Pharmaceutical composition for treating hypertrophic scars and application

Abstract

The invention provides a pharmaceutical composition for treating hypertrophic scars and application, and belongs to the technical field of traditional Chinese medicine, the pharmaceutical composition is obtained by screening traditional Chinese medicine centella asiatica and rheum officinale through an immobilized Notch1 receptor chromatography model, and the pharmaceutical composition is added into optimized blank ointment to prepare an anti-hypertrophic scar external cream preparation. A hypertrophic scar animal model is copied to evaluate the drug effect of the traditional Chinese medicine composition, and it is found that the screened traditional Chinese medicine composition has a good hypertrophic scar resisting effect.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine technology, and in particular relates to a pharmaceutical composition and its application for treating hypertrophic scars. Background Technology

[0002] Hypertrophic scars are abnormal fibrous reactions following skin trauma, inflammation, surgery, or burns. They result from excessive repair and abnormal proliferation of the skin after injury, primarily manifested as excessive proliferation of fibroblasts and excessive deposition of extracellular matrix. Hypertrophic scars often affect a patient's appearance and are accompanied by itching and pain. Furthermore, the contractures they cause can lead to varying degrees of functional impairment, such as tendon contractures, joint dislocations, and motor dysfunction, severely reducing quality of life. Literature reports that hypertrophic scars are related to factors such as genetics, trauma, regulation by multiple cytokines, and inflammatory responses; therefore, hypertrophic scars can be inhibited through these different pathways. Currently, the main treatments for hypertrophic scars include surgical excision combined with skin grafting, which carries a high risk of recurrence; radiotherapy, which uses superficial X-rays or electron beams to inhibit scar hyperplasia by killing fibroblasts and disrupting collagen synthesis and degradation, but it can have systemic or local effects and should be used with caution; cryotherapy, but complications such as increased pigmentation and mild skin atrophy can occur during the cryotherapy process; and pressure therapy, suitable for large scar areas that are difficult to treat with medication or radiotherapy, although surgical treatment combined with pressure therapy has good results, the treatment time is long. In addition, injectable drug therapies such as imiquimod and rapamycin, while able to inhibit scar hyperplasia to some extent, still have a high long-term recurrence rate.

[0003] Recent research indicates that hypertrophic scarring is related to the Notch1 signaling pathway. The Notch1 signaling pathway is not only a crucial signal for the proliferation and differentiation of keratinocytes, but also a key regulatory signal for the formation and regeneration of skin appendages, and it participates in angiogenesis and formation. The Notch1 signaling pathway is involved in cell proliferation, differentiation, and apoptosis. Activation of the Notch1 signaling pathway can induce epidermal stem cells to differentiate into fibroblasts. Fibroblasts secrete cytoplasmic matrix, and excessive activation of the Notch1 signaling pathway leads to excessive accumulation of cytoplasmic matrix, resulting in hypertrophic scarring. Inhibiting the Notch1 signaling pathway may inhibit the formation of hypertrophic scars. Traditional Chinese medicine believes that scars are caused by the interplay of qi stagnation and blood stasis, meridian obstruction, and phlegm-dampness accumulation. Clinical treatment often employs methods such as promoting blood circulation and removing blood stasis, softening and dispersing masses, and clearing the meridians and relieving pain, combining internal and external therapies. However, there are currently no treatments targeting the Notch1 signaling pathway specifically for hypertrophic scarring.

[0004] Medications or topical preparations for scarring still require further research. Summary of the Invention

[0005] Therefore, the object of the present invention is to provide a pharmaceutical composition and application for treating hypertrophic scars.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a pharmaceutical composition for treating hypertrophic scars, comprising the following raw materials in parts by weight:

[0008] 0.5-2 parts of asiaticoside, 0.5-2 parts of hydroxyasiaticoside, 0.5-2 parts of aloe-emodin, 1-3 parts of rhein, and 0.5-2 parts of emodin.

[0009] Preferably, the raw materials include the following parts by weight:

[0010] Centella asiatica 0.7–1.5 parts, hydroxycentella asiatica 0.7–1.5 parts, aloe-emodin 0.7–1.5 parts, rhein 1.5–2.5 parts, and emodin 0.7–1.5 parts.

[0011] Preferably, the raw materials include the following parts by weight:

[0012] 1 part asiaticoside, 1 part hydroxyasiaticoside, 1 part aloe-emodin, 2 parts rhein, 1 part emodin.

[0013] This invention provides the use of the pharmaceutical composition described herein in the preparation of a medicament for treating hypertrophic scars.

[0014] This invention provides an ointment for treating hypertrophic scars, the ointment comprising the following components in the following mass ratio:

[0015] The pharmaceutical composition is as follows: stearic acid, glyceryl monostearate, liquid paraffin, white petrolatum, triethanolamine, glycerin, water, ethylparaben = 2-10: 5-7: 5-15: 20-25: 5-15: 5-10: 5-15: 30-40: 0.01-0.2.

[0016] This invention provides a method for preparing the ointment, comprising the following steps:

[0017] 1) The pharmaceutical composition, stearic acid, glyceryl monostearate, liquid paraffin, and white petrolatum are mixed to obtain an oil phase;

[0018] 2) Triethanolamine, glycerol, ethylparaben, and water were mixed to obtain an aqueous phase;

[0019] 3) Heat the aqueous phase and oil phase separately and then mix them to obtain the ointment.

[0020] This invention provides the use of the ointment or the ointment obtained by the preparation method in the preparation of a medicament for treating hypertrophic scars.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention provides a pharmaceutical composition and its application for treating hypertrophic scars. The traditional Chinese medicines Centella asiatica and rhubarb are extracted separately by heating and reflux with ethanol. The anti-hypertrophic scar components are then screened using an immobilized Notch1 receptor chromatographic model to obtain compounds that target the Notch1 receptor. Compared to other methods, this approach has the advantages of high target specificity and a clear mechanism of action. Combining the screened compounds yields a superior pharmaceutical composition for treating hypertrophic scars. Furthermore, this invention optimizes the composition of the blank ointment. After mixing the blank ointment with the pharmaceutical composition, the resulting ointment for treating hypertrophic scars also exhibits a significant anti-scar hyperplasia effect. Attached Figure Description

[0023] Figure 1 Preparation route for an ointment (Centella asiatica / rhubarb anti-hypertrophic scar cream) for treating hypertrophic scars;

[0024] Figure 2 SDS-PAGE analysis was performed to purify Notch1 receptor expression, where A represents uninduced cells; L represents induced cells; P represents the supernatant obtained after cell lysis using cell lysis buffer; and P1 represents Ni. 2+ Column purification of the target protein; P2 represents the target protein after DEAE column purification.

[0025] Figure 3 Preparation of an ointment for treating hypertrophic scars (Centella asiatica / rhubarb anti-hypertrophic scar cream);

[0026] Figure 4 Active ingredients of Centella asiatica for treating hypertrophic scars screened using Notch1 receptor chromatographic columns;

[0027] Figure 5 Active components of rhubarb for treating hypertrophic scars screened using Notch1 receptor chromatographic columns

[0028] Figure 6 The images show the final products of different creams, where A is the Centella Asiatica cream, B is the Rhubarb cream, C is the low-dose compound cream, D is the medium-dose compound cream, and E is the high-dose compound cream.

[0029] Figure 7 Evaluation results of treatment in different groups;

[0030] Figure 8 HE staining results for different groups: A is blank control group, B is model group, C is positive drug group, D is Centella asiatica cream, E is rhubarb cream, F is compound low-dose cream, G is compound medium-dose cream, and H is compound high-dose cream.

[0031] Figure 9 Masson staining results for different groups: A is the blank control group, B is the model group, C is the positive drug group, D is the Centella asiatica group, E is the rhubarb group, F is the low-dose compound group, G is the medium-dose compound group, and H is the high-dose compound group. Detailed Implementation

[0032] This invention provides a pharmaceutical composition for treating hypertrophic scars, comprising the following raw materials in parts by weight:

[0033] Centella asiatica glycoside 0.5-2 parts, preferably 0.7-1.5 parts, more preferably 1 part;

[0034] Hydroxyacin is added in the amount of 0.5 to 2 parts, preferably 0.7 to 1.5 parts, and more preferably 1 part;

[0035] Aloe-emodin: 0.5-2 parts, preferably 0.7-1.5 parts, more preferably 1 part;

[0036] Rhein, 1-3 parts, preferably 1.5-2.5 parts, more preferably 2 parts;

[0037] The amount of emodin is 0.5 to 2 parts, preferably 0.7 to 1.5 parts, and even more preferably 1 part.

[0038] The present invention provides the use of the pharmaceutical composition described herein in the preparation of a medicament for treating hypertrophic scars, wherein the medicament can effectively reduce the scar area, decrease the scar height, and restore the skin color at the scar site.

[0039] This invention provides an ointment for treating hypertrophic scars, wherein the ointment preferably comprises the following components in the following mass ratio:

[0040] The pharmaceutical composition comprises: stearic acid, glyceryl monostearate, liquid paraffin, white petrolatum, triethanolamine, glycerin, water, and ethylparaben in a ratio of 2–10: 5–7: 5–15: 20–25: 5–15: 5–10: 5–15: 30–40: 0.01–0.2, more preferably 3–7: 5.5–6.5: 7–12: 21–23: 7–12: 6–8: 7–12: 33–38: 0.05–0.15, and even more preferably 4.65: 6.25: 9: 22: 10: 7.5: 10: 35.25: 0.1.

[0041] The present invention provides a method for preparing the ointment, preferably comprising the following steps:

[0042] 1) The pharmaceutical composition, stearic acid, glyceryl monostearate, liquid paraffin, and white petrolatum are mixed to obtain an oil phase;

[0043] 2) Triethanolamine, glycerol, ethylparaben, and water were mixed to obtain an aqueous phase;

[0044] 3) Heat the aqueous phase and oil phase separately and then mix them to obtain the ointment.

[0045] In this invention, the mixing temperature in step 1) is preferably 60-90°C, more preferably 70-85°C, and even more preferably 80°C; the mixing speed is preferably 5-100 rpm, more preferably 10-60 rpm, and even more preferably 30 rpm; and the mixing time is preferably 15-30 min, more preferably 17-25 min, and even more preferably 25 min.

[0046] In this invention, the mixing temperature in step 2) is preferably 60-90°C, more preferably 70-85°C, and even more preferably 80°C; the mixing speed is preferably 50-500 rpm, more preferably 100-400 rpm, and even more preferably 300 rpm; and the mixing time is preferably 15-30 min, more preferably 17-25 min, and even more preferably 25 min.

[0047] In this invention, the heating temperature in step 3) is preferably 70-90°C, more preferably 75-85°C, and even more preferably 80°C; the mixing time is preferably 5-30 min, more preferably 10-25 min, and even more preferably 15 min; the mixing speed is preferably 50-500 rpm, more preferably 100-400 rpm, and even more preferably 300 rpm; and the mixing method is preferably to slowly pour the heated aqueous phase into the heated oil phase.

[0048] This invention provides the use of the ointment or the ointment obtained by the preparation method in the preparation of a medicament for treating hypertrophic scars.

[0049] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Source of materials

[0051]

[0052] Example 1

[0053] (1.1) Technical route of topical cream for anti-hypertrophic scars

[0054] By constructing an immobilized Notch1 receptor chromatographic model, the active ingredients of Centella asiatica and rhubarb for anti-proliferative scarring were screened. The optimal preparation process of the blank cream was optimized. An appropriate amount of the screened active ingredients was added to the blank cream to prepare an anti-proliferative scar topical cream. For details of the technical route, see [link to technical details]. Figure 1 .

[0055] (1.2) Establishment of an immobilized Notch1 receptor chromatographic model

[0056] The DNA fragment of the Notch1 ligand-binding region was inserted into the pET vector via double enzyme digestion, and then transformed into *E. coli* BL21(DE3) competent cells by heat shock transformation. Expression was induced in a compound self-induction medium at 37°C and 220 rpm. OD... 600 When the pH reached 0.6-0.8, the bacteria were collected by low-temperature centrifugation. The bacterial cells were resuspended in a 1:10 mass-to-volume ratio of cell-lysis buffer (the lysis buffer was 20 mM PB; the specific preparation method for 20 mM PB is as follows: accurately weigh 0.288 g Na2HPO4·2H2O and 0.059 g NaH2PO4·2H2O, dissolve them, and bring the volume to 100 mL, calibrating the pH to 7.4 with a pH meter to obtain 20 mM PB buffer). The cells were then sonicated at 60% power for 3 seconds on and 5 seconds off, repeated twice, at a maximum temperature of 10℃ for 20 minutes. After purification using a nickel ion column, the buffer containing the target protein was collected and desalted using a G25 desalting column to remove excess NaCl and imidazole, followed by purification using a DEAE anion exchange column to obtain pure Notch1 receptor. SDS-PAGE was used to detect Notch1 receptor expression; the results are shown below. Figure 2 .

[0057] The purified receptor was reacted with activated aminopropyl silica gel at room temperature for 2 hours. After washing repeatedly with 20 mM phosphate buffer, the immobilized receptor was obtained. The column was then packed with 20 mM phosphate buffer as a homogenizer and displacement agent to obtain the immobilized Notch1 receptor chromatographic column.

[0058] The activated aminopropyl silica gel is prepared by mixing 2g of silica gel (5μm, Add 25 ml of methanol and 25 ml of concentrated hydrochloric acid to a round-bottom flask, stir mechanically for 30 min, filter, wash with water 4 times, then wash with anhydrous ethanol 4 times, add 25 ml of concentrated sulfuric acid and continue stirring for 30 min, filter, wash with water 4 times, then wash with anhydrous ethanol 4 times, and dry in an oven at 60℃.

[0059] (1.3) Screening of effective components of Centella Asiatica / Rhubarb in preventing scar hyperplasia

[0060] One part of Centella asiatica and one part of rhubarb were weighed separately, ground into powder using a mortar and pestle, and extracted twice by heating and reflux with 50 mL of 60% ethanol. The extracts were then filtered, the residue discarded, and the filtrates were filtered through a microporous membrane and injected into immobilized Notch1 receptor chromatographic columns to screen for the effective components targeting the Notch1 receptor in Centella asiatica / rhubarb. Mass spectrometry identification revealed the components to be asiaticoside, hydroxyasiaticoside, emodin, aloe-emodin, and rhein. Figure 4 , Figure 5 As shown.

[0061] (1.4) Preparation and evaluation of blank matrix for cream formulation

[0062] Using color, spreadability, appearance, stability, and cold and heat resistance as indicators, the preparation process of the cream was optimized through single-factor experiments, Plackett-Burman experiments, and response surface methodology. The single-factor experiments determined the content range of each component in the blank cream matrix (per 40g of cream matrix): stearic acid 2-4g, glyceryl monostearate 3-5g, liquid paraffin 8-12g, white petrolatum 4-6g, triethanolamine 3-5g, and glycerin... 2-6g; Plackett-Burman experiments identified stearic acid, glyceryl monostearate, and liquid paraffin as the main influencing factors in the blank matrix of the cream. Response surface methodology was used to further optimize these three factors, ultimately determining the optimal values ​​for the blank matrix of the cream to be 6.25% stearic acid, 9% glyceryl monostearate, 22% liquid paraffin, 10% white petrolatum, 7.5% triethanolamine, 10% glycerin, 35.25% water, and 0.1% ethylparaben.

[0063] The specific preparation is as follows (40g cream): Weigh 2.5g stearic acid, 3.6g glyceryl monostearate, 8.8g liquid paraffin, and 4g white petrolatum into a 250mL beaker. Place the beaker on an 80℃ constant-temperature magnetic stirrer, set the stirring speed to 300rpm, and continue heating and stirring until all components are completely melted. Aqueous phase preparation: Weigh 3g triethanolamine, 4g glycerin, 0.04g ethylparaben, and 14.1g purified water into another 250mL beaker. Place both beakers on an 80℃ constant-temperature magnetic stirrer and stir at 300rpm until all components are completely dissolved. Under constant temperature of 80℃, maintain a stirring speed of 300rpm and slowly and uniformly add the aqueous phase component dropwise to the oil phase system. After the addition is complete, continue stirring at a constant temperature for 15min to ensure complete emulsification. Then stop heating and continue stirring until the system cools naturally to room temperature (25±2℃) to obtain the final blank base cream.

[0064] (1.5) Preparation of ointments for treating hypertrophic scars

[0065] The preparation of the oil and aqueous phases is the same as the optimal blank cream described above. Under constant temperature of 80℃ and stirring speed of 300 rpm, the aqueous phase component is slowly and uniformly added dropwise to the oil phase system. After the addition is complete, stirring is continued at a constant temperature for 15 minutes to ensure complete emulsification. The system is then cooled to 60℃, and a 10% citric acid solution is slowly added to adjust the pH to 6.0±0.1. Stirring is continued for 10 minutes to homogenize the system. The system temperature is then further cooled to 45℃, and asiaticoside, asiaticoside, aloe-emodin, rhein, and emodin are added sequentially. Stirring is maintained until the active ingredients are completely and uniformly dispersed. Heating is then stopped, and stirring is continued until the system naturally cools to room temperature (25±2℃). This yields the ointment for treating hypertrophic scars. The preparation process is detailed below. Figure 3 .

[0066] Example 2

[0067] A compound low-dose cream was prepared by emulsification. The preparation method of the cream is the same as that in Example 1 (1.4-1.5), except that the effective Chinese medicine ingredients in step (1.5) were replaced with 0.11g of asiaticoside, 0.32g of hydroxyasiaticoside, 0.006g of aloe-emodin, 0.384g of rhein, and 0.11g of emodin.

[0068] Example 3

[0069] The compound medium-dose cream was prepared by emulsification. The preparation method of the cream was the same as that in Example 1 (1.4-1.5), except that the effective Chinese medicine ingredients in step (1.5) were replaced with 0.22g of asiaticoside, 0.64g of hydroxyasiaticoside, 0.012g of aloe-emodin, 0.768g of rhein, and 0.22g of emodin.

[0070] Example 4

[0071] The compound high-dose cream was prepared by emulsification. The preparation method of the cream was the same as that in Example 1 (1.4-1.5), except that the effective Chinese medicine ingredients in step (1.5) were replaced with 0.44g of asiaticoside, 1.28g of hydroxyasiaticoside, 0.024g of aloe-emodin, 1.536g of rhein, and 0.44g of emodin.

[0072] Comparative Example 1

[0073] A cream made from Centella asiatica was prepared by emulsification. The preparation method of the cream was the same as that in Example 1 (1.4-1.5), except that the effective ingredients of traditional Chinese medicine in step (1.5) were replaced with 0.22g of asiaticoside and 0.64g of hydroxyasiaticoside.

[0074] Comparative Example 2

[0075] Rhubarb cream was prepared by emulsification. The preparation method of the cream is the same as that in Example 1 (1.4-1.5), except that the effective Chinese medicine ingredients in step (1.5) were replaced with 0.012g of aloe-emodin, 0.768g of rhein, and 0.22g of emodin.

[0076] Experimental Example 1

[0077] Male New Zealand white rabbits, weighing 2.5±0.2 kg, were selected and acclimatized for one week. The rabbits were divided into eight groups: a blank control group, a model group, a positive control group, a Centella asiatica group, a rhubarb group, a low-dose group, a medium-dose group, and a high-dose group. Except for the blank control group, the remaining seven groups underwent model creation. The model creation method was as follows: 24 hours before surgery, the hair in the surgical area was removed, the rabbits were weighed before surgery, and they were fixed in a rabbit restraint device. A circular skin defect with a diameter of 0.8 cm was drawn on the side of the ear with a marker, with six marking points on each ear. The distance between the wounds was no less than 1 cm. The surgical area was disinfected twice with 75% alcohol, strictly following aseptic technique. 3% sodium pentobarbital was slowly administered via the marginal ear vein for anesthesia (maximum 1.0 mL / kg; initially, 0.5 mL / kg was injected, with subsequent small injections depending on the anesthesia progress). The rabbits' reactions were carefully observed, and local massage was given to promote the onset of anesthesia. After the rabbits had lost their pain response, the model was created on the ventral side of the ear, avoiding the major blood vessels in the ear, following the long axis. A 0.8cm skin punch was used to make incisions at the marked locations. The entire layer of skin and perichondrium was gently peeled away until the cartilage was exposed, at which point the peeling stopped (applying pressure as needed to stop bleeding and clean the wound). The rabbits were returned to their original cages and kept individually in their original environment. The wound condition of each rabbit was observed regularly. By the 9th day after modeling, the wound had largely completed epithelialization (the scab had completely fallen off).

[0078] Blank control group: Normal diet;

[0079] Model group: Apply blank matrix cream and maintain normal diet;

[0080] Positive drug group: Apply asiaticoside cream twice a day, gently massage until absorbed, continue for 42 days, normal diet;

[0081] Centella Asiatica group: Apply the cream prepared in Comparative Example 1 twice a day, gently massage until absorbed, and continue for 42 days with a normal diet;

[0082] Rhubarb group: Apply the cream prepared in Comparative Example 2 twice a day, gently massage until absorbed, administer continuously for 42 days, with normal diet;

[0083] Low-dose compound group: Apply the cream prepared in Example 2 twice a day, gently massage until absorbed, and continue for 42 days while maintaining a normal diet;

[0084] Medium-dose compound group: Apply the cream prepared in Example 3 twice a day, gently massage until absorbed, and continue for 42 days with a normal diet;

[0085] High-dose compound group: Apply the cream prepared in Example 4 twice a day, gently massage until absorbed, and continue for 42 days with a normal diet;

[0086] Take photos daily to document the condition of the scars. See details of the prepared cream. Figure 6 For specific treatment effects, please see [link / reference]. Figure 7 , Figure 7 The results showed that the model group had visible scar elevation, while the treatment group had smaller scar area, lower scar height, and skin color that was close to normal skin.

[0087] To further illustrate the effects of different treatment groups, HE staining and Masson staining were performed. Specific sections were fixed as follows:

[0088] Tissue fixation: The scar tissue was longitudinally divided from the highest point of the hyperplastic mass, and the harvested rabbit ear scar tissue was fixed in 4% paraformaldehyde general tissue fixation solution for 24 hours.

[0089] Tissue dehydration: After fixation, the rabbit ear scar tissue was removed, smoothed, and placed in a dehydration box. The dehydration box was then placed in a tissue dehydrator and dehydrated with alcohol solutions of progressively increasing concentrations: 75% alcohol for 4 hours, 85% alcohol for 2 hours, 90% alcohol for 2 hours, 95% alcohol for 1 hour, and then dehydrated twice more with anhydrous ethanol, each time for 30 minutes.

[0090] Tissue clearing: After dehydration, the tissue is first cleared with a 1:1 mixture of anhydrous ethanol and xylene for 10 minutes, then cleared twice more with xylene, each time for 10 minutes. Clearing makes the internal structure of the tissue clearer, facilitating subsequent microscopic observation.

[0091] Tissue paraffin infiltration: After clearing, the tissue is first immersed in melted soft paraffin for 2 hours, then immersed twice more in hard paraffin with a higher melting point, each time for 2 hours. The entire process is carried out in a constant temperature incubator or water bath at 60℃. The paraffin infiltration process helps the tissue maintain a certain degree of hardness and toughness, making it easier to section later.

[0092] Tissue embedding: Pour molten paraffin wax into a mold, and quickly place the wax-impregnated tissue into the center of the mold using preheated tweezers, adjusting its position so that the cut surface faces down. Then place the mold on a freezing stage to allow the wax to solidify rapidly, completing the embedding process.

[0093] The specific steps for HE staining are as follows:

[0094] Sectioning and mounting: Place the embedded wax block into a microtome and cut it into thin sections with a thickness of 3μm. Place the thin sections into a spreader with 40℃ warm water to spread them out, then use a glass slide to lift them out and place them in a 60℃ oven for 30 minutes to bake the sections firmly onto the glass slide.

[0095] Dewaxing and hydration: The sections were dewaxed three times in xylene for 15 minutes each time, and then soaked twice in anhydrous ethanol for 5 minutes each time to complete the dewaxing. Subsequently, they were hydrated in 95% ethanol, 85% ethanol and 75% ethanol in sequence for 5 minutes each time, and finally placed in water for 5 minutes to complete the hydration.

[0096] Hematoxylin staining: Immerse the slides in hematoxylin staining solution for 5 minutes to ensure uniform staining of the cell nuclei, resulting in a blue-purple or dark purple color. Rinse with running water for 2 minutes after staining.

[0097] Differentiation: Differentiate the sections with 1% hydrochloric acid solution, wash with running water, then invert blue with 0.5% ammonia solution for 30 seconds, and finally rinse with running water for 1 minute to complete the staining.

[0098] Eosin staining: Immerse the prepared tissue sections in eosin staining solution for cytoplasmic staining, with the staining time controlled at 3 minutes. After staining, rinse thoroughly with running water to remove unbound dye molecules, resulting in a characteristic pink staining of the cytoplasm and extracellular matrix.

[0099] Dehydration: Perform gradient ethanol dehydration, first with 70% ethanol for 5 min, then with 85% ethanol for 5 min, then with 95% ethanol for 5 min, and finally with anhydrous ethanol for 5 min.

[0100] Clearing: After dehydration, transfer to xylene solution for clearing. The first clearing takes 5 minutes, and then the clearing is repeated twice, each time for 5 minutes, until the section is completely transparent.

[0101] Mounting: Apply a suitable amount of neutral resin mounting medium to the cleared section surface and carefully cover with a coverslip. Ensure the section surface is smooth and free of air bubbles. Gently press to ensure the mounting medium covers the surface evenly and squeeze out any excess.

[0102] Microscopic examination: HE-stained sections of rabbit ear scars were observed using a Nikon NIS-Elements microscope.

[0103] For detailed HE staining results, please refer to... Figure 8 .

[0104] Figure 8The results showed a significant increase in the number of fibroblasts in the model group, indicating that tissue repair or fibrosis processes were significantly activated. Furthermore, the collagen fibers in the model group were tightly and disordered, while those in the control group were loosely and regularly arranged, reflecting the structural state of normal tissue. The collagen fiber arrangement in the drug-treated group was more regular and looser than that in the model group, indicating that the drug treatment had a certain ameliorative effect on the tissue repair process. In terms of tissue thickness, both the epidermis and dermis in the model group were significantly thicker than those in the control group, possibly related to inflammatory response, edema, or fibrosis. Further observation of scar tissue sections revealed significant inflammatory infiltration in the scar area of ​​the model group, and the rabbit ear periosteum also showed significant inflammatory infiltration. Overall, both the model group and the drug-treated group exhibited scar hyperplasia, but the low-dose combination showed the most significant effect. The medium-dose and high-dose combination groups showed more pronounced scar hyperplasia. The therapeutic effect was not dose-dependent, suggesting that at low doses, the active ingredients of Centella asiatica-rhubarb may exert their therapeutic effect through specific targets, while at medium and high doses, the drug may activate other non-target pathways or produce toxic effects, leading to decreased efficacy. Simultaneously, the low-dose combination group showed significantly reduced periosteal inflammatory infiltration in rabbit ears compared to the positive control group, consistent with the strong anti-inflammatory activity of rhubarb.

[0105] Masson staining is as follows:

[0106] Sectioning and mounting: Place the embedded wax block into a microtome and cut it into thin sections with a thickness of 3μm. Place the thin sections into a spreader with 40℃ warm water to spread them out, then use a glass slide to lift them out and place them in a 60℃ oven for 30 minutes to bake the sections firmly onto the glass slide.

[0107] Dewaxing and hydration: The sections were dewaxed three times in xylene for 15 minutes each time, and then soaked twice in anhydrous ethanol for 5 minutes each time to complete the dewaxing. Subsequently, they were hydrated in 95% ethanol, 85% ethanol and 75% ethanol in sequence for 5 minutes each time, and finally placed in water for 5 minutes to complete the hydration.

[0108] Potassium dichromate staining: Immerse the sections in 2.5% potassium dichromate solution at room temperature overnight (about 15 hours), then wash with tap water for 30 seconds until the yellow color fades from the tissue.

[0109] Ponceau S Acid Fuchs Staining: Slightly drain excess water from the sections, immerse the sections in Ponceau S Acid Fuchs solution for 6 minutes. At this time, the tissue will appear bright red. If the red is too light, the staining time can be extended appropriately. Rinse with running water until the remaining water is colorless.

[0110] Phosphomolybdic acid staining: Slightly drain the water from the sections (do not use dry sections), then soak them in a phosphomolybdic acid aqueous solution for about 1 minute. This step is for differentiation; differentiation continues until the collagen fibers turn light red and the fibers turn red.

[0111] Aniline blue staining: After phosphomolybdic acid, do not wash with water, directly immerse in aniline blue staining solution for 6-30 seconds.

[0112] Differentiation: Rinse the slices in 1% glacial acetic acid for 30 seconds, then rinse with running water for 1 minute to complete the differentiation.

[0113] Dehydration: The stained sections were placed in 70%, 85%, 95%, and 100% ethanol solutions in sequence for 5 minutes each time.

[0114] Transparency: Immerse the dehydrated sections in xylene solution three times for 5 minutes each time until the sections are completely transparent.

[0115] Mounting: Place a drop of neutral resin on the cleared section and cover with a coverslip. Ensure the section surface is smooth and free of air bubbles. Gently press to ensure the mounting medium covers the section evenly and squeeze out any excess.

[0116] Microscopic examination: Masson-stained sections of rabbit ear scars were observed and photographed using a Nikon NIS-Elements microscope. See attached image for details. Figure 9 .

[0117] Figure 9 The results showed that the model group had excessive collagen deposition and dense arrangement. The drug-treated group showed reduced collagen deposition compared to the model group, with a smaller density product and fewer collagen fibers. Masson staining results were consistent with HE staining results.

[0118] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for treating hypertrophic scars, characterized in that, The raw materials include the following parts by weight: 0.5-2 parts of asiaticoside, 0.5-2 parts of hydroxyasiaticoside, 0.5-2 parts of aloe-emodin, 1-3 parts of rhein, and 0.5-2 parts of emodin.

2. The pharmaceutical composition according to claim 1, characterized in that, The raw materials include the following parts by weight: Centella asiatica 0.7–1.5 parts, hydroxycentella asiatica 0.7–1.5 parts, aloe-emodin 0.7–1.5 parts, rhein 1.5–2.5 parts, and emodin 0.7–1.5 parts.

3. The pharmaceutical composition according to claim 1, characterized in that, The raw materials include the following parts by weight: 1 part asiaticoside, 1 part hydroxyasiaticoside, 1 part aloe-emodin, 2 parts rhein, 1 part emodin.

4. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a medicament for treating hypertrophic scars.

5. A cream for treating hypertrophic scars, characterized in that, The ointment comprises the following components in the following mass ratio: The pharmaceutical composition according to any one of claims 1 to 3: stearic acid, glyceryl monostearate, liquid paraffin, white petrolatum, triethanolamine, glycerin, water, ethylparaben = 2 to 10: 5 to 7: 5 to 15: 20 to 25: 5 to 15: 5 to 10: 5 to 15: 30 to 40: 0.01 to 0.

2.

6. The method for preparing the ointment according to claim 5, characterized in that, The following steps are involved: 1) The pharmaceutical composition, stearic acid, glyceryl monostearate, liquid paraffin, and white petrolatum are mixed to obtain an oil phase; 2) Triethanolamine, glycerol, ethylparaben, and water were mixed to obtain an aqueous phase; 3) Heat the aqueous phase and oil phase separately and then mix them to obtain the ointment.

7. The use of the ointment of claim 5 or the ointment obtained by the preparation method of claim 6 in the preparation of a medicament for treating hypertrophic scars.

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