Traditional Chinese medicine in-situ temperature-sensitive gel for treating specific dermatitis and preparation method of traditional Chinese medicine in-situ temperature-sensitive gel

Abstract

The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a traditional Chinese medicine in-situ temperature-sensitive gel for treating specific dermatitis (AD) and a preparation method thereof. The traditional Chinese medicine in-situ temperature-sensitive gel comprises the following components: an original medicine extracting solution, a gel matrix, polyethylene glycol PEG2000 and water. The raw medicine extracting solution is prepared from the following raw materials: radix angelicae sinensis, radix rehmanniae, radix saposhnikoviae, periostracum cicada, herba portulacae, cortex moutan, radix sophorae flavescentis, fructus arctii, radix glycyrrhizae and cortex dictamni. A novel natural deep eutectic (NADES) extraction method is adopted in the raw medicine extracting solution, the content of effective components of the medicine can be effectively increased, and better transdermal absorption can be achieved. The proportion of the two components of the NADES and the components forming the traditional Chinese medicine in-situ temperature-sensitive gel are subjected to orthogonal tests, the simplest and optimal effective auxiliary materials are selected, and the prepared traditional Chinese medicine in-situ temperature-sensitive gel is good in drug effect and extremely high in market value.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a traditional Chinese medicine in-situ thermosensitive gel for treating atopic dermatitis and a preparation method thereof. Background Art

[0002] Atopic dermatitis (AD) is a chronic, recurrent, inflammatory skin disease that affects 5%-10% of adults and 15%-20% of children worldwide. It is characterized by severe, persistent itching and extensive skin damage. The accompanying itching symptoms seriously lead to mental burdens such as sleep disorders and depression. Atopic dermatitis harms human physical and mental health and has become a global public health issue that needs to be addressed urgently.

[0003] The etiology of AD is complex, and its pathogenesis may be closely related to genetics, immune abnormalities, and skin barrier dysfunction, but the specific mechanism is not yet fully understood. With the continuous deepening of research, the cross-correlations between various molecules and signaling pathways that affect immune function have been discovered, and multi-mechanism and multi-target intervention has become the key to breakthroughs in AD treatment. The "multi-component, multi-target, and comprehensive treatment" treatment method of traditional Chinese medicine preparations undoubtedly provides a more comprehensive and in-depth solution for the treatment of complex diseases.

[0004] Traditional Chinese medicine is mainly extracted to obtain active ingredients, which are mainly water extraction and alcohol extraction. Water extraction is the most widely used method in clinical practice, that is, the active ingredients are extracted by decoction. However, there are some factors in the decoction process that will affect the extraction of active ingredients, such as the amount of water added, the length of time, the degree of temperature control, and the factors such as auxiliary materials, which can be continuously superimposed, resulting in a large reduction in the content of active ingredients, thereby affecting the efficacy and therapeutic effect. Therefore, it is urgent to find new extraction methods to replace traditional water extraction methods to increase the content of active ingredients and improve the treatment of AD. Summary of the invention

[0005] In view of the above-mentioned defects in the background technology, the main purpose of the present invention is to provide a traditional Chinese medicine in situ thermosensitive gel for treating AD and a preparation method thereof. The gel has a higher content of active ingredients and a better transdermal effect. When applied to the skin epidermis, the drug has good transdermal penetration effect and high drug bioavailability.

[0006] The present invention is based on the classic prescription "Xiaofengsan", combined with the latest research results of natural medicines for the treatment of AD, to improve the prescription to obtain "Xiaofengsan Improved Prescription (MXFS)", further prepare traditional Chinese medicine in situ thermosensitive gel, and use NADES extraction method to increase the content of active ingredients in the drug and improve the transdermal absorption rate.

[0007] The compatibility principle of traditional Chinese medicine is "select medicines according to law, the main and the subordinate are in order, the auxiliary and the counter-complement form the system, and the prescription and the syndrome are combined". This means that certain principles must be followed when formulating traditional Chinese medicine. The prescription composed after compatibility has a typical structure, including four parts: monarch, minister, adjuvant, and messenger. In the "Improved Xiaofengsan Prescription (MXFS)" of the present invention, Saposhnikovia divaricata, Arctium lappa, and Chan Tuo can dispel wind and evil, dispel wind and relieve itching, and are monarch medicines; Moutan bark, Portulaca oleracea, Sophora flavescens, and Dactylic acid peel can clear away heat and detoxify, and are minister medicines; Angelica sinensis and Rehmannia glutinosa can nourish blood and cool blood, and are adjuvant medicines; Licorice can clear away heat and detoxify and harmonize various medicines, and is a messenger medicine. The whole prescription has the functions of dispelling wind and relieving itching, clearing away heat and detoxifying, and is a good prescription for treating AD.

[0008] The present invention adopts the following technical solution:

[0009] A traditional Chinese medicine in situ thermosensitive gel for treating AD, the traditional Chinese medicine in situ thermosensitive gel comprising the following components: original medicine extract, gel matrix, polyethylene glycol PEG2000 and water;

[0010] The raw materials for preparing the original drug extract include the following components: angelica (A), rehmannia root (B), siler (C), cicada shell (D), purslane (E), peony bark (F), sophora flavescens (G), burdock fruit (H), liquorice (I), and white moss peel (J).

[0011] Furthermore, the weight ratio of the components is: original drug extract: gel matrix: polyethylene glycol PEG2000: water = (55-65): (16-20): (5-15): 100.

[0012] Furthermore, the gel matrix is ​​poloxamer 407 (P407), poloxamer 188 (P188) and sodium chloride.

[0013] Preferably, the weight ratio of P407:P188:sodium chloride in the gel matrix is ​​17:1.3:0.7.

[0014] Furthermore, the weight ratio of the raw materials for preparing the original drug extract is: Angelica sinensis: Rehmannia root: Saposhnikovia divaricata: Periostracum cicadae: Portulaca oleracea: Paeonia suffruticosa: Sophora flavescens: Arctium lappa: Licorice: Dictamni peel = 2:2:2:2:2:2:2:2:2:1:1.

[0015] Another object of the present invention is to provide a method for preparing the traditional Chinese medicine in-situ temperature-sensitive gel, comprising the following steps:

[0016] 1) weighing the raw materials for preparing the original drug extract, grinding them into original drug powder, adding an extractant, and subjecting them to ultrasonic treatment and then centrifugation; filtering the supernatant after centrifugation (preferably filtering three times through Whatman No. 1 filter paper) to obtain the original drug extract, and storing it at 4° C.;

[0017] 2) The in situ thermosensitive gel of traditional Chinese medicine was prepared by the "cold dissolution method": PEG2000 was dissolved in water, and the original drug extract obtained in step 1) was added, and it was completely dissolved by ultrasound. The gel matrix was added under an ice water bath and stirred evenly, and then placed in a refrigerator at 4-10°C to allow it to fully swell, thereby obtaining a clear and evenly dispersed in situ thermosensitive gel of traditional Chinese medicine.

[0018] Furthermore, in step 1), the extractant is NADES, and the NADES is a liquid mixture of citric acid: glycerol: water in a mass ratio of 4:1:5;

[0019] The preparation method of NADES is as follows: keeping a mixture of citric acid and glycerol in a mass ratio of 4:1 at 25° C. for 24 hours, then mixing it evenly with a magnetic stirrer at 70° C., adding distilled water equal to the total mass of citric acid and glycerol, and cooling to obtain a stable, uniform, transparent liquid NADES.

[0020] Furthermore, in the step 1), the solid-liquid ratio of the original drug powder to the extractant is 10 mg: 1 mL.

[0021] Furthermore, the conditions for ultrasonic treatment in step 1) are: ultrasonic treatment at 300W power for 90 minutes at 60°C; and the conditions for centrifugation are: centrifugation at 4000rpm for 8 minutes.

[0022] The present invention also provides application of the traditional Chinese medicine in-situ temperature-sensitive gel in preparing a medicine for treating atopic dermatitis.

[0023] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] (1) The traditional Chinese medicine in situ thermosensitive gel prepared by the present invention is a gel, which can be applied topically to avoid first-pass elimination and reduce adverse reactions. At the same time, it is in a liquid state in vitro, easy to apply, has a long retention time at the administration site, and can continuously release the drug. In addition, the traditional Chinese medicine in situ thermosensitive gel of the present invention has a good drug percutaneous penetration effect, which can further improve the bioavailability of the drug.

[0025] (2) The raw material for preparing the original drug extract used in the present invention is the "Xiaofengsan Modified Formula (MXFS)" obtained by modifying the classic formula "Xiaofengsan". Six herbs with duplicate efficacy and poor effect were removed from the formula, and three herbs with the latest anti-AD effect were added. The overall formula was simplified to achieve precise medication. Pharmacodynamic evaluation showed that the "Xiaofengsan Modified Formula" was more effective than "Xiaofengsan" in anti-specific dermatitis.

[0026] (3) The present invention optimizes the ratio of citric acid to glycerol in the natural deep eutectic extract, thereby improving the extraction rate of the active ingredients of the drug, avoiding the use of organic reagents to cause organic residues, and avoiding the use of permeation enhancers in the gel. The excipients in the formula are greatly simplified, avoiding the superposition of multiple excipients to interfere with the absorption of the main drug ingredients. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the in vitro release curve of the traditional Chinese medicine in situ thermosensitive gel in Example 4;

[0028] Figure 2 This is a trend diagram of pH value variation of the traditional Chinese medicine in-situ thermosensitive gel in Example 4;

[0029] Figure 3 The organ coefficients of mice in each group on the 19th day of the experiment in Example 5;

[0030] Figure 4 This is the effect of MXFS in Example 5 on DNFB-induced AD skin symptoms. Figure 4 (A) Effects of MXFS on DNFB-induced AD skin symptoms, representative dorsal skin photos of each treatment group, showing the comparison of AD-like skin lesions on days 9 and 18; Figure 4 (B) is the skin lesion score, which is the average of all individual scores in each group on days 9 and 18; Figure 4 (C) The sum of the number of scratching behaviors of mice within 15 minutes on days 9 and 18 of each group was calculated, and the results were expressed as mean ± standard error;

[0031] Figure 5 This is the effect of MXFS in Example 5 on the epidermal thickness and mast cell number of AD mice. Figure 5 (A) Hematoxylin and eosin (H&E) staining (scale bar = 100 μm); Figure 5 (B) Toluidine blue staining of dorsal skin (scale bar = 100 μm); Figure 5 (C) is the result of mast cell counting analysis, and the results are expressed as mean ± standard error; Figure 5 (D) is the measurement result of epidermal thickness;

[0032] Figure 6 The values ​​are the levels of IgE, IL-10 and IL-4 in the serum of each group of mice on the 19th day of the experiment in Example 5.

[0033] Figure 4-Figure 6 In the table, ns>0.05 indicates not significant, *p<0.05 indicates relatively significant difference, **p<0.01 indicates significant difference, ***p<0.005 indicates very significant difference, ****p<0.001 indicates extremely significant difference. DETAILED DESCRIPTION

[0034] The embodiment of the present invention provides a traditional Chinese medicine in-situ thermosensitive gel adapted from a classic formula drug and having an optimal preparation process, with Chinese medicinal materials including angelica, rehmannia root, siler, cicada shell, purslane, peony bark, sophora flavescens, burdock fruit, liquorice, and white moss peel as important drug ingredients, good transdermal absorption, the drug is in the form of a gel, and is applied to the skin in the form of a gel, with high bioavailability. The local external use of this gel can avoid first-pass elimination and reduce adverse reactions, is easy to operate, has a long retention time at the administration site, can continuously release the drug, and has a better anti-specific dermatitis effect than "Xiaofengsan".

[0035] Unless otherwise specified, the materials, reagents, instruments, etc. used in the examples of the present invention can be obtained from commercial sources, and the names of materials, reagents, reactions, instruments, etc. are all conventional terms in the art.

[0036] The above contents are further described in detail below, but the scope of the present invention is not limited to the following embodiments. Without departing from the content of the present invention, any changes based on the conventional technical knowledge and means in the art shall fall within the protection scope of the present invention.

[0037] Xiaofengsan, a well-known Chinese medicine prescription, has been used in the clinic to treat dermatitis. However, with further research on Chinese medicine ingredients, Chinese medicine prescriptions with multiple chemical components have shown additive and synergistic advantages in the treatment of AD and have shown excellent anti-AD effects. In order to further adopt precise drug delivery for atopic dermatitis, combined with the etiology and pathogenesis of AD analyzed by Chinese medicine, we established a clinical modified formula based on "Xiaofengsan", named "Xiaofengsan Modified Prescription (MXFS)".

[0038] The herbal dosages of MXFS are shown in Table 1.

[0039] Table 1 Herbal formula of MXFS

[0040]

[0041]

[0042] MXFS is composed of 10 herbs, of which 7 main ingredients of "Xiaofengsan" are retained, including Angelica sinensis, Rehmannia root, Saposhnikovia divaricata, Periostracum cicadae, Sophora flavescens, Arctium lappa, and Licorice, and three new herbs are added that can repair the skin barrier, lower the level of inflammation, and significantly improve the symptoms of AD: Portulaca oleracea, Cortex Moutan, and Cortex Dactylis chinensis. Among them, Portulaca oleracea can significantly upregulate the expression of Filaddrin and caspase-14 in the skin barrier system of AD animals, suggesting that Portulaca oleracea can accelerate the dephosphorylation of Profiladdrin into Filaddrin by promoting the expression of Filaddrin and (caspase)-14 genes, and then accelerate the hydrolysis of Filaddrin into various natural moisturizing factors to maintain the water content of the stratum corneum, participate in skin hydration, and maintain the skin barrier; Paeonol, the main component of Paeonia suffruticosa, can improve the symptoms of atopic dermatitis by inhibiting the production of inflammatory cytokines, inducing Foxp3 expression, increasing intestinal barrier integrity and changing the composition of intestinal microbiota; Dictyophora diffusa (DC) is a Chinese herbal medicine with the effect of dispelling wind and relieving itching, and is the most popular single herbal medicine for the treatment of atopic dermatitis. Dictyophora extract (DCE) can significantly improve AD-like symptoms in AD-like mice by inhibiting mast cell infiltration, inhibiting the production of Th2-related cytokines (IL-4) and pro-inflammatory cytokines (TNF-α), and enhancing the protein expression of filaggrin by inhibiting MAPKs and NF-KB pathways. In addition, DCE inhibits mast cell degranulation by reducing the level and inactivation of intracellular Ca2+Lyn, Syk, and PLCγs, indicating that DCE can regulate mast cell-mediated allergic reactions and significantly improve AD symptoms.

[0043] The traditional Chinese medicine in situ thermosensitive gel for treating AD in the embodiment of the present invention comprises the following components in weight ratio: original medicine extract: poloxamer 407 (P407): poloxamer 188 (P188): polyethylene glycol PEG2000: sodium chloride: water = 58:17:1.3:12:0.7:100.

[0044] The raw materials for preparing the above-mentioned original drug extract include Angelica sinensis (A), Rehmannia root (B), Saposhnikovia divaricata (C), Periostracum cicadae (D), Portulaca oleracea (E), Paeonia suffruticosa (F), Sophora flavescens (G), Arctium lappa (H), Licorice (I), and Dictamni (J), and the weight ratio is: A:B:C:D:E:F:G:H:I:J=2:2:2:2:2:2:2:2:2:1:1.

[0045] Example 1

[0046] Screening of the Optimal Process for Extraction of Drug Solution

[0047] Required reagents: 65% ethanol (ethanol concentrations are all volume concentrations), 70% ethanol, 75% ethanol, water, NADES solution with a citric acid to glycerol mass ratio of 2:1, NADES solution with a citric acid to glycerol mass ratio of 3:1, and NADES solution with a citric acid to glycerol mass ratio of 4:1.

[0048] In situ thermosensitive gel extraction process of traditional Chinese medicine: After using single factor to investigate the factors affecting the extraction of flavonoid compounds (i.e., matrine in Sophora flavescens, paeonol in Paeonia suffruticosa, and arctiin in Arctium lappa), the optimal extraction process was investigated using five-factor three-level orthogonal method.

[0049] Specific steps of orthogonal experiment:

[0050] Weigh Chinese Angelica (A), Rehmannia (B), Saposhnikovia (C), Periostracum Cicadae (D), Portulaca Oleracea (E), Paeonia Suffruticosa (F), Sophora flavescens (G), Arctium lappa (H), Licorice (I), and Dictamni (J) in the weight ratio of A:B:C:D:E:F:G:H:I:J=2:2:2:2:2:2:2:2:1:1, 1 portion is 3 g, and grind into original drug powder.

[0051] Extraction method: different extractants (solid-liquid ratio of 10 mg: 1 mL) were added to the above-mentioned original drug powder, and ultrasonic treatment was performed using a digital ultrasonic extractor at different temperatures and different powers for 90 minutes, and then centrifuged at 4000 rpm for 8 minutes. The supernatant obtained after centrifugation was filtered three times through Whatman No. 1 filter paper to obtain the original drug extract.

[0052] The extraction methods using ethanol, pure water and NADES as extraction agents were investigated respectively. The preparation method of NADES was as follows: 20 g of a mixture of citric acid and glycerol in different mass ratios was placed in a 50 mL conical flask, kept at 25°C for 24 hours, then mixed evenly with a magnetic stirrer at 70°C, and then 20 g of distilled water was added and cooled to obtain a stable, uniform and transparent liquid NADES.

[0053] A single-factor experiment was used to investigate the effects of ethanol volume concentration, the mass ratio of citric acid and glycerol in NADES, ultrasonic temperature, and ultrasonic power on the extraction rate. The temperature and power were set according to different schemes. The specific schemes are shown in Table 2.

[0054] HPLC high performance liquid chromatography and external standard method were used to detect and calculate the contents of matrine, paeonol and arctiin in the original drug extract. (Chromatographic conditions: Shimadzu LC-16 automatic injection high performance liquid chromatograph, AQ-C18 chromatographic column, 250mm×4.6mm, 5μm; flow rate: 1.0mL / min; column temperature: 35℃; injection volume 5μL, the mobile phase for matrine detection was methanol-0.025% phosphoric acid solution (5:95), and the detection wavelength was 220nm; the mobile phase for paeonol detection was methanol-water solution (65:35), and the detection wavelength was 274nm; the mobile phase for arctiin detection was methanol-water solution (1:1.2); the detection wavelength was 280nm), the results are shown in Tables 3, 4, and 5.

[0055] Table 2 Factor level design table of ethanol extraction method, pure water extraction method and NADES extraction method

[0056]

[0057]

[0058] Table 3 Results of orthogonal test of NADES extraction method

[0059]

[0060] Note: K value represents the sum average of the indicators at different levels of each factor; R represents the range, which is an important indicator to measure the influence of factors on experimental results. K1~K3, R1 corresponds to matrine; K4~K6, R2 corresponds to paeonol; K 7～ K9 and R3 correspond to arctiin.

[0061] Table 4 Results of orthogonal experiment of ethanol extraction

[0062]

[0063] Note: K value represents the sum average of the indicators at different levels of each factor; R represents the range, which is an important indicator to measure the influence of factors on experimental results. K1~K3, R1 corresponds to matrine; K4~K6, R2 corresponds to paeonol; K 7～ K9 and R3 correspond to arctiin.

[0064] Table 5 Results of orthogonal test of water extraction method

[0065]

[0066] Note: K value represents the sum average of the indicators at different levels of each factor; R represents the range, which is an important indicator to measure the influence of factors on experimental results. K1, K2, and R1 correspond to matrine; K3, K4, and R2 correspond to paeonol; and K5, K6, and R3 correspond to arctiin.

[0067] Comparing Tables 3, 4, and 5, it can be seen that the NADES extraction rate > ethanol extraction rate > water extraction rate. Under the conditions of NADES extraction, it can be seen from Table 3 that the content of effective substances extracted is the highest when the ultrasonic temperature is 60°C, the power is 300W, and the mass ratio of citric acid to glycerol is 4:1.

[0068] Therefore, the best extraction process was selected as the ultrasound-assisted NADES extraction method, that is, ultrasound (power of 300 W) was performed in a 60°C water bath with NADES having a mass ratio of citric acid to glycerol of 4:1 for 90 min.

[0069] Example 2

[0070] Screening of the optimal composition of the Chinese medicine in situ thermosensitive gel matrix: The Chinese medicine in situ thermosensitive gel for the treatment of AD is prepared according to the following conditions, including the following components in weight proportions: original drug extract: P407: P188: polyethylene glycol PEG2000: sodium chloride: water = 58: (16-18): (1.3-1.5): 12: (0.6-0.8): 100.

[0071] After obtaining the original drug extract according to the optimal conditions in Example 1, the "cold dissolution method" was used to prepare the traditional Chinese medicine in situ thermosensitive gel: PEG2000 was dissolved in water, and the original drug extract was added, and it was completely dissolved by ultrasound. P407, P188 and sodium chloride were slowly added in turn under an ice water bath and stirred evenly to obtain a gel solution, which was then placed in a refrigerator at 4-10°C for storage.

[0072] The gelation temperature and gelation time are determined by the "stirring bar method". The gel solution of different weight proportions stored at 4-10°C is placed in a vial, the vial is placed in a water bath pot with a constant temperature heating magnetic stirrer, a magnetic stirrer (size 0.6cm×1.2cm) is added, and the probe of the thermometer is completely immersed in the gel solution; the initial water bath is controlled at a low temperature (10°C), the stirring switch is turned on, the stirring speed is 200r / min, and the water bath temperature is controlled to continue to slowly rise at a rate of 1°C to 2°C / min; the temperature when the magnetic stirrer stops rotating completely is observed, and this temperature is defined as the gelation temperature, and the time from adding the magnetic stirrer to the time when the magnetic stirrer stops rotating completely is defined as the gelation time. The gelation temperature and time of each formula are measured in parallel 3 times, and the average value is taken as the final gelation temperature.

[0073] After using single factor to investigate the influencing factors on gelation time and gelation temperature, the optimal extraction process was investigated through three-factor three-level orthogonal method.

[0074] Specific steps of orthogonal experiment: P188, P407 and sodium chloride with different mass ratios were selected to prepare 9 kinds of gel matrices as shown in Table 6, and their gel properties were investigated. The results of the orthogonal test of gel matrices are shown in Table 7.

[0075] Table 6 Design of factors affecting gel properties

[0076]

[0077] Table 7 Gel matrix orthogonal test results

[0078]

[0079]

[0080] It can be seen from Table 7 that when 17% P407, 1.3% P188, and 0.7% sodium chloride are selected, the gelation temperature is 35.55°C, which is slightly lower than the skin epidermal temperature, and the gelation time is shorter at this time. Therefore, 17% P407, 1.3% P188, and 0.7% sodium chloride are used as the gel matrix.

[0081] Example 3

[0082] Preparation of Chinese medicine in situ thermosensitive gel: The Chinese medicine in situ thermosensitive gel is prepared by "cold dissolution method": 12g PEG2000 is dissolved in 100mL of water, and 58g of the original drug extract obtained by the optimal extraction process in Example 1 is added, and it is completely dissolved by ultrasound. 17g P407, 1.3g P188 and 0.7g sodium chloride are slowly added in an ice-water bath and stirred evenly, and then placed in a 4-10°C refrigerator for 24h to fully swell, and a clear, uniformly dispersed Chinese medicine in situ thermosensitive gel (i.e., formula 3) is obtained. On the basis of formula 3, azone (azone accounts for 1.5% of the mass of formula 3) is added to obtain formula 2.

[0083] Except that the extractant is water, the other preparation conditions of Formula 4 are the same as those of the preparation of the in-situ thermosensitive gel of traditional Chinese medicine. Based on Formula 4, azone (azone accounts for 1.5% of the mass of Formula 4) is added to obtain Formula 1.

[0084] Except that the extractant of Formula 5 is 65% ethanol, the other preparation conditions are the same as those of the preparation of the in-situ thermosensitive gel of traditional Chinese medicine. Based on Formula 5, azone (azone accounts for 1.5% of the mass of Formula 5) is added to obtain Formula 6.

[0085] Preparation of Formula 8: Weigh 6g each of Angelica sinensis, Rehmannia root, Saposhnikovia divaricata, Periostracum cicadae, Rhizoma Anemarrhenae, Sophora flavescens, Sesame, Nepeta tenuifolia, Atractylodes lancea, Fructus arctii, Gypsum, and 3g each of Licorice and Akebia. After obtaining the original drug extract with XFS as the main drug according to the optimal extraction process in Example 1, obtain Formula 8 according to the preparation conditions of the in-situ thermosensitive gel of traditional Chinese medicine. On the basis of Formula 8, add azone (azone accounts for 1.5% of the mass of Formula 8) to obtain Formula 7.

[0086] Preparation of ex vivo pig skin: Take pig skin, remove pig hair, scrape off subcutaneous fat tissue, make the thickness of all pig skin uniform, and store in refrigerator for later use.

[0087] The effect of transdermal absorption was studied after adding different penetration enhancers to the in-situ thermosensitive gel of traditional Chinese medicine in this embodiment: a Franz diffusion cell was used for transdermal test. In group experiments, pigskin was fixed between the diffusion chamber and the receiving chamber of the transdermal diffusion instrument, with the inner side of the pigskin facing the receiving chamber and the stratum corneum of the skin facing the diffusion chamber to make it in close contact. 1.2g of different formula drugs (specific drug components are shown in Table 8) were covered on the two groups of pigskin diffusion chambers, and physiological saline receiving liquid was injected into the receiving chamber. The liquid surface just contacted the inner layer of the skin, and the electromagnetic stirrer was started to stir at a constant speed and the temperature was kept at 37°C. The transdermal absorption liquid was collected at 1.5, 3, 4.5, 6, and 7.5h respectively, and its absorbance was measured by ultraviolet spectrophotometry using matrine as a marker, and the cumulative penetration amount was calculated. Table 9 shows the cumulative penetration amount at each time point.

[0088] Table 8 Specific drug components of each formula drug

[0089]

[0090] Table 9 Cumulative permeation of each formula

[0091]

[0092] As shown in Table 9, there is no statistically significant difference in the experimental results between formula 2 and 3. Considering the simplification of the formula, formula 3 was selected. Therefore, the final composition of the gel was determined to be the following components in the following weight ratio: original drug extract: P407: P188: polyethylene glycol PEG2000: sodium chloride: water = 58: 17: 1.3: 12: 0.7: 100.

[0093] Example 4 Performance Test

[0094] 1. pH value determination

[0095] The pH value of the traditional Chinese medicine in situ thermosensitive gel with the best transdermal effect obtained in Example 3 (i.e., Formula 3) was determined according to the method in General Chapter 0631 of Part 4 of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The experiments were conducted three times in parallel, and the average value was 5.64, which is within the normal pH range of human skin (4.5-6.5), so the gel is safe.

[0096] 2. Dissolution

[0097] The Franz diffusion cell was used to conduct a transdermal test to determine the cumulative release rate: the pigskin was fixed between the diffusion chamber and the receiving chamber of the transdermal diffusion instrument, with the inner side of the pigskin facing the receiving chamber and the skin stratum corneum facing the diffusion chamber, so that it was in close contact. 1.2g of the best transdermal Chinese medicine in-situ thermosensitive gel (i.e., formula 3) obtained in Example 3 was covered on the two groups of pigskin diffusion chambers, and physiological saline receiving liquid was injected into the receiving chamber, and the liquid surface just contacted the inner layer of the skin. The electromagnetic stirrer was started to stir at a constant speed and the temperature was kept at 37°C. The transdermal absorption liquid was collected at 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, and 720 minutes, respectively. Matrine was used as a marker, and the content was determined by HPLC, and the cumulative release rate was calculated. The cumulative release rate at different time points was calculated according to the following formula:

[0098] Q = the concentration of matrine in the absorption solution measured at different time points Mt / the concentration of matrine after complete release M∞*100%.

[0099] Where M∞ is the concentration of matrine in the absorption solution measured at 720 min.

[0100] The drug release curve was obtained by plotting the cumulative release rate Q against the release time t. The origin software Allometric1 distribution model fitted the equation Q = Mt / M∞*100% = 93.47 (1-e -0.003t ), the results are shown in Figure 1 (A): Fitting equation Q = Mt / M∞*100% = 3.80*(t^(1 / 2))-12.18 using Higuchi distribution model. The results are shown in Figure 1 (B).

[0101] Depend on Figure 1 It can be seen that the release rate of the in-situ temperature-sensitive gel of traditional Chinese medicine was not very stable in the early stage. The cumulative release rate was 28.20% in 120 minutes, 56.14% in 300 minutes, and about 78.00% after 600 minutes, which was close to stability. The results show that the release rate of the in-situ temperature-sensitive gel of traditional Chinese medicine is relatively slow. In summary, in the prescribed release medium, the in-situ temperature-sensitive gel of traditional Chinese medicine can slowly and non-constantly release drugs according to time changes, which can reduce the frequency of administration. The in-situ temperature-sensitive gel of traditional Chinese medicine shows obvious sustained-release characteristics and can achieve complete release.

[0102] 3. Stability test

[0103] 30 g of the best transdermal Chinese medicine in-situ thermosensitive gel sample (Formula 3) prepared in Example 3 was sealed in a transparent glass bottle and placed at a temperature of 25±2°C and a relative humidity of 60±5% for 6 months. Samples were taken 5 times at the 0th, 1st, 2nd, 3rd and 6th month of the test period, and the pH value changes were recorded. Figure 2 , Figure 2 There was no significant change in pH value.

[0104] At the same time, the samples after being placed for 6 months were placed at 25°C and 37°C for 15 minutes and then centrifuged (2000r / min, 30min) to observe whether there were unstable phenomena such as stratification. The results showed that the gel solution did not undergo stratification after being placed at 25°C and 37°C and centrifuged, indicating that the quality of the gel was stable under these conditions, and the traditional Chinese medicine in situ thermosensitive gel prepared by the present invention could be effectively and stably stored for a long time.

[0105] Example 5 Animal Experiment

[0106] 1. Construction of animal model

[0107] In this experiment, 40 female mice were randomly divided into 5 groups (n=8): (1) normal control group (Normal); (2) DNFB model group (Model); (3) dexamethasone (DEX) positive control group (999 dermatitis flat); (4) MXFS group; (5) XFS group. After one week of adaptive feeding, the back hair of the mice was shaved (about 3cm×2cm) one day before the experiment. Then, on the first day of the experiment, the normal control group used 100 μL of matrix solution (mixed solvent of acetone: olive oil volume ratio = 3:1, the same below) to evenly apply on the mouse depilatory area, and the other groups used 100 μL of 1wt% 2,4-dinitrofluorobenzene DNFB solution (solvent is a mixture of acetone: olive oil volume ratio = 3:1, the same below) to apply sensitization. On the second day of the experiment, each group had no special treatment. From the third day, the normal control group applied 100 μL of matrix solution every 3 days, and the other groups applied 100 μL of 0.5wt% DNFB solution every 3 days to induce dermatitis, once every three days. From the fourth day of the experiment, the model group, DEX, MXFS, and XFS groups were administered with corresponding drugs at the same time every day. The drug of the MXFS group was the traditional Chinese medicine in situ thermosensitive gel (formula 3) with the best transdermal effect obtained in Example 3, and the drug of the XFS group was formula 8 in Example 3, once in the morning and evening, 0.15 g each time, and administered continuously for 15 days.

[0108] 2. Drug safety verification

[0109] The weight of mice, daily food and water intake were recorded every day in the experiment, and the average values ​​were taken as shown in Table 10. It can be seen that there was no significant difference in the weight of mice in each group.

[0110] To further verify the safety, mice in each group were killed by cervical dislocation on the 19th day of the experiment, and their thymus and spleen tissues were dissected and removed. The residual blood was removed with filter paper and then weighed immediately to calculate the organ coefficient: organ index (g / g) = 100 × organ wet weight / mouse body weight. Figure 3It can be seen that the drug has no obvious effect on the organ indexes of mice, so the drug is considered safe.

[0111] Table 10 Mouse data records

[0112]

[0113]

[0114] 3. Pharmacodynamic Verification

[0115] 1. Skin lesion score and scratching frequency measurement

[0116] In the above experiment, mice were sensitized with 100 μL 0.5 wt% 2,4-dinitrofluorobenzene DNFB for 30 min on days 9 and 18, respectively, and then placed in an observation box (18×24×30 cm) for 15 min of adaptive activity. The mice were then recorded with a camera for 15 min, and the video was played back to record scratching behavior (the mouse scratched the trunk and back with its hind paws, and bit all parts of the body with its mouth, and continuous scratching was counted as 1 time).

[0117] Clinical symptoms were scored on the 9th and 18th days of the experiment to assess the severity of dorsal dermatitis. The specific scoring criteria were divided into four parts: erythema / hemorrhage, exudation / scab, epidermal exfoliation / erosion, and desquamation / dryness, and each part was scored as follows: 0 (none), 1 (mild), 2 (moderate), and 3 (severe). The maximum score for each mouse was 12 points.

[0118] Figure 4 (A) Effects of MXFS on DNFB-induced AD skin symptoms, representative dorsal skin photos of each treatment group, showing the comparison of AD-like skin lesions on days 9 and 18; Figure 4 (B) is the skin lesion score, which is the average of all individual scores in each group on days 9 and 18; Figure 4 (C) The average number of scratching behaviors of mice within 15 minutes on the 9th and 18th days of each group was calculated, and the results were expressed as mean ± standard error.

[0119] Figure 4 (A) It can be seen that on the 18th day of modeling, the skin of mice in the Model group was significantly thickened, with obvious erosion, swelling, scabs and scaling, accompanied by scratches of varying degrees. The skin lesions of mice in the MXFS, XFS and DEX treatment groups were significantly alleviated. Figure 4 (B) and 4(C) show that compared with the Normal group mice, the scratching times of the Model group mice within 15 minutes increased significantly, while the scratching times of the MXFS, XFS and DEX groups decreased significantly, and the MXFS group was superior to the XFS group, and the skin lesion score was significantly reduced.

[0120] 2. Histopathological observation

[0121] On the 19th day, mice in each group were killed by cervical dislocation, and the lesional skin on the back of the mice was cut off. Part of the skin tissue was fixed with 4% paraformaldehyde solution, embedded in paraffin, and sliced ​​for HE staining and toluidine blue staining. Five fields of view were randomly selected for each section, and the epidermal thickness and number of mast cells in each field of view were measured by ImageJ, and the mean was calculated.

[0122] Figure 5 The effect of MXFS on epidermal thickness and mast cell number in AD mice. Figure 5 (A) Hematoxylin and eosin (H&E) staining (scale bar = 100 μm); Figure 5 (B) Toluidine blue staining of dorsal skin (scale bar = 100 μm); Figure 5 (C) is the result of mast cell counting analysis, and the results are expressed as mean ± standard error; Figure 5 (D) is the measurement result of epidermal thickness.

[0123] Depend on Figure 5 (A) It can be seen that under HE staining, the skin structures of the Normal group were intact, the boundary between the dermis and the epidermis was clear, and there was no obvious inflammatory cell infiltration. Compared with the Normal group, the epidermis of the Model group was incompletely keratinized and hyperkeratinized, the spinous layer was obviously thickened, and there was a large amount of inflammatory cell infiltration in the upper superficial layer of the dermis, accompanied by capillary dilation. The epidermal thickness of the Model group was significantly higher than that of the Normal group. The inflammatory cell infiltration of the dermis in the skin tissue of the MXFS group, DEX group, and XFS group was significantly reduced, and the epidermal thickening phenomenon was also significantly alleviated, and the difference was statistically significant (P<0.01).

[0124] Figure 5 (B) and Figure 5 As shown in (D), the mast cells in the skin lesions of each group were stained with toluidine blue. The number of mast cells infiltrating in the back skin lesions of mice in the Model group increased significantly. The mast cell infiltration in the MXFS group, XFS group, and DEX group was significantly reduced, and the difference was statistically significant (P<0.01).

[0125] 3. Determination of inflammatory factor levels

[0126] On the 19th day of the experiment, the right eyeballs of the mice were removed before the mice were killed by cervical dislocation and blood was collected. After being placed at room temperature for 2 hours, the blood was centrifuged at 3500r / min for 15 minutes, and the serum was collected and stored in a -20℃ refrigerator for testing. The ELISA method was used to determine the levels of IgE, IL-10, and IL-4 in the serum of each group of mice, and the operation was strictly in accordance with the instructions of the IgE, IL-10, and IL-4 detection kits (purchased from Wuhan ABconal Technology Co., Ltd.). The experimental results are shown in the figure. Figure 6 .

[0127] From the experimental results, it is found that Figure 6 As shown in (A), the inflammatory factor IL-4 in the serum of mice in the Model group increased significantly, while MXFS, XFS and DEX significantly reduced the total serum IL-4 level of mice, and the difference was statistically significant. Figure 6 As shown in (B), the serum total IL-10 levels in the MXFS, XFS and DEX groups were significantly higher than those in the Model group, and the differences were statistically significant (p<0.05).

[0128] In addition, the large amount of IgE in serum has been considered a typical sign of AD. In the experiment, after mice were sensitized with DNFB, Figure 6 As shown in (C), the total serum IgE level of mice in the Model group was significantly increased, while MXFS, XFS and DEX significantly reduced the total serum IgE level of mice, and the difference was statistically significant (p<0.05).

[0129] Therefore, it is believed that the traditional Chinese medicine in situ thermosensitive gel prepared based on MXFS in the present invention has good efficacy and extremely high market value.

[0130] The above content has been described in detail with general explanations and specific implementation results of the present invention, but it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. A traditional Chinese medicine in situ thermosensitive gel for treating atopic dermatitis AD, characterized in that: The traditional Chinese medicine in-situ thermosensitive gel comprises the following components: original medicine extract, gel matrix, polyethylene glycol PEG2000 and water; The raw materials for preparing the original drug extract include the following components: angelica, rehmannia root, siler, cicada shell, purslane, peony bark, sophora flavescens, burdock fruit, liquorice and white moss peel.

2. The Chinese medicine in situ thermosensitive gel according to claim 1, characterized in that: The weight ratio of the components is: original drug extract: gel matrix: polyethylene glycol PEG2000: water = (55-65): (16-20): (5-15):

100.

3. The Chinese medicine in situ thermosensitive gel according to claim 1 or 2, characterized in that: The gel matrix is ​​poloxamer 407, poloxamer 188 and sodium chloride.

4. The traditional Chinese medicine in situ thermosensitive gel according to claim 1, characterized in that: The weight ratio of the raw materials for preparing the original drug extract is: Angelica sinensis: Rehmannia root: Saposhnikovia divaricata: Periostracum cicadae: Portulaca oleracea: Paeonia suffruticosa: Sophora flavescens: Arctium lappa: Licorice: Dictamni peel = 2:2:2:2:2:2:2:2:2:1:

1.

5. A method for preparing the traditional Chinese medicine in situ thermosensitive gel according to any one of claims 1 to 4, comprising the following steps: 1) Weigh the raw materials for preparing the original drug extract according to any one of claims 1 to 4, grind them into original drug powder, add the extractant, perform ultrasonic treatment and then centrifuge; filter the supernatant after centrifugation to obtain the original drug extract, and store it at 4°C; 2) The in situ thermosensitive gel of traditional Chinese medicine was prepared by the "cold dissolution method": PEG2000 was dissolved in water, and the original drug extract obtained in step 1) was added, and it was completely dissolved by ultrasound. The gel matrix was added in an ice water bath and stirred evenly, and then placed in a refrigerator at 4-10 ℃ to allow it to fully swell, thereby obtaining a clear and evenly dispersed in situ thermosensitive gel of traditional Chinese medicine.

6. The preparation method according to claim 5, characterized in that: The extractant in step 1) is NADES, which is a mixture of citric acid: glycerol: water in a mass ratio of 4:1:

5.

7. The preparation method according to claim 6, characterized in that: The preparation method of NADES is as follows: keeping a mixture of citric acid and glycerol in a mass ratio of 4:1 at 25° C. for 24 hours, then mixing it evenly with a magnetic stirrer at 70° C., adding distilled water equal to the total mass of citric acid and glycerol, and cooling to obtain a stable, uniform, transparent liquid NADES.

8. The preparation method according to claim 5, characterized in that: In the step 1), the material-liquid ratio of the original drug powder to the extractant is 10 mg:1 mL.

9. The preparation method according to claim 5, characterized in that: The conditions for the ultrasonic treatment in step 1) are: ultrasonic treatment at 60° C. and 300 W power for 90 min; the conditions for the centrifugation are: centrifugation at 4000 rpm for 8 min.

10. Use of the traditional Chinese medicine in situ thermosensitive gel according to claims 1 to 4 or the traditional Chinese medicine in situ thermosensitive gel obtained by the preparation method according to claims 5 to 9 in preparing a drug for treating atopic dermatitis.

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