to form a thermodynamic stable, isotropic and clear appearance. It can enhance the solubility of poorly soluble drugs and enhance the stability of drugs. However, due to the strong flowability of microemulsion, it is not easy to be coated and retained on the skin when applied to transdermal drug delivery, while the preparation of microemulsion gel can effectively improve the viscosity of microemulsion and play a sustained-release role.

[0005] The present application combines the effective Wenqing Yin extract with microemulsion technology and gel technology to prepare a microemulsion gel for transdermal administration to treat AD. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis and a preparation method thereof to solve the problems of the prior art.

[0007] The present application provides a traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis, comprising a Wenqingyin microemulsion and a gel framework, wherein the Wenqingyin microemulsion is loaded on the gel framework.

[0008] The gel framework is carbomer-940 (CP940).

[0009] The Wenqingyin microemulsion comprises 2.546% of oleic acid, 12.727% of castor oil polyoxyethylene ether, 12.727% of 1,2-pentanediol, and 72% of an aqueous phase.

[0010] Preferably, the microemulsion gel comprises 2%-4.5% of CP940, 0%-15% of glycerol, and 0.5%-2% of triethanolamine.

[0011] Preferably, the microemulsion gel comprises 3.5% of CP940, 10% of glycerol, and 1% of triethanolamine.

[0012] Another technical solution of the present application is a preparation method of a traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis, comprising the following steps:

[0013] S1. Extraction and concentration of a Wenqingyin water extract;

[0014] S2. Preparing a Wenqingyin microemulsion by taking the Wenqingyin water extract extracted in step S1 as an aqueous phase;

[0015] S3. Adding the Wenqingyin microemulsion prepared in step S2 to a gel framework CP940 to make it completely swell and prepare a microemulsion gel.

[0016] Preferably, step S1 specifically comprises weighing angelica, chuanxiong, white peony root, prepared rehmannia, scophularia, coptis, golden cypress, and gardenia pieces, soaking them in 47% ethanol for 30 min at a solid-liquid ratio of 1:25, refluxing and extracting for 115 min, filtering the hot extract through four layers of gauze, repeating the extraction for two more times, finally combining the extract, and concentrating the extract by rotary evaporation at 60°C to obtain an aqueous phase for the microemulsion.

[0017] Preferably, step S2 specifically comprises:

[0018] S2.1. Screening of an oil phase, a surfactant, and a co-surfactant;

[0019] S2.2. The surfactant and co-surfactant are mixed and stirred uniformly from Km = 1:1 to both sides (3:1, 2:1, 1:1, 1:2, 1:3), and then mixed with the oil phase in a mass ratio of 1:9-9:1, and then prepared into Wenqingyin microemulsion by titration with distilled water while stirring;

[0020] S2.3. The Wenqingyin extract is centrifuged at 4000 r / min for 10 min, the supernatant is collected, and the supernatant is concentrated under reduced pressure to an appropriate amount as the microemulsion aqueous phase, and 0.75 g / mL water extract is determined as the optimal water extract concentration.

[0021] Preferably, the oil phase in the S2.1 step is oleic acid, the surfactant is castor oil polyoxyethylene ether, and the co-surfactant is 1,2-pentanediol.

[0022] Preferably, the heating temperature in the microemulsion preparation of step S2.2 is 35°C, the stirring speed is 600 r / min, and the stirring time is 0.5 h.

[0023] Preferably, the step S3 is specifically:

[0024] S3.1. An appropriate amount of Wenqingyin microemulsion is taken, and gel matrix CP940 is added to completely swell;

[0025] S3.2. 1% triethanolamine is added dropwise and continuously stirred to form a gel;

[0026] S3.2. 10% glycerol is added and continuously stirred to form a finished gel.

[0027] Compared with the prior art, the present application achieves the following technical effects:

[0028] The Wenqingyin extract with definite curative effect is innovatively combined with microemulsion technology and gel technology to prepare a microemulsion gel for transdermal administration to treat AD. The Chinese medicine compound in the present application has remarkable effects and small side effects in treating atopic dermatitis. The microemulsion gel has the advantages of convenient administration and discontinuation, avoidance of liver first-pass effect, and reduction of gastrointestinal irritation. BRIEF DESCRIPTION OF DRAWINGS

[0029] For ease of illustration, the present application is described in detail by the following specific embodiments and drawings.

[0030] Figure 1 The figure is a selection diagram of surfactants.

[0031] Figure 2 The figure is a selection diagram of co-surfactants.

[0032] Figure 3 The figure is a selection diagram for determining Km value.

[0033] Figure 4 Figure for clear drink microemulsion to present transparent and clear state.

[0034] Figure 5 Figure for microemulsion type dyeing method identification.

[0035] Figure 6 Figure for microemulsion type dilution method identification.

[0036] Figure 7 Figure for microemulsion microscopic morphology.

[0037] Figure 8 Figure for glycerol moisturizing rate determination results at different concentrations.

[0038] Figure 9 Figure for glycerol water loss rate determination results at different concentrations.

[0039] Figure 10 Figure for microemulsion gel appearance.

[0040] Figure 11 Figure for in vitro transdermal absorption results.

[0041] Figure 12 Figure for modeling and administration time arrangement.

[0042] Figure 13 Figure for mouse back skin condition and dermatitis severity score.

[0043] Figure 14 Figure for comparison of spleen size and index of each group of mice.

[0044] Figure 15 Figure for mouse skin histopathological changes.

[0045] Figure 16 Figure for mouse serum inflammatory factor levels, where (n=6) (p*<0.05, p**<0.01, p***<0.001).

[0046] Figure 17 Figure for mouse skin tissue inflammatory factor levels, where (n=6) (p*<0.05, p**<0.01, p***<0.001).

[0047] Figure 18 Flowchart of the present application. DETAILED DESCRIPTION

[0048] The following is a specific embodiment of the present application and in conjunction with the drawings, the technical solutions of the present application are further described, but the present application is not limited to these embodiments; in the following description, specific details such as specific configurations are provided only to help a comprehensive understanding of the embodiments of the present application. Therefore, those skilled in the art should understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present application.

[0049] It should be noted that the embodiments and features in the embodiments can be combined with each other without conflict.

[0050] The materials, practices and experimental equipment involved in the embodiments of the present application, if not specifically stated, are consistent with the commercially available products in the relevant chemical and biotechnology fields.

[0051] Example 1 Extraction and concentration of Wenqing drink water extract

[0052] Take 3g of each of angelica, chuanxiong, white peony root, prepared rehmannia, scutellaria, coptis, golden cypress, and gardenia slices, soak in 47% ethanol for 30min with a solid-liquid ratio of 1:25, reflux extraction for 115min, filter with four layers of gauze while hot, repeat extraction for 2 times, finally combine the extract, concentrate at 60℃ by rotary evaporation, as the water phase of the microemulsion. Among them, through the previous research of the research group, the total content of 5 index components of gardenoside, ferulic acid, baicalin, berberine hydrochloride and wogonoside is determined as the effective components of Wenqing drink, and the liquid phase conditions are determined as follows: Agilent Polaris 3C18-A chromatographic column (250mm x 4.6mm, 5μm); mobile phase 0.2% phosphoric acid (A)-acetonitrile (B), gradient elution (0-8min, 5%-20% B; 8-30min, 20%-38% B; 30-40min, 38%-50% B; 40-50min, 50%-90% B); volume flow rate 0.6mL / min; column temperature 40℃; detection wavelength 254nm; injection volume 10μL.

[0053] Example 2 Preparation of Wenqing drink microemulsion

[0054] 2.1 Selection of oil phase

[0055] The oil phase is a crucial component in microemulsion formation. Within a certain range, the smaller the relative molecular mass of the oil phase, the stronger its drug-dissolving ability. Large-molecule oil phases are less likely to embed into surfactants to form interfacial films, while small-molecule oil phases are more prone to embedding. Therefore, to increase drug solubility and enlarge the microemulsion formation area, a short-chain oil phase is preferable. Commonly used oil phases for microemulsion preparation include soybean oil, oleic acid, ethyl oleate, olive oil, and isopropyl palmitate. Oleic acid is a naturally occurring fatty acid with a composition similar to human cell membranes, exhibiting good biocompatibility and low interfacial tension. Its unsaturated double bonds contribute to the formation of a stable microemulsion structure, improving both drug solubility in the oil phase and increasing the microemulsion formation area. Therefore, after preliminary screening, oleic acid was selected as the oil phase.

[0056] 2.2 Screening of Surfactants

[0057] Tween-60, Tween-80, Span-80, and castor oil polyoxyethylene ether (EL-40) were mixed with anhydrous ethanol at a ratio of Km = 1:1. Figure 1 As shown, after being stirred evenly under a magnetic stirrer, it is then mixed evenly with oleic acid at a mass ratio of 1:9 to 9:1. Microemulsions are prepared by titration with distilled water while stirring. The critical point for microemulsion formation is defined as the change of the system from clear to turbid. The amount of water added at the critical point is recorded. A pseudo-ternary phase diagram is drawn using Origin software to screen out the best surfactant.

[0058] The results showed that EL-40 formed the largest microemulsion area when used as a surfactant, so EL-40 was selected as the surfactant.

[0059] 2.3 Screening of co-surfactants

[0060] Anhydrous ethanol, 1,2-propanediol, glycerol, 1,2-pentanediol, butylene glycol, and polyethylene glycol 400 monolaurate (PEG-400) were mixed with the screened surfactant EL-40 at a ratio of Km = 1:1. Figure 2 As shown in the figure, after being magnetically stirred evenly, it is then mixed with oleic acid at a mass ratio of 1:9 to 9:1. Microemulsions are prepared by titration with distilled water while stirring. The critical point for microemulsion formation is defined as the change of the system from clear to turbid. The amount of water added at the critical point is recorded. A pseudo-ternary phase diagram is drawn using Origin software to screen out the best co-surfactant.

[0061] The results showed that EL-40 and glycerol would separate into layers when mixed. When anhydrous ethanol, 1,2-propanediol, 1,2-pentanediol, butanediol and PEG-400 were used as co-surfactants, 1,2-pentanediol formed the largest microemulsion area. Therefore, 1,2-pentanediol was selected as the co-surfactant.

[0062] 2.4 Selection of Km value

[0063] The surfactant (EL-40) and the co-surfactant (1,2-pentanediol) were mixed and stirred uniformly from Km = 1:1 to both sides (3:1, 2:1, 1:1, 1:2, 1:3) (as shown in the table), and then mixed uniformly with oleic acid at a mass ratio of 1:9-9:1, respectively. The microemulsion was prepared by titration with distilled water while stirring. The critical point of the formation of the microemulsion was recorded when the system changed from clear to turbid. Then, the mass percentage of the mixed surfactant, the oil phase, and the water phase in the system was calculated through the amount of water added. The pseudo-ternary phase diagram was drawn using the origin software, the size of the microemulsion region was compared, and the Km value was determined. Figure 3

[0064] The results showed that when Km was 1:1, the area of the formed microemulsion region was the largest, so the Km value of the surfactant and the co-surfactant was selected to be 1.

[0065] 2.5 Determination of the concentration of the extract

[0066] The extract of Wenqingyin was centrifuged at 4000 r / min for 10 min, the supernatant was collected, and the supernatant was concentrated under reduced pressure to an appropriate amount as the water phase of the microemulsion. The preliminary determination of the composition ratio of the microemulsion prescription was as follows: oil phase: mixed surfactant = 1:9, Km = 1, and water phase 72%. The water phase was replaced with different mass concentrations (0.25, 0.5, 0.75, 1, 1.25 g / mL) of the medicinal material extract to investigate the effect of different mass concentrations of the water extract on the particle size and drug loading capacity of the microemulsion, and then the optimal extract concentration was determined.

[0067] Table 1 Effect of different extract concentrations on the particle size and drug loading capacity of the microemulsion

[0068]

[0069] From the above results, it can be seen that the water extract of different mass concentrations has a greater effect on the particle size of the microemulsion, so under the premise of ensuring the drug loading capacity, 0.75 g / mL of the water extract was selected as the optimal water extract concentration.

[0070] 2.6 Determination of the optimal prescription

[0071] ​The determination of the optimal prescription of microemulsion mainly needs to consider the stability of microemulsion. Therefore, in the pseudo-ternary phase diagram of Km = 1, the appropriate proportion is selected, and the selected point is as far as possible from the critical value, otherwise the microemulsion may be stratified or not formed due to the change of environment or other factors. Therefore, the points in the center of the microemulsion region and several points near the points (oil phase: mixed surfactant is 1:6, 1:7, 1:8, 1:9, 1:10, 1:11) are selected to prepare microemulsion, and the particle size and drug loading are evaluated.

[0072] Table 2 Effect of different ratios of oil phase and mixed surfactant on particle size and drug loading of microemulsion

[0073]

[0074] The results show that when the oil phase: mixed surfactant = 1:10, the drug loading of the formed microemulsion is moderate, and the particle size is the smallest. Therefore, the oil phase: mixed surfactant = 1:10 is determined.

[0075] In summary, the final prescription of Wenqingyin microemulsion is as follows: oleic acid 2.546%, castor oil polyoxyethylene ether 12.727%, 1,2-pentanediol 12.727%, and water phase 72%.

[0076] 2.7 Selection of preparation process conditions

[0077] Taking the particle size and drug loading of microemulsion as the index, the effects of temperature, stirring speed and stirring time on the preparation process of microemulsion are investigated by single factor test method.

[0078] 2.7.1 Investigation of temperature

[0079] First, fix the stirring speed at 400 r / min and the stirring time at 0.5 h, and prepare the compound Wenqingyin microemulsion at four different temperatures of 25±2℃, 35±2℃, 45±2℃ and 55±2℃, respectively, to investigate the effects of temperature on the particle size and drug loading of microemulsion.

[0080] Table 3 Effect of different temperatures on particle size and drug loading of microemulsion

[0081]

[0082] After 35℃, with the increase of temperature, the drug loading increases, but the particle size of microemulsion also increases. The drug loading at 45℃ increases slightly compared with that at 35℃, but the particle size of microemulsion at 35℃ is smaller than that at other temperatures. Therefore, the heating temperature is selected as 35℃.

[0083] 2.7.2 Investigation of stirring speed

[0084] Compound Wenqingyin microemulsion was prepared at a temperature of 35℃ and a stirring time of 0.5h at four different speeds of 200r / min, 400r / min, 600r / min, and 800r / min. The effect of speed on microemulsion particle size and drug loading was investigated.

[0085] Table 4. Effects of different stirring speeds on microemulsion particle size and drug loading.

[0086]

[0087] As the stirring speed increases, the drug loading increases and the microemulsion particle size decreases. When the rotation speed increases to 800 r / min, the drug loading increases significantly, but the particle size also increases significantly. Therefore, a stirring speed of 600 r / min is selected.

[0088] 2.7.3 Investigation of stirring time

[0089] Compound Wenqingyin microemulsion was prepared at a temperature of 35℃ and a rotation speed of 600 r / min for four different time groups of 0.5h, 1h, 1.5h and 2h to investigate the effect of time on microemulsion particle size and drug loading.

[0090] Table 5. Effects of different stirring times on microemulsion particle size, PdI, and drug loading.

[0091]

[0092] Before 1.5 hours, extending the stirring time had little effect on the drug loading and particle size of the microemulsion. When the time was increased to 2 hours, the drug loading increased significantly, but the particle size also increased significantly. For the sake of high efficiency and energy saving, the stirring time was selected to be 0.5 hours.

[0093] In summary, the optimal conditions for microemulsion preparation are: temperature 35℃, rotation speed 600 r / min, and time 0.5 h.

[0094] 2.8 Investigation of the physicochemical properties of microemulsions

[0095] 2.8.1 Characteristics

[0096] Observe whether the Wenqingyin microemulsion is transparent and clear and whether the Tyndall effect exists.

[0097] Depend on Figure 4 It can be seen that the Wenqingyin microemulsion is transparent and clear, and exhibits the Tyndall effect.

[0098] 2.8.2 Microemulsion Type Identification

[0099] Methods for identifying microemulsion types include centrifugation, dilution, and staining.

[0100] (1) Centrifugation method: Take a certain amount of Wenqingyin microemulsion according to the prescription, place it in a centrifuge tube, and centrifuge at 10,000 r / min for 15 min. Observe whether the sample is layered.

[0101] After centrifugation, the Wenqingyin microemulsion did not show layering.

[0102] (2) Staining method (as shown in Figure Figure 4 ): Take an equal amount of Wenqingyin microemulsion, add an appropriate amount of methylene blue and Sudan red III respectively, observe the diffusion speed and color change of the two dyes in the microemulsion, and determine the type of the microemulsion.

[0103] The results show that, since methylene blue is a water-soluble dye, it can rapidly diffuse in the microemulsion and form a uniform blue solution. Sudan red III, being a fat-soluble dye, floats on the upper layer when added to the microemulsion, showing obvious layering.

[0104] (3) Dilution method: Take a certain amount of Wenqingyin microemulsion according to the prescription, dilute it with water to different multiples and place it overnight, and observe the solution state.

[0105] The results show that after dilution with water to different multiples, the Wenqingyin microemulsion did not show layering or turbidity, and the solutions of different dilution multiples did not show layering or precipitation after being placed overnight, indicating that the Wenqingyin microemulsion was a uniform and stable solution after being diluted with water.

[0106] In summary, the prepared microemulsion is of O / W type.

[0107] 2.8.3 Particle size distribution, zeta potential and viscosity of Wenqingyin microemulsion

[0108] Particle size is one of the most important characteristics of microemulsion, and the size of microemulsion particle size will directly affect the quality of microemulsion preparation; the zeta potential of microemulsion is an important indicator of microemulsion stability, and generally the higher the homogeneity of microemulsion charge, the better the stability, and studies have shown that the negative charge on the surface of microemulsion is beneficial to the penetration of droplets through the skin. The particle size and distribution of microemulsion will affect its viscosity, generally the smaller the particle size and the more uniform the distribution, the lower the viscosity of the microemulsion, and in drug delivery systems, microemulsion with low viscosity is more likely to penetrate the skin or mucosal barrier, thereby improving the absorption rate and bioavailability of drugs. Therefore, in this experiment, laser particle size analyzer was used to determine the particle size distribution and Zeta potential of microemulsion, and rotary viscometer was used to determine the viscosity of microemulsion.

[0109] The experiment measured the particle size of Wenqingyin microemulsion to be 72.93 ± 2.93 nm, which meets the microemulsion particle size range of 1-100 nm; the Zeta potential was measured to be -16.29 ± 0.63 mv, indicating that the microemulsion system has a negative charge in the potential distribution, and the charge homogeneity is good, the microemulsion stability is good. The rotary viscometer measured the viscosity of Wenqingyin microemulsion to be 40.48 ± 1.55 mpa·s.

[0110] 2.8.4 Investigation of microemulsion micro-morphology

[0111] The sample was prepared by negative staining method: take the microemulsion, drop on the copper grid with Formvar support film, negative staining with 2% phosphotungstic acid solution for 1-2 min, absorb the excess liquid on the copper grid with filter paper and dry under natural conditions, observe the microemulsion morphology under TEM (transmission electron microscope).

[0112] It can be seen that the Wenqingyin microemulsion is spherical or nearly spherical, smooth surface, uniformly distributed in the field of view and no aggregation phenomenon. Figure 7

[0113] 2.8.5 Stability test of microemulsion

[0114] (1) centrifuge the microemulsion sample including blank microemulsion and drug-loaded microemulsion at 10000 rpm for 30 min, observe whether the phases are separated, whether the microemulsion morphology is well maintained and whether the clarity is maintained.

[0115] (2) take 3 batches of microemulsion samples, place them in a -4°C refrigerator for 12 h, restore to room temperature after taking out, and observe whether there are phenomena such as separation, oil floating, oil-water separation, etc.

[0116] (3) similarly, take 3 batches of microemulsion samples, place them in a 40°C oven for 12 h, restore to room temperature after taking out, and observe whether there are phenomena such as separation, oil floating, oil-water separation, etc.

[0117] The stability experiment results show that the Wenqingyin microemulsion does not appear to be separated after high-speed centrifugation, cold resistance experiment and heat resistance experiment, and can still maintain a clear state, indicating that the prepared microemulsion has good thermodynamic stability.

[0118] Example 3 Preparation of Wenqingyin microemulsion gel

[0119] The microemulsion drug delivery system can improve the drug loading capacity of poorly soluble drugs and promote the transdermal absorption of drugs. However, the viscosity of Wenqingyin microemulsion as a skin drug preparation is low, the flowability is large, and the adhesion to the skin is poor. The microemulsion can be effectively improved in viscosity by using a suitable gel matrix excipient, so that it is easy to adhere to the skin and exert the drug effect.

[0120] 3.1 Selection of gel matrix

[0121] Select commonly used gel matrices such as carbomer-940 (CP-940), sodium polyacrylate (NP-700), hydroxypropyl methylcellulose (HPMC) and sodium carboxymethyl cellulose (CMC-Na), respectively, swell in a small amount of water, prepare a solution with a certain concentration, and slowly add an appropriate amount of Wenqingyin microemulsion solution to it while stirring, observe the appearance of the formed microemulsion gel, and select the appropriate gel matrix.

[0122] ​Table 6 Screening of gel matrix

[0123]

[0124] In order to more clearly observe the state and color of the four kinds of matrix materials after swelling, the Wendingyin extract with deep color was not added during the screening of the matrix. Compared with NP-700, HPMC, CMC-Na and other external matrix, the swelling speed of CP940 was relatively fast, the appearance state was good, and the gel could be quickly formed at a lower concentration after being neutralized with alkali. Therefore, CP940 was used as the gel material.

[0125] 3.2 Determination of gel concentration

[0126] 3.2.1 Preparation of microemulsion gel

[0127] 4g of Wendingyin microemulsion was taken and added with gel skeleton CP940 to completely swell, and microemulsion gels with different concentrations (2%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%) were prepared. Then 0.5% of triethanolamine was added dropwise and continuously stirred to form a gel. The appearance, viscosity, spreadability and centrifugal stability of the Wendingyin microemulsion gel were comprehensively scored to determine the concentration of the microemulsion gel.

[0128] 3.2.2 Evaluation index

[0129] ①Observe the smoothness, uniformity and fineness of the formed microemulsion gel; ②Determine the viscosity of the microemulsion gel using a rotary viscometer; ③Weigh 0.1 g of sample, press it with 100 g weight on two glass slides, and let it stand for about 5 min. Measure the diameter of the formed circle to compare the spreadability of each microemulsion gel; ④Centrifuge at 6000 r / min for 15 min, and observe whether the microemulsion gel is layered. The scoring standard is developed according to the above evaluation index, as shown in Table 3-2.

[0130] Table 7 Scoring standard of microemulsion gel

[0131]

[0132] 3.2.3 Results

[0133] Table 8 Comprehensive scoring results of microemulsion gel

[0134]

[0135] From the table, when the amount of CP940 is 3.5%, the comprehensive score of the microemulsion gel is higher, so 3.5% CP940 is selected as the gel matrix.

[0136] 3.3 Determination of glycerol concentration

[0137] 3.3.1 Preparation of microemulsion gel

[0138] The water content of the aqueous gel base is large, and it is easy to lose water when the storage conditions are poor or the storage time is too long, which makes it difficult to spread on the skin, so a suitable humectant is often added. Commonly used humectants include propylene glycol, glycerol, sorbitol, betaine, etc. Studies have shown that compared with betaine, sorbitol and other humectants, propylene glycol and glycerol have more stable moisturizing performance and more lasting moisturizing ability. Compared with propylene glycol, glycerol has good moisturizing effect and less skin irritation, so considering safety and adaptability, glycerol is selected as the humectant. Since the commonly used amount of glycerol in the gel is 5% to 15%, the concentrations of 0%, 5.0%, 10%, and 15% are selected as the investigation concentrations of the added amount of glycerol, and the Wenqingyin microemulsion gel is prepared under the same conditions. The microemulsion gel evaluation standards, the moisture retention rate and the water loss rate in Table 3-2 are used as evaluation indexes to screen the appropriate glycerol concentration range.

[0139] 3.3.2 Moisture retention rate determination

[0140] The moisture retention rate is the percentage of the mass of water retained by the microemulsion gel at different time points. An appropriate amount of microemulsion gel is accurately weighed into a glass dish that has been dried to a constant mass (m). The total mass is recorded as m0, and the microemulsion gel is evenly applied to a glass slide. The glass slide is placed in a dry oven at room temperature and a relative humidity of 43%. The mass of the glass dish and the microemulsion gel is measured at 0, 6, 12, 24, and 36 h (m t ), and the moisture retention rate is calculated using the following formula.

[0141] Moisture retention rate (%) = (m t -m) / (m0-m) x 100%

[0142] 3.3.3 Water loss rate determination

[0143] A glass dish that has been dried to a constant mass is taken, and the mass is recorded as M2. An appropriate amount of microemulsion gel is then placed on the dried glass dish, and the total mass of the microemulsion gel and the glass dish is recorded as M1. The glass dish is placed in a 60°C oven for 1 h, then removed and allowed to cool in a dry oven. The total mass after drying is recorded as M3. The water loss rate of the microemulsion gel with different concentrations of glycerol is calculated using the following formula.

[0144] Water loss rate (%) = (M3-M2) / (M1-M2) x 100%

[0145] 3.3.4 Results (as shown in Figure 8 , Figure 9 )

[0146] Table 9 Comprehensive evaluation results of microemulsion gel

[0147]

[0148] In summary, with the increase of glycerol concentration, the moisture retention rate increases and the water loss rate decreases. When the glycerol concentration is 10%, the microemulsion gel has good moisturizing effect and comfortable skin feel without stickiness. When the glycerol concentration continues to increase to 15%, the gel is too sticky and has an oily feeling. Therefore, the glycerol concentration is determined to be 10%.

[0149] 3.4 Determination of triethanolamine concentration

[0150] 4g of Wenqingyin microemulsion was taken, 3.5% CP940 was added to make it completely swollen, then 10% glycerol was added, and finally 0.5%, 1%, 1.5%, and 2.0% triethanolamine was added respectively, and constant stirring was carried out to form a gel. The appearance, viscosity, spreadability, and centrifugal stability of the Wenqingyin microemulsion gel were comprehensively scored to determine the concentration of the microemulsion gel.

[0151] Table 10 Comprehensive score results of microemulsion gel

[0152]

[0153] As can be seen from the results, with the increase of triethanolamine concentration, the microemulsion gel will appear liquefaction, and the centrifugation will have drug liquid precipitation. When the triethanolamine concentration is 1%, the comprehensive score is the highest and the spreadability is the best, so the triethanolamine concentration is determined to be 1%.

[0154] The appearance of the Wenqingyin microemulsion gel was observed, its viscosity, pH value, and total content of index components were determined, and its preliminary stability, transdermal performance, and skin irritation were investigated.

[0155] 3.5.1 Appearance

[0156] As shown in Figure 10 , the Wenqingyin microemulsion gel is yellow-brown, uniform in texture, and smooth in consistency.

[0157] 3.5.2 Determination of viscosity, pH value, and total content of index components of microemulsion gel

[0158] The rotary viscometer was used to measure the viscosity of the Wenqingyin microemulsion gel, which was 43700.00±0.23 mpa·s; the pH meter measured the pH value of the microemulsion gel, which was 5.19±0.22, which met the optimal skin pH range of human body (4.5-6.5); HPLC measured the total content of index components of the microemulsion gel, which was 8.760±0.913 mg / g.

[0159] 3.5.3 Stability investigation of microemulsion gel

[0160] The microemulsion gel was first centrifuged at 4000 r / min for 30 min, and whether the phases were separated and the morphology of the microemulsion gel was good was observed.

[0161] Then the microemulsion gel was alternately placed in room temperature 25℃, 4℃ refrigerator environment, -20℃ refrigerator environment, 40℃ oven environment, each time about 60 min, and then the separation of each phase under different temperature environment changes was observed, and whether the microemulsion gel morphology was destroyed under room temperature environment was observed, so as to determine the stability of the microemulsion under different temperatures. Finally, in order to verify the long-term stability of the preparation, each microemulsion gel that completed the above test was sealed and stored in a desiccator at room temperature and a relative humidity of 43% for 1 month, and then centrifugation was performed again (parameters were the same as before), and whether the phases were separated and whether the morphology was maintained well was observed.

[0162] The results show that the microemulsion gel does not appear to be layered after centrifugation, and after temperature environment changes and long-term stability tests, it can still maintain the original state, and the texture is uniform and delicate when applied, indicating that the prepared microemulsion gel has good stability.

[0163] 3.5.4 In vitro transdermal absorption experiment

[0164] During the experiment, the isolated rat skin was thawed in physiological saline, the water on the surface of the skin was absorbed with filter paper, the treated stratum corneum was placed flat on the receiving pool with the stratum corneum facing up, the diffusion pool was fixed on the receiving pool with iron clips, 30% ethanol-containing physiological saline was added to the receiving pool as the receiving liquid, a magnetic stirrer was added, and finally 0.3g of Wenqingyin microemulsion, Wenqingyin gel, and Wenqingyin microemulsion gel was added to the supply pool, respectively. The prepared diffusion pool was placed in the in vitro transdermal diffusion instrument, the water bath temperature was adjusted to (35±1)℃, and the constant speed was 300r·min -1 . After 1, 2, 4, 6, 8, 10, 12, 24h of the experiment, 1ml of sample was taken from the receiving pool, and an equal volume of blank receiving liquid was immediately supplemented. The total content of the index component was measured by HPLC method. The cumulative penetration amount of the index component per unit area Q n (μg / cm 2 ) was calculated according to the following formula. After 24h of transdermal experiment, the skin was taken out, the surface drug was washed with physiological saline, and the water was absorbed with filter paper. Then the skin was cut into small pieces and placed in a centrifuge tube, and an appropriate amount of methanol was added. After ultrasonic treatment for 30min, centrifugation was performed at 3500r / min for 10min, and the supernatant was measured by HPLC method to measure the total content of the index component, so as to calculate the skin retention amount Q s (μg / cm 2 ).

[0165]

[0166] Among them, C n represents the mass concentration of the drug measured at the nth sampling point, V represents the total volume of the receiving liquid in the receiving pool (15mL), and A represents the effective diffusion area (1.539cm2 ), C i represents the mass concentration of the drug in the receiving liquid at the ith sampling, V i represents the volume of each sampling (1 mL); V s represents the total volume of the skin extract, and C is the concentration of the drug in the skin extract.

[0167] At 24 h after administration, the cumulative permeation amounts Q n of Wenqingyin microemulsion, Wenqingyin gel and Wenqingyin microemulsion gel were (1916.84 ± 1.46) μg·cm -2 , (1685.34 ± 1.22) μg·cm -2 and (1995.93 ± 1.57) μg·cm -2 , respectively; the skin retention amounts Q s of Wenqingyin microemulsion, Wenqingyin gel and Wenqingyin microemulsion gel were (23.76 ± 0.48) μg·cm -2 , (17.62 ± 1.79) μg·cm -2 and (21.56 ± 1.13) μg·cm -2 , respectively. The results showed that the ordinary gel had the lowest transdermal amount and skin retention amount; because the microemulsion is a thermodynamically stable nanosystem with small particle size and good fluidity, and contains surfactants and co-surfactants, it reduces the skin barrier resistance to some extent, so its transdermal rate is fast, the speed of diffusion into the skin surface layer is also fast, and its skin retention amount is the most; while the microemulsion gel has the structure of microemulsion, which can promote the drug into the skin, but at the same time, because the viscosity of the microemulsion gel is larger than that of the microemulsion, the drug needs to overcome more resistance to release from the preparation into the skin, so the skin retention amount will be less than that of the microemulsion, but the content of the two will be higher than that of the ordinary gel. It was also found through the cumulative transdermal amount that the cumulative transdermal amount of the microemulsion was higher than that of the microemulsion gel before 12 h, while the cumulative transdermal amount of the microemulsion gel increased slowly after 12 h, and was finally higher than that of the microemulsion, which also showed that the effective ingredient in the microemulsion presented the characteristics of too fast release, while after being prepared into a microemulsion gel, the release of each component was more constant, had a certain sustained-release effect, and basically did not affect the in vitro release of the drug.

[0168] The release mechanism of Wenqingyin microemulsion gel was explored through model fitting of transdermal data (see Table 11). If the drug is released at a constant rate, independent of concentration, it conforms to a zero-order kinetic model, commonly used to describe the release process of controlled-release formulations. If the drug release rate is proportional to the drug concentration and slows down as the drug concentration decreases, it conforms to a first-order kinetic model. If drug release is mainly diffusion-based, it conforms to the Higuchi kinetic model, and these two models are commonly used to describe the release process of sustained-release formulations. If drug release is simultaneously controlled by diffusion and matrix erosion, it conforms to the Ritger-Peppas kinetic model, which requires determining whether the drug release is sustained-release or controlled-release based on the mechanism. The results show that the in vitro release of Wenqingyin microemulsion gel within 24 hours best conforms to the first-order kinetic model, indicating that Wenqingyin microemulsion gel has a certain sustained-release effect.

[0169] Table 11. In vitro transdermal transdermal volume-time model fitting of Wenqingyin microemulsion gel.

[0170]

[0171] 3.5.5 Skin Irritation Assessment

[0172] 3.5.5.1 Handling of laboratory animals

[0173] Six rats were used. Following the "Technical Guidelines for Research on Irritation and Hemolytic Properties of Traditional Chinese Medicine and Natural Drugs," a self-comparison was conducted using left and right sides of the same animal. The right side represented normal skin, and the left side represented damaged skin. The same side was divided using a marker; the upper end was the control group coated with pure water, and the lower end was the experimental group coated with Wenqingyin microemulsion gel. Hair was removed 24 hours before the experiment, with each side measuring 3cm × 3cm. Damaged skin was defined as a crisscross pattern drawn with sandpaper after hair removal, until bleeding was observed. A scoring standard was established based on the "Technical Guidelines for Research on Irritation and Hemolytic Properties of Traditional Chinese Medicine and Natural Drugs."

[0174] Table 12 Skin Irritation Response Scoring Criteria

[0175]

[0176]

[0177] Table 13 Evaluation Criteria for Skin Irritation Intensity

[0178]

[0179] 3.5.5.2 Skin irritation assessment after a single dose

[0180] 0.5 ml Wenqingyin microemulsion gel was directly applied to the lower end of the rat skin which had been depilated on both sides, then covered with two layers of gauze (3 cm x 3 cm), and fixed with non-irritating adhesive tape. The upper end of the rat was used as control with pure water. After 4 h, the drug application site was cleaned with warm water, and the presence or absence of erythema and edema was observed and recorded at 24, 48 and 72 h.

[0181] Table 14 Comparison of results of single-dose skin irritation experiment

[0182]

[0183]

[0184] 3.5.5.3 Investigation of multiple-dose skin irritation

[0185] 0.5 ml Wenqingyin microemulsion gel was directly applied to the lower end of the rat skin which had been depilated on both sides, and the upper end was used as control with pure water. The drug was continuously administered for 7 days. One hour before each administration, the residual drug on the skin of the rat was cleaned with warm water, and the presence or absence of erythema and edema was observed at 24 h after administration.

[0186] Table 15 Comparison of results of multiple-dose skin irritation experiment

[0187]

[0188] From the above experimental results, in the single-dose skin irritation test and the multiple-dose skin irritation test, no skin irritation reactions such as erythema and edema were observed at the drug application sites of the intact skin and the damaged skin of the rat, indicating that the Wenqingyin microemulsion gel developed in this project has good safety. The results of this experiment can also provide a basis for safety in further experimental research and product development in the later stage.

[0189] Example 4 Pharmacodynamic study of Wenqingyin microemulsion gel

[0190] 4.1 Animal grouping

[0191] 50 healthy female BALB / c mice, 8 weeks old, SPF level, body weight (18 ± 2) g, were randomly divided into 5 groups: blank group, model group, positive group, negative group and treatment group.

[0192] 4.2 Modeling and administration method

[0193] As Figure 12As shown, 50 mice were acclimatized for one week. The day before the experiment, the hair on the back of each mouse was shaved (area 2cm × 3cm) using a shaver. Sensitizing agents were prepared by completely dissolving DNCB in a matrix solution (acetone: olive oil = 3:1) to prepare 0.5% and 1% DNCB solutions. Except for the blank control group, on days 1, 4, and 7 of the experiment, 100μL of 1% DNCB solution was evenly applied to the back skin of the mice using a pipette to induce sensitization. Then, from day 9 to day 25 of the experiment, 100μL of 0.5% DNCB solution was applied to the back of the mice every other day for resensitization. The appearance of AD-like symptoms such as erythema, scaling, and crusting on the back of the mice in the early stages was considered a successful model establishment. Simultaneously, from day 13 to the end of the experiment, the drug (0.5 ml / mouse) was applied once daily to the hairless area on the back of the mice. The blank control group and the model group did not receive the drug. The positive control group received hydrocortisone butyrate cream, the negative control group received blank microemulsion gel matrix, and the treatment group received Wenqingyin microemulsion gel. On the day that dermatitis was induced, the drug was administered again after 4 hours.

[0194] 4.3 Assessment of the severity of dermatitis

[0195] On days 12, 19, and 28, the skin of the mice was photographed to compare the treatment progress. The EASI scoring system was used to observe and score symptoms such as erythema, edema, and dryness (desquamation) on the backs of the mice. Each parameter was scored independently from 0 to 3 (0 = normal, 1 = mild, 2 = moderate, 3 = severe), resulting in a cumulative score from 0 to 9. The assessment was performed independently by three researchers. The skin condition scoring criteria are shown in Table 16.

[0196] Table 16 Skin Condition Scoring Criteria

[0197]

[0198] like Figure 13 As shown, after DNCB sensitization, mice gradually developed symptoms such as epidermal thickening, erythema, and desquamation. After one week of treatment with Wenqingyin microemulsion gel, the skin inflammation gradually subsided, and the epidermal thickening was alleviated. After two weeks of continuous treatment, the epidermal thickness of the mice basically returned to normal, and the desquamation was significantly improved. Furthermore, the treatment effect was close to that of the positive group. 4.4 Determination of the spleen index in mice, such as... Figure 14 As shown

[0199] To assess treatment-related systemic adverse reactions, the body weight and spleen of mice in each group were weighed and the data were analyzed. Spleen index (mg / g) = spleen weight (mg) / body weight (g) × 100%.

[0200] Compared with the control group, the spleen index of the model group (Model) and the negative group (Blank ME-gel) mice increased (p<0.001), indicating that atopic dermatitis can cause excessive activation of immune cells in the spleen, infiltration of inflammatory cells, and splenomegaly. After treatment with Wenqingyin microemulsion gel (WQYME-gel), the spleen index of AD mice was significantly reduced (p<0.001), basically close to the blank group. The spleen index of mice treated with hydrocortisone butyrate cream (Positive) also decreased significantly, but was lower than the blank group, indicating that glucocorticoids not only inhibit local reactions, but also may excessively weaken the body's immune defense function. This further indicates that traditional Chinese medicine preparations can restore spleen function through immune regulation rather than broad suppression, so they may have better safety in long-term treatment.

[0201] 4.5 Histopathological examination of skin lesions, Figure 15 as shown

[0202] The skin lesion tissues of mice in each group were fixed with 10% formaldehyde solution, dehydrated with ethanol, transparentized with xylene, and embedded with paraffin. Sections were stained with HE and TB, mounted with neutral resin, and observed under a microscope for pathological changes in the back skin.

[0203] As can be seen from the above pictures, the blank group (Control) is normal skin tissue, with regular epidermis and dermis layers. The HE staining results of the model group (Model) and the negative group (Blank ME-gel) show that the mouse skin is hyperkeratotic, the epidermis is thickened, and the epidermis is protruding downward, which are all typical symptoms of atopic dermatitis. The TB staining results also show that the number of mast cells in the dermis layer of mice significantly increases. The Wenqingyin microemulsion gel treatment group (WQYME-gel) shows that the mouse keratinization degree is greatly improved, the epidermis and protruding downward phenomenon are significantly reduced, the skin has a trend of returning to normal, and the number of mast cells in the dermis layer is also reduced compared with the model group. The results are similar to those of the positive group, indicating that Wenqingyin microemulsion gel can significantly improve the skin condition of AD mice and promote recovery.

[0204] 4.6 ELISA detection of inflammatory factor levels in mouse serum and back skin, Figure 16 、 Figure 17 as shown

[0205] (1) Mice were taken blood from the eye orbit, and after 2 hours of standing at room temperature, centrifuged at 3000 r / min for 15 min, and the serum was collected and stored at -20°C. The IL-4, IL-6, IL-1β, TNF-α and IgE levels in each group were detected by ELISA. The coated microwells were pre-coated with mouse plasma antibodies, and then the samples, standards, and HRP-labeled detection antibodies were added in turn, incubated and washed thoroughly. Color development was performed with substrate TMB, which was converted to blue under the catalysis of peroxidase and to the final yellow under the action of acid. The color depth was positively correlated with the concentration of mouse plasma inflammatory cytokines in the sample. The absorbance (OD value) was measured at 450 nm wavelength by a microplate reader, and the inflammatory factor concentration was calculated according to the standard curve.

[0206] (2) After homogenizing the mouse back tissue and centrifuging to obtain the supernatant, an enzyme-labeled plate pre-coated with specific capture antibodies was used, and after washing, BSA was used to block non-specific sites. Then gradient-diluted standards and samples were added, incubated to allow the target factor to bind to the capture antibody, and after washing, HRP-labeled detection antibodies were added in turn, and after each incubation, thorough washing was performed to remove non-binders. Color development was performed by adding TMB substrate, and after stopping the reaction, the absorbance (OD value) was measured at 450 nm, and the inflammatory factor concentration was calculated according to the standard curve.

[0207] The results showed that the IgE, IL-4, IL-6, IL-1β and TNF-α levels in the serum and skin tissue of the model group mice were significantly higher than those of the blank group mice, and the difference was statistically significant (P<0.05); after treatment with Wenqing Drink microemulsion gel, the IgE level in the serum and skin tissue was significantly reduced, the expression of Th2-type immune factors IL-4 and IL-6 was inhibited, and the concentration of pro-inflammatory mediator IL-1β was decreased, and the differences were statistically significant (P<0.05). However, after treatment with Wenqing Drink microemulsion gel, only the concentration of TNF-α in the serum was significantly reduced, and the concentration of TNF-α in the skin was not reduced.

[0208] 4.7 Western blot detection of the expression of PI3K / AKT and MAPK signaling pathway-related proteins in the back tissue

[0209] About 100 mg of skin tissue sample was weighed, ground, and homogenate was added to lysis buffer and lysed on ice for 30 min; the lysate was collected in a 1.5 mL EP tube and centrifuged at 10000 rpm for 15 min at 4°C, and the supernatant was collected, aliquoted, and stored at -80°C for later use; the protein content was measured by BCA method, and the protein bands were separated by SDS-PAGE method; the proteins on the gel were transferred to a PVDF membrane, which was blocked with 5% skim milk for 1 h, and then incubated; finally, ECL chemiluminescence reagent was used for imaging.

[0210] Those skilled in the art can make various modifications or supplements to the specific embodiments described or adopt similar ways to replace, but will not deviate from the inventive concept of the present application or exceed the scope defined by the appended claims.

Claims

1. A traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis, characterized in that, The Wenqingyin microemulsion and the gel skeleton, the Wenqingyin microemulsion is loaded on the gel skeleton, and the gel skeleton is carbomer-940; The Wenqingyin microemulsion comprises 2.546% of oleic acid, 12.727% of castor oil polyoxyethylene ether, 12.727% of 1,2-pentanediol, and 72% of a water phase which is a Wenqingyin water extract; The microemulsion gel agent comprises 3.5% of carbomer-940, 10% of glycerol, and 1% of triethanolamine; The preparation method of the Wenqingyin microemulsion gel agent for treating atopic dermatitis comprises the following steps: S1. Extraction and concentration of the Wenqingyin water extract; S2. Preparing the Wenqingyin microemulsion by taking the Wenqingyin water extract extracted in step S1 as the water phase; S3. Adding the Wenqingyin microemulsion prepared in step S2 into the gel skeleton carbomer-940 to make it completely swell and prepare the microemulsion gel agent; The step S1 is specifically as follows: weighing angelica, chuanxiong, white peony root, prepared rehmannia, scute, coptis, golden cypress, and gardenia slices, soaking them in 47% ethanol for 30 min at a material-liquid ratio of 1:25, refluxing and extracting for 115 min, filtering the hot extract through four layers of gauze, repeating the extraction for two times, finally combining the extract, and concentrating the extract by rotary evaporation at 60℃, wherein the concentration of the water extract is 0.75 g / mL, and the water extract is used as the water phase of the microemulsion.

2. The preparation method of the traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis according to claim 1, characterized in that, The preparation method comprises the following steps: S1. Extraction and concentration of the Wenqingyin water extract; S2. Preparing the Wenqingyin microemulsion by taking the Wenqingyin water extract extracted in step S1 as the water phase; S3. Adding the Wenqingyin microemulsion prepared in step S2 into the gel skeleton carbomer-940 to make it completely swell and prepare the microemulsion gel agent.

3. The preparation method of the traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis according to claim 2, characterized in that, The step S2 comprises: Mixing castor oil polyoxyethylene ether, 1,2-pentanediol, and oleic acid uniformly, and then preparing the Wenqingyin microemulsion by titration with distilled water while stirring.

4. The preparation method of the traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis according to claim 3, characterized in that, The heating temperature for preparing the microemulsion in step S2 is 35℃, the stirring speed is 600 r / min, and the stirring time is 0.5 h.

5. The preparation method of the traditional Chinese medicine compound microemulsion gel for treating atopic dermatitis according to claim 4, characterized in that, The step S3 is specifically as follows: S3.

1. Taking an appropriate amount of the Wenqingyin microemulsion, and adding the gel skeleton carbomer-940 to make it completely swell; S3.

2. Adding 1% of triethanolamine while continuously stirring to form the gel; S3.

2. Adding 10% of glycerol while continuously stirring to form the finished gel agent.

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