Lamiophlomis rotata gel and application thereof

Abstract

The invention belongs to the field of pharmaceutical preparations, and particularly relates to lamiophlomis rotata gel and application thereof. The lamiophlomis rotata gel is an external preparation prepared from the following raw and auxiliary materials in parts by weight: 1.25 to 5 parts of lamiophlomis rotata extract, 0.5 to 1 part of carbomer 940, 0.01 to 0.05 part of ethylparaben, 0.5 to 1 part of triethanolamine, 1 to 2 parts of glycerol and 1.5 to 2 parts of 1, 2-propylene glycol. The curative effect of the lamiophlomis rotata gel is equivalent to that of the lamiophlomis rotata extract with the same dosage when the lamiophlomis rotata gel is used for repairing acute wounds; however, when the composition is used for repairing infectious wounds, the curative effect of the composition is obviously superior to that of the same dosage of lamiophlomis rotata extract, and is also superior to that of fusidic acid cream and compound cortex phellodendri liquid which are commonly used clinically at present, so that the composition has practical popularization and application values.

Description

Technical Field

[0001] The invention belongs to the field of traditional Chinese medicine preparations, and particularly relates to a Lamiophlomis gel and application thereof. Background Art

[0002] Lamiophlomis rotata (Benth.) Kudo, the only species of the genus Lamiophlomis in the Lamiaceae family, is also known as Dubutong (Dubatong) and, in Tibetan, as "Daba" or "Dabuba." It is a commonly used Tibetan medicine with significant analgesic, hemostatic, and anti-inflammatory properties, with minimal side effects, and a wide range of clinical applications. Tablets, capsules, and granules made from Lamiophlomis rotata have been reported to be used primarily for the treatment of various pain conditions in gynecology, surgery, internal medicine, dermatology, oncology, and ENT departments.

[0003] Pharmaceutical gels refer to thick liquid or semisolid preparations that combine a drug with a gel-forming excipient to form a solution, suspension, or emulsion. As a new dosage form, pharmaceutical gels have become a hot topic in pharmaceutical research in recent years, and their advantages are gradually becoming apparent in clinical use. Patent CN 1739609A discloses a Lamiophlomis gel with significant analgesic effects. However, as an oral preparation, the gel undergoes hepatic metabolism, resulting in drug conversion in the liver, which reduces its bioavailability. Topical preparations are directly absorbed through the skin or mucous membranes, avoiding the first-pass effect of oral drugs through the gastrointestinal tract and liver, thereby improving drug bioavailability. Because topical preparations absorb less drug, systemic exposure is lower, and the incidence of adverse reactions is also lower. However, there are currently no reports of gels made with Lamiophlomis as the sole active ingredient that significantly improve skin pathology when applied topically to the skin. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a Lamiophlomis gel, which is an external preparation prepared from raw materials and auxiliary materials in the following parts: 1.25-5 parts by weight of Lamiophlomis lappa extract, 0.5-1 parts by weight of Carbomer 940, 0.01-0.05 parts by weight of ethylparaben, 0.5-1 parts by volume of triethanolamine, 1-2 parts by volume of glycerol, and 1.5-2 parts by volume of 1,2-propylene glycol; The Lamiophlomis lappa extract is a water extract of Lamiophlomis lappa; Each 1g of the water extract is equivalent to 3-5g of Radix Angelicae Pubescentis; The carbomer 940 is dissolved in water to form a solution with a concentration of 3-3.8%; The ethyl hydroxybenzoate is dissolved in anhydrous ethanol to form a solution with a concentration of 3-3.5%.

[0005] Furthermore, it is an external preparation prepared from raw materials and auxiliary materials including the following parts: 2.5 parts by weight of Lamiophlomis lappa extract, 0.875 parts by weight of Carbomer 940, 0.025 parts by weight of ethylparaben, 0.75 parts by volume of triethanolamine, 1.875 parts by volume of glycerol, and 1.875 parts by volume of 1,2-propylene glycol; The carbomer 940 is dissolved in water to form a solution with a concentration of 3.5%; The ethyl paraben is dissolved in anhydrous ethanol to form a solution with a concentration of 3.3%; Each 1g of the water extract is equivalent to 4.8g of Radix Angelicae Pubescentis.

[0006] The present invention also provides a method for preparing the aforementioned Lamiophlomis gel, comprising the following steps: 1) Weigh the raw materials and excipients according to the ratio. Take carbomer 940 and dissolve it in water to obtain a carbomer 940 solution. Take ethyl paraben and dissolve it in ethanol to obtain an ethyl paraben solution. 2) Take the carbomer 940 solution obtained in step 1), place it at 2-8°C to swell and defoam, then add triethanolamine, glycerol, 1,2-propylene glycol and the ethylparaben solution obtained in step 1), mix well, and finally add the Lamiophlomis radiata extract and stir well to obtain the product.

[0007] Furthermore, in step 1), the concentration of the Carbomer 940 solution is 3.5%.

[0008] Furthermore, in step 1), the ethanol is anhydrous ethanol; and the concentration of the ethyl hydroxybenzoate solution is 3.3%.

[0009] The present invention also provides a use of the aforementioned Lamiophlomis gel in preparing a medicine for repairing wounds.

[0010] Furthermore, the medicine is a medicine for repairing acute wounds.

[0011] Furthermore, the medicine is a medicine for repairing infectious wounds.

[0012] Furthermore, the drug has the effects of anti-inflammation, analgesia, promoting angiogenesis, promoting granulation tissue formation and / or promoting collagen deposition.

[0013] The Lamiophlomis gel of the present invention combines a Lamiophlomis extract with specific excipients, thereby not only achieving a high drug loading of the Lamiophlomis extract but also improving the stability and fluidity of the gel itself. Animal experiments have confirmed that the Lamiophlomis gel of the present invention is as effective as an equivalent dose of Lamiophlomis extract in repairing acute wounds; and significantly superior to an equivalent dose of Lamiophlomis extract in repairing infected wounds. It is also superior to fusidic acid cream and compound phellodendron liquid currently commonly used in clinical practice, demonstrating its practical application value.

[0014] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0015] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 cumulative pain score; Figure 2 Initial screening of concentration of Duyiwei gel to promote acute wound healing - wound healing; Figure 3 Comparison of the effects of Lamiophlomis gel and Lamiophlomis extract on promoting acute wound healing - Wound healing; Figure 4 Comparison of the effects of Lamiophlomis gel and Lamiophlomis extract on promoting acute wound healing - HE staining; Figure 5 Comparison of the effects of Lamiophlomis gel and Lamiophlomis extract on promoting acute wound healing - Masson staining; Figure 6 Study on the activity of Duyiwei gel in promoting acute wound healing - wound healing; Figure 7 Study on the activity of Duyiwei gel in promoting acute wound healing - HE staining; Figure 8 Study on the activity of Duyiwei gel in promoting acute wound healing - Masson staining; Figure 9 Study on the activity of Duyiwei gel in promoting acute wound healing - CD31 immunohistochemistry; Figure 10 Study on the activity of Duyiwei gel in promoting acute wound healing - TNF-α immunohistochemistry; Figure 11 Study on the activity of Duyiwei gel in promoting acute wound healing - α-SMA immunofluorescence; Figure 12 Study on the activity of Duyiwei gel in promoting acute wound healing - MPO immunofluorescence; Figure 13 Initial screening of concentration of Duyiwei gel to promote healing of infected wounds - wound healing; Figure 14 Comparison of the effects of Lamiophlomis gel and Lamiophlomis extract on promoting healing of infected wounds - wound healing; Figure 15Comparison of the effects of Lamiophlomis gel and Lamiophlomis extract on promoting healing of infected wounds - HE staining; Figure 16 Comparison of the effects of Lamiophlomis gel and Lamiophlomis extract on promoting healing of infected wounds - Masson staining; Figure 17 Study on the activity of Lamiophlomis gel in promoting the healing of infected wounds - wound healing; Figure 18 Study on the activity of Lamiophlomis gel in promoting the healing of infected wounds - HE staining; Figure 19 Study on the activity of Lamiophlomis gel in promoting the healing of infected wounds - Masson staining; Figure 20 Study on the activity of Duyiwei gel in promoting the healing of infected wounds - CD31 immunohistochemistry; Figure 21 Study on the activity of Duyiwei gel in promoting the healing of infected wounds - TNF-α immunohistochemistry; Figure 22 Study on the activity of Duyiwei gel in promoting acute wound healing - α-SMA immunofluorescence; Figure 23 Study on the activity of Duyiwei gel in promoting acute wound healing-MPO immunofluorescence. DETAILED DESCRIPTION

[0017] The raw materials, reagents and equipment used in the specific embodiment of the present invention are all purchased from the market, among which the Lamiophlomis lappa extract and Lamiophlomis lappa capsules are both provided by Kangxian Lamiophlomis Bio-Pharmaceutical Co., Ltd.; the preparation method of the Lamiophlomis lappa extract is as follows: crush the Lamiophlomis slices, add water and boil them three times for 1 hour each time, combine the decoctions, filter, concentrate the filtrate, dry it below 80°C, and crush it to obtain the Lamiophlomis lappa extract; each 1g of the Lamiophlomis lappa extract is equivalent to 4.8g of the slices. Example 1: Preparation of the Lamiophlomis gel of the present invention Formula: 2.5g of Lamiophlomis chinensis extract, 0.875g of Carbomer 940, 0.025g of ethylparaben, 0.75ml of triethanolamine, 1.875ml of glycerol, 1.875ml of 1,2-propylene glycol; Preparation method: 1) Take Carbomer 940 and dissolve it in 25 ml of water to prepare a 3.5% (g / ml) Carbomer 940 solution. Take ethyl paraben and dissolve it in 0.75 ml of anhydrous ethanol to prepare a 3.3% (g / ml) ethyl paraben solution. 2) The carbomer 940 solution obtained in step 1) is dissolved and defoamed at 4°C, and then triethanolamine, glycerol, 1,2-propylene glycol, and the ethylparaben solution obtained in step 1) are added at room temperature and mixed evenly, and then the extract of Lamiophlomis lappa is added and stirred evenly to obtain the product.

[0018] The beneficial effects of the present invention are described below by way of test examples: Test Example 1 Study on Lamiophlomis gel 1. Prescription screening and preparation process research of Duyiwei gel 1 Experimental Purpose Prepare a Lamiophlomis gel with excellent stability and excipient properties to improve the therapeutic effect of Lamiophlomis.

[0019] 2 Prescription design Gels contain a wide variety of excipients, and their composition and ratio affect not only the gel's formation but also the efficacy of the active ingredients. Based on preliminary experimental results, a gel containing carbomer, methylcellulose, hydroxypropyl methylcellulose, sodium phosphomethylcellulose, alginate, tragacanth gum, gelatin, starch, laurocapram, menthol, pH adjusters, moisturizers, thickeners, transdermal enhancers, and preservatives was prepared by uniformly mixing carbomer 940 as the primary matrix with appropriate amounts of glycerol, 1,2-propylene glycol, triethanolamine, ethyl parahydroxybenzoate (Ethylparaben), and Lamiophlomis extract powder. This resulted in a well-formed gel and is expected to maximize the efficacy of the Lamiophlomis extract.

[0020] At the same time, regarding the dosage of Carbomer 940, referring to the dosage concentration of 0.5% to 2% recommended in the Handbook of Pharmaceutical Excipients, a gel with good excipient effect has not been obtained. Therefore, based on the component characteristics of the aqueous extract of Du Yiwei, the dosage of Carbomer 940 was further adjusted in order to make the gel have ideal rheological properties, improve the patient experience and fully exert the efficacy of Du Yiwei.

[0021] 3 Prescription Investigation The optimal prescription was screened by mixing different concentrations of Lamiophlomis extract and matrix ratios.

[0022] 4. Investigation of property stability The properties and stability of the Lamiophlomis gels with different drug loadings were observed.

[0023] 5. Results 5.1 Blank Gel Formulation Investigation Gel matrices with varying carbomer concentrations were prepared according to Table 1. Triethanolamine, glycerol, and 1,2-propylene glycol were added to 2 mL of gel matrix at a time to produce blank gels. Observation of the gel morphology revealed that the addition of glycerol, 1,2-propylene glycol, and ethylparaben did not affect the gel's properties. The resulting gel was a colorless, transparent, semisolid that maintained a consistent shape.

[0024] Table 1 Blank gel prescription 5.2 Study on the prescription of Duyiwei gel Blank gels were prepared according to 5.1. 2 mL of blank gel was added to different masses of Lamiophlomis extract powder. The properties of Lamiophlomis gels with different drug loadings (0, 5, 8, 10, and 15%) were investigated (Table 2). As shown in Table 2, the Lamiophlomis extract significantly affected the gel properties. When the carbomer concentration was 4% and the Lamiophlomis concentration was 8-10%, the excipient effect was better and the drug loading was greater.

[0025] Table 2 Screening of prescriptions for Lamiophlomis gel 5.3 Investigation on the stability of Lamiophlomis gel To meet the stability requirements of gel formulations, after formula review and extensive trial and error, the corresponding parameters were further adjusted based on the results. Specifically, 25 mL of Lamiophlomis gels with varying drug loadings were prepared according to Table 3. First, a 3.5% CP940 solution was prepared. Once completely dissolved, it was placed in a 4°C refrigerator to allow swelling and defoaming. Then, triethanolamine, glycerol, 1,2-propylene glycol, and ethylparaben in anhydrous ethanol were added at room temperature, in the order shown in the table below, and stirred thoroughly to obtain a blank gel matrix. Appropriate amounts of Lamiophlomis extract powder were added to the blank gels and stirred thoroughly to obtain Lamiophlomis gels with varying drug loadings. Their properties were then preliminarily evaluated. Finally, the Lamiophlomis gels were transferred to squeezable plastic bottles and allowed to stand for stability observation.

[0026] Table 3 Optimization of the prescription of Lamiophlomis gel The prepared Lamiophlomis gels with different drug loading amounts were transferred into squeezable plastic bottles and stored at 4°C to observe their stability. The results are shown in Table 4.

[0027] Table 4 Investigation on the stability of the appearance of Lamiophlomis gel As shown in Table 4, when the drug loading is 10%, the color, odor, excipient properties, extrusion from the soft bottle, and fluidity in the soft bottle of the gel are optimal, and the drug loading is high and the stability is good. The preparation method of the gel of Group 2 with a drug loading of 10% is as follows: Take 0.875g of carbomer 940, add 25ml of water to dissolve it to prepare a carbomer 940 solution with a concentration of 3.5%, g / ml; take 0.025g of ethyl hydroxybenzoate, add 0.75ml of anhydrous ethanol to dissolve it to prepare a 3.3% concentration, g / ml ethyl hydroxybenzoate solution; take 25ml of the prepared carbomer 940 solution, place it in a 4°C refrigerator to swell and defoam, then add 0.75ml of triethanolamine, 1.875ml of glycerol, 1.875ml of 1,2-propylene glycol and the prepared ethyl hydroxybenzoate solution in sequence and mix them evenly, finally add 2.5g of Lamiophlomis ulmoides extract and stir evenly to obtain the product.

[0028] 2. Pharmacodynamics Evaluation (1) Evaluation of postoperative analgesic effect in rats 1 Experimental Purpose A rat foot incision pain model was established, and the analgesic effect of topical Du Yiwei gel (Gels with Du Yiwei concentrations of 10%, 15%, and 20%, respectively, prepared according to the optimized process in Table 3) was evaluated and compared with compound polymyxin B ointment and oral Du Yiwei capsules.

[0029] 2 Experimental methods At least 90 adult male Sprague-Dawley rats (8-10 weeks old, weighing 200-220 g) were selected and fed a standard diet every 12 hours with free access to water. The room temperature was maintained at approximately 25°C. The 90 rats were randomly divided into a control group, an acute pain model group, a Duyiwei capsule group (at different doses), a compound polymyxin B ointment group, and a Duyiwei gel group (at different doses), with 10 rats in each group. The rats were placed in observation cages for two days to acclimate to the environment before the experiment.

[0030] All rats in the experimental groups, except the control group, underwent left plantar incision surgery (the most common model of postoperative pain). All rats, except the control group, were placed in a transparent induction chamber and induced with 3% isoflurane inhalation. After falling asleep, the rats were removed and maintained with 1.5% isoflurane inhalation via a nasal mask at an oxygen flow rate of 0.3 L / min. The plantar surface of the left paw was disinfected with alcohol. A 1-cm longitudinal incision was made 0.5 cm from the heel edge, penetrating the skin, fascia, and plantar muscle. The skin was then closed with two 5-0 antibacterial absorbable sutures. Pressure was applied to stop bleeding, and the wound was cleaned and disinfected to establish an acute rat plantar incisional pain model.

[0031] The model group was gavaged with normal saline (1 mL / 200 g) after the sole of the foot was incised. The Duyiwei capsule group was gavaged with the capsule contents (dissolved in normal saline at concentrations of 3%, 6%, and 9%, respectively, at a dose of 1 mL / 200 g). The compound polymyxin B ointment group was smeared with compound polymyxin B ointment (0.04 mL / 200 g) on ​​the incision. The Duyiwei gel group was smeared with Duyiwei gel (0.04 mL / 200 g) on ​​the incision.

[0032] One hour after the pain model was established, all rats were placed in a glass box (40 cm × 40 cm × 40 cm) and the "cumulative pain score" method was used to observe the rats' hind paw touching the ground and bearing weight on the surgical side. A score of 2 was assigned if the surgical side's hind paw was raised due to pain and not touching the ground; a score of 1 was assigned if the surgical side's hind paw touched the ground but did not bear weight (i.e., the hind paw did not turn white); a score of 0 was assigned if the surgical side's hind paw touched the ground and bore weight. The non-surgical side was scored the same as the surgical side. Starting from one hour after surgery, observations were conducted every 5 minutes for 1 minute each. The most common standing posture adopted by the rat within 1 minute was used as the standard for a total of 1 hour. The cumulative pain score (0-24 points) was calculated using the following formula: Cumulative pain score = surgical side pain score - non-surgical side pain score 3. Results The lower the cumulative pain score, the better the analgesic effect. Figure 1 As shown, the 6% and 9% Duyiwei capsule groups had the lowest cumulative pain scores, at 5.00 and 5.33, respectively; the 20% Duyiwei gel group had the lowest cumulative pain score, at 6.33. Although there was no statistically significant difference between the gel and model groups, the cumulative pain scores showed that the gel group had an analgesic effect and was superior to the compound polymyxin B group. Compound polymyxin B contains lidocaine hydrochloride, which has analgesic effects, suggesting that Duyiwei gel also has a pain-relieving effect, and its analgesic effect is superior to compound polymyxin B.

[0033] Table 5 Pain cumulative score table Note: Compared with the control group, a P<0.05; compared with the model group, b P<0.05; compared with the compound polymyxin B group, c P<0.05 (2) Pharmacodynamic evaluation of the activity of Duyiwei gel in promoting acute wound healing 1 Experimental Purpose An acute wound model was established in rats, and the wound repair activity of Du Yiwei gel (gels with Du Yiwei concentrations of 10%, 15%, and 20%, respectively, prepared according to the optimized process in Table 3) was evaluated through pharmacodynamic experiments and compared with Du Yiwei extract, Kangfuxin solution, and recombinant human epidermal growth factor gel.

[0034] 2 Experimental methods 2.1 Initial screening of concentrations of Duyiwei gel for promoting acute wound healing Six rats were randomly selected and anesthetized with 2% sodium pentobarbital (60 mg / kg). The dorsal hair was then removed with an electric shaver and further removed at the base using a safe, non-irritating depilatory cream to fully expose the area on either side of the dorsal midline. The dorsal midline was disinfected with 75% ethanol, and then covered with a disposable sterile surgical drape. Four circular, full-thickness skin defects, each 8 mm in diameter, were created on either side of the dorsal midline using a skin biopsy punch, reaching the fascia.

[0035] Each rat received four wound treatments as follows: 1) an untreated blank control group; 2-4) groups treated with 10%, 15%, and 20% Lamiaceae gel. During the initial screening of gel concentrations, the gel's air permeability and exudate adsorption properties were evaluated. The healing of the rats' back wounds was observed daily for 14 consecutive days and photographed to determine the optimal concentration of Lamiaceae gel for promoting wound healing.

[0036] 2.2 Comparison of the effects of Lamiophlomis gel and extract on promoting acute wound healing Six rats were randomly selected and anesthetized with 2% sodium pentobarbital (60 mg / kg). The dorsal hair was then removed with an electric shaver and further removed at the base using a safe, non-irritating depilatory cream to fully expose the area on either side of the dorsal midline. The dorsal midline was disinfected with 75% ethanol, and the wounds were covered with disposable sterile surgical drapes. Four circular, full-thickness skin defects with a diameter of 8 mm were created on the back of each rat using a skin biopsy punch, extending to the fascia. These wounds were evenly distributed in a triangular pattern. The four wounds in each rat were treated as follows: 1) untreated blank control group; 2-3) 10% Lamiaceae gel group; and 4) 10% Lamiaceae extract group.

[0037] The wound healing of the rats' backs was observed daily for 14 consecutive days, and photographs were taken. Wound healing was calculated on days 3, 8, and 14. Rats were sacrificed on days 3, 8, and 14. Skin samples from the wounds were collected and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 4 μm thickness. The sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome. Microscopic photographs were taken, and the distribution of epidermal regeneration and collagen deposition around the wounds was observed in the tissue sections.

[0038] 2.3 Study on the activity of Duyiwei gel in promoting acute wound healing After identifying the optimal concentration of Duyiwei gel to promote wound healing, ten rats were anesthetized with 2% sodium pentobarbital (60 mg / kg). The dorsal hair was then removed with an electric shaver and further removed at the base using a safe, non-irritating depilatory cream to fully expose the areas on both sides of the dorsal midline. The dorsal midline was disinfected with 75% ethanol, and the wounds were covered with disposable sterile surgical drapes. Four circular, full-thickness skin defects with a diameter of 8 mm were created on both sides of the dorsal midline using a skin biopsy punch, reaching the fascia. The four wounds in each rat were treated as follows: 1) untreated blank control group; 2) optimal dose Duyiwei gel group; 3) Kangfuxin liquid group; and 4) recombinant human epidermal growth factor gel group.

[0039] The wound healing was observed on days 0, 3, 8, and 14, and wound photos were taken. The wound area was measured using ImageJ software, and the wound healing rate was calculated using the following formula: Wound shrinkage (%) = (A0 − At) / A0 × 100% A0 represents the wound area on day 0, and At represents the wound area at different times.

[0040] The effects of Du Yiwei gel on epidermal regeneration, inflammation, angiogenesis, and oxidative stress in the wound area were evaluated. Rats were sacrificed on days 3, 8, and 14. Skin samples from the wound area were collected and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 4 μm thickness. The sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome. All sections were analyzed and photographed microscopically. The distribution of epidermal regeneration and collagen deposition around the wound area was observed in the photographed tissue sections.

[0041] Immunohistochemistry was used to analyze the expression levels of the cytokines CD31 and TNF-α in the regenerated skin at the wound site. Furthermore, skin tissue sections from days 3, 8, and 14 were immunofluorescently stained for α-SMA (smooth muscle actin) and MPO (myeloperoxidase). Cell nuclei were then stained with a fixative containing DAPI, and the slides were observed under an inverted fluorescence microscope.

[0042] 3. Results 3.1 Preliminary screening results of the concentration of Duyiwei gel for promoting acute wound healing Table 6 Results of wound healing rate determination based on initial screening concentration Based on the observation results of wound healing process during the experiment, from Table 6 and Figure 2It can be seen that the different gel concentration groups showed no significant differences in promoting wound healing. However, after comprehensively considering the preparation characteristics of the initial gel formulation, stability assessment, and the deposition behavior of the gel after application to the wound, it was found that the 15% and 20% concentrations of Lamiophlomis gels exhibited excessive viscosity, resulting in significant adhesion to the wound site. Therefore, the 10% concentration of Lamiophlomis gel was selected for subsequent wound healing promotion experiments.

[0043] 3.2 Comparison of the effects of Lamiophlomis gel and extract on promoting acute wound healing Table 7 Results of wound healing rate determination compared with the extract According to the analysis of wound surface recovery, from Table 7 and Figure 3 It can be seen that compared with the control group, the wound healing rates of the Lamiophlomis gel group and the Lamiophlomis extract group were higher on the 3rd and 14th days; the healing rate of the Lamiophlomis extract group was higher than that of the Lamiophlomis gel group on the 3rd and 8th days, and the healing rates of the Lamiophlomis extract group and the Lamiophlomis gel group were equivalent on the 14th day. This shows that the efficiency of Lamiophlomis gel and Lamiophlomis extract in repairing acute wounds is comparable.

[0044] Table 8 Granulation tissue gap determination results Note: Compared with the control group, a P<0.001; compared with the extract group, b P<0.01 The wound refilling, granulation tissue formation and epithelial reconstruction were evaluated according to the results of HE section staining. Figure 4 It can be seen that during the treatment process, the gaps between the granulation tissues in each group gradually decreased, indicating that the wound was healing. However, the drug-treated group had smaller gaps between the granulation tissues and a large amount of new granulation tissue, and the quality of tissue healing was better. On the 14th day, the surface skin structure of each tissue had gradually become complete and thickened. More obvious hair follicles and new blood vessels could be observed in the drug-treated group, and there was no significant difference between the Lamiophlomis gel group and the Lamiophlomis extract group.

[0045] Table 9 Collagen deposition measurement results Note: Compared with the control group, a P<0.001; compared with the extract group, b P<0.01 Masson staining of sections was used to assess collagen deposition and tissue healing. Figure 5On days 8 and 14 of treatment, significantly greater collagen fiber density and thickness were observed in the Lamiophlomis gel and extract groups than in the control group. These results indicate that both the Lamiophlomis gel and extract promote collagen deposition at a faster rate. Furthermore, the treatment groups exhibited more abundant, structurally intact, and discernible angiogenesis, further demonstrating the significant effectiveness of the Lamiophlomis gel and extract in promoting tissue healing and angiogenesis.

[0046] 3.3 Study on the activity of Lamiophlomis gel in promoting acute wound healing Table 10 Results of wound healing rate compared with positive drugs Note: Compared with the control group, a P<0.001; compared with the gel group, b P<0.01 According to the analysis of wound surface recovery, from Table 10 and Figure 6 The wound healing rate was higher in the Kangfuxin Liquid group in the early stage, likely due to the characteristics of the Kangfuxin Liquid liquid formulation. It was slightly lower in the Duyiwei Gel group in the middle stage, and there was no significant difference in healing rates between the groups in the later stages. Overall, there was no significant difference in wound healing rates between the Duyiwei Gel group, the rhEGF group, and the control group.

[0047] Table 11 Granulation tissue gap measurement results Note: Compared with the control group, a P<0.001; compared with the gel group, b P<0.05 The wound refilling, granulation tissue formation and epithelial reconstruction were evaluated according to the results of HE section staining. Figure 7 It can be seen that each medication group showed good wound healing performance, the granulation tissue gap was smaller than that of the control group, the tissue adhesion was strong, and there was more angiogenesis. The wound tissue granulation tissue of the medication group recovered better, but the healing effect of Duyiwei gel was not as good as that of Kangfuxin liquid.

[0048] Table 12 Collagen deposition measurement results Note: Compared with the control group, a P<0.001; compared with the gel group, b P<0.001 Masson staining was used to evaluate collagen deposition and tissue healing. Figure 8As can be seen, the treatment group showed greater collagen fiber deposition than the control group, with a faster rate of collagen deposition and more complete and clearly discernible angiogenesis. Duyiwei Gel initially performed inferior to the two positive drug groups. Based on the HE and Masson results, both treatment groups effectively promoted the recovery of skin appendages, but the gel group was slightly less effective than the Kangfuxin Liquid and recombinant human epidermal growth factor groups.

[0049] Table 13 CD31 positive expression determination results Note: Compared with the control group, a P<0.001; compared with the gel group, b P<0.001 CD31 is an intercellular adhesion molecule found on endothelial cells and is used to assess angiogenesis and stability. Figure 9 As can be seen, during the overall wound healing process, CD31 expression showed a gradual increase from the early to mid-stages, followed by a decrease in the later stages. Compared to the control group, the treatment group showed significantly higher CD31 expression in the healing skin, effectively enhancing early vascularization in the wound and promoting the healing of tissue appendages. Furthermore, the effect of Duyiwei Gel was similar to that of Kangfuxin Liquid and superior to that of recombinant human epidermal growth factor.

[0050] Table 14 TNF-α positive expression determination results Note: Compared with the control group, a P<0.05 Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine secreted by M1 macrophages and is used to assess the inflammatory status during wound healing. Figure 10 As can be seen: On day 3, the expression of TNF-α in the control group was significantly higher than that in the other drug-treated groups. On days 8 and 14, the expression of TNF-α in the control group decreased slightly but was still higher than that in the drug-treated groups, indicating that the inflammatory response in the control group was more severe during treatment. Duyiwei gel had a comparable anti-inflammatory effect as the positive drug. Combined with the expression of CD31 and TNF-α, it can be seen that Duyiwei gel, Kangfuxin liquid, and recombinant human epidermal growth factor groups had stronger angiogenesis abilities and smaller inflammatory responses, but the gel group was less effective than the positive drug group.

[0051] Table 15 α-SMA positive expression determination results Note: Compared with the control group, a P<0.001 α-Smooth muscle actin (α-SMA) is mainly used to mark smooth muscle fibroblasts. Figure 11The results showed that the control group had lower α-SMA positive staining, indicating the least blood vessel formation. Both the Duyiwei gel group and the positive drug group showed significantly higher α-SMA positive staining than the control group, proving that the drug-treated group has the ability to enhance α-SMA expression and promote angiogenesis, which is beneficial to wound tissue healing, and the effect of Duyiwei gel is close to that of the positive drug.

[0052] Table 16 MPO positive expression determination results Note: Compared with the control group, a P<0.001 Myeloperoxidase (MPO) is a marker of systemic inflammation. MPO is mainly used to detect wound inflammation. Table 16 and Figure 12 The results showed that the control group had significantly higher MPO expression than the other treatment groups. This suggests that the control group experienced a more severe inflammatory response during the wound recovery phase, while the Lamiaceae gel and positive drug groups effectively reduced the inflammatory response at the wound site, thereby ensuring normal wound tissue repair. The Lamiaceae gel was as effective as the positive drug. Combining the α-SMA and MPO test results, it can be concluded that the drug-treated group not only had a stronger ability to promote angiogenesis but also significantly reduced the inflammatory response.

[0053] The above results show that Du Yiwei gel can promote the healing of acute wounds, and its overall healing effect is comparable to that of Du Yiwei extract, but inferior to Kangfuxin solution and recombinant human epidermal growth factor currently commonly used in clinical practice.

[0054] (3) Study on the effect of Duyiwei gel on promoting healing of infected wounds 1. Experimental Purpose A bacterial infection rat wound model was established to evaluate the antibacterial and wound repair-promoting activity of Du Yiwei gel (gels with Du Yiwei concentrations of 10%, 15%, and 20% prepared according to the optimized process in Table 3). The antibacterial and wound repair-promoting effects were compared with those of compound phellodendron chinense solution and fusidic acid cream.

[0055] 2. Experimental methods 2.1 Initial screening of concentrations of Duyiwei gel for promoting healing of infected wounds Six rats were randomly selected and each rat was anesthetized with 2% sodium pentobarbital (60 mg / kg). The hair on the back was then removed with an animal electric shaver, and the hair at the root was further removed with a safe and non-irritating depilatory cream to completely expose the area on both sides of the midline of the back. The areas on both sides of the midline of the back were disinfected with 75% ethanol, and then covered with a disposable sterile surgical sheet. Four circular full-thickness skin defect wounds with a diameter of 8 mm were punched on both sides of the midline of the back of each rat with a skin biopsy punch until the depth reached the fascia. 50 μL of bacterial suspension (Staphylococcus aureus, 108 CFU / mL), and the wound was covered with sterile PU film for 24 h to prepare a bacterial infection wound model.

[0056] Each rat received four wound treatments as follows: 1) an untreated blank control group; 2-4) groups treated with 10%, 15%, and 20% Lamiaceae gel. During the initial screening of gel concentrations, the air permeability of the sterile PU membrane, the gel's air permeability, and its ability to absorb exudate were evaluated. For 15 consecutive days, the healing of the rats' back wounds was observed daily and photographed to determine the optimal concentration of Lamiaceae gel for promoting wound healing.

[0057] 2.2 Comparison of the effects of Lamiophlomis gel and extract on promoting healing of infected wounds Ten rats were randomly selected and each rat was anesthetized with 2% sodium pentobarbital (60 mg / kg). The hair on the back was then removed with an animal electric shaver, and the hair at the root was further removed with a safe and non-irritating depilatory cream to completely expose the areas on both sides of the midline of the back. The areas on both sides of the midline of the back were disinfected with 75% ethanol, and then covered with a disposable sterile surgical sheet. Four circular full-thickness skin defect wounds with a diameter of 8 mm were punched on the back of each rat with a skin biopsy punch until the depth reached the fascia, and the wounds were evenly distributed in a triangular shape on the back of the rat. 50 μL of bacterial suspension (Staphylococcus aureus, 10 8 CFU / ml), and the wound was covered with sterile PU film for 24 h to prepare a bacterial infection wound model.

[0058] The four wounds of each rat were treated as follows: 1) untreated blank control group; 2-3) 10% Lamiophlomis gel group; 4) 10% Lamiophlomis extract group.

[0059] For 15 consecutive days, the wound healing of the rats' backs was observed and photographed daily. Wound healing was calculated on days 3, 9, and 15. Rats were sacrificed on days 3, 8, and 14 of administration. Skin samples from the wounds were collected, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 4 μm thickness. The sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome. Microscopic photographs were taken, and the tissue sections were used to observe the formation of granulation tissue and the distribution of collagen deposition in the wounds.

[0060] 2.3 Study on the activity of Duyiwei gel in promoting the healing of infected wounds After screening the optimal concentration of Duyiwei gel that promotes the healing of bacterial wounds, 10 rats were anesthetized with 2% sodium pentobarbital (60 mg / kg), and then the hair on the back was removed with an animal electric shaver, and the hair at the root was further removed with a safe and non-irritating depilatory cream to completely expose the area on both sides of the midline of the back. The areas on both sides of the midline of the back were disinfected with 75% ethanol, and then covered with disposable sterile surgical sheets. Four circular full-thickness skin defect wounds with a diameter of 8 mm were punched on both sides of the midline of the back of each rat with a skin biopsy punch until the depth reached the fascia. 50 μl of bacterial suspension (Staphylococcus aureus, 10 8 CFU / ml), and the wound was covered with sterile PU film for 24 h to prepare a bacterial infection wound model.

[0061] Each rat received four wounds treated as follows: 1) untreated blank control; 2) 10% Du Yi Wei gel; 3) fusidic acid cream; and 4) compound Huang Bo liquid. Wound healing was observed on days 3, 9, and 15, and wounds were photographed. Wound area was measured using ImageJ software, and wound healing rates were calculated. Rats were sacrificed on days 3, 8, and 14 after treatment. Regenerated skin tissue samples were collected, fixed with 4% paraformaldehyde, dehydrated with graded alcohols, and embedded in paraffin. The wounds were cut into 3 μm-thick sections and then stained with hematoxylin-eosin (H&E) and Masson's trichrome as histological evaluation indicators, platelet-endothelial cell adhesion molecule (CD31) and tumor necrosis factor α (TNF-α) as immunohistochemical evaluation indicators, α-smooth muscle actin (α-SMA) and myeloperoxidase (MPO) as immunofluorescence evaluation indicators, and cell nuclei were stained with DAPI fixative to comprehensively evaluate the granulation tissue formation, collagen deposition, tissue angiogenesis, and tissue inflammation of the contaminated wounds.

[0062] 3. Results 3.1 Preliminary screening results of the concentration of Duyiwei gel in promoting healing of infected wounds Table 17 Results of wound healing rate determination based on initial screening concentration Based on the observation results of wound healing during the experiment (Table 17 and Figure 13 ), no significant differences were observed between the different gel concentration groups in promoting wound healing. Based on the properties and stability investigated during the initial gel preparation, as well as the gel deposition on the wound during the experiment, the 10% Lamiophlomis gel group was selected for the subsequent infection-promoting wound healing experiment.

[0063] 3.2 Comparison of the effects of Lamiophlomis gel and extract on promoting healing of infected wounds Table 18 Comparison of wound healing rate with extracts The wound healing rate was calculated and analyzed based on the experimental record chart. The analysis results are shown in Table 18 and Figure 14 As shown, compared with the control group, the wound healing rate of the Lamiophlomis gel group was higher overall during the observation period, while the Lamiophlomis extract group had a higher healing rate only on day 15. Compared with the Lamiophlomis extract group, the Lamiophlomis gel group had a higher healing rate on days 3 and 8, and a similar healing rate on day 15. This indicates that Lamiophlomis gel is more effective in repairing infected wounds than Lamiophlomis extract.

[0064] Table 19 Granulation tissue gap determination results Note: Compared with the control group, a P<0.001; compared with the extract group, b P<0.001 The wound refilling, granulation tissue formation and epithelial reconstruction were evaluated according to the results of HE section staining, as shown in Table 19 and Figure 15 As shown in the analysis chart, during the healing process, there were significant differences between the Lamiophlomis gel group and the extract group and the control group. On the 14th day, the gel group showed the smallest tissue gap and was significantly different from the Lamiophlomis extract group. This phenomenon indicates that the Lamiophlomis gel group is better than the control group and the extract group in promoting wound healing.

[0065] Table 20 Collagen deposition measurement results Note: Compared with the control group, a P<0.05; compared with the extract group, b P<0.001 Masson staining of sections was used to assess collagen deposition and tissue healing. Figure 16 It can be seen that during the healing process, compared with the control group, the Lamiophlomis gel group and the extract group showed higher collagen deposition capacity, and the collagen deposition capacity of the gel group was better than that of the extract group, and the difference was significant. This shows that the Lamiophlomis gel group can significantly accelerate collagen deposition in the wound site.

[0066] 3.3 Study on the activity of Lamiophlomis gel in promoting the healing of infected wounds Table 21 Results of wound healing rate compared with positive drugs The wound healing rate was calculated and analyzed based on the experimental record chart. The analysis results are shown in Table 21 and Figure 17As shown in the figure, on the 3rd and 15th days, the wound healing rates of the drug-treated group were higher than those of the control group, and on the 15th day, the wound healing effect of the drug-treated group was equivalent.

[0067] Table 22 Granulation tissue gap determination results Note: Compared with the control group, a P<0.001; compared with the fusidic acid cream group, b P<0.05; compared with the compound Huangbai liquid group, c P<0.05 The wound refilling, granulation tissue formation and epithelial reconstruction were evaluated according to the results of HE section staining. Figure 18 It can be seen that there were significant differences between the drug-treated group and the control group on the 8th and 14th days, and on the 14th day, the Duyiwei gel group showed the smallest tissue gap, which was better than the positive drug group.

[0068] Table 23 Collagen deposition measurement results Note: Compared with the control group, a P<0.001; compared with the fusidic acid cream group, b P<0.001; compared with the compound Huangbai liquid group, c P<0.05 Masson section staining was used to evaluate collagen deposition and tissue healing. Figure 19 The analysis chart showed that there were significant differences among the Duyiwei gel group, the fusidic acid cream group, and the compound phellodendron liquid group during the healing process, that is, the ability of collagen deposition in the Duyiwei gel group was better than that in the positive drug group.

[0069] Table 24 CD31 positive expression determination results Note: Compared with the control group, a P<0.05; compared with the fusidic acid cream group, b P<0.05; compared with the compound Huangbai liquid group, c P<0.05 CD31 is an intercellular adhesion molecule found on endothelial cells and is used to assess blood vessel formation and stability. Figure 20 The results showed that compared with the control group, the expression of CD31 in the healing skin of the drug-treated group was significantly increased, and the expression of CD31 in the Duyiwei gel group was comparable to that in the compound Huangbo liquid group. This phenomenon indicates that Duyiwei gel can effectively enhance the early vascularization of infected wounds.

[0070] Table 25 TNF-α positive expression determination results Note: Compared with the control group, a P<0.01; compared with the fusidic acid cream group, b P<0.05; compared with the compound Huangbai liquid group, c P<0.05 Tumor necrosis factor α (TNF-α) is a pro-inflammatory factor secreted by M1 macrophages and is used to assess the inflammatory status during wound healing. Figure 21 As can be seen, compared with the control group, the drug-treated groups showed varying degrees of reduction in TNF-α on days 3, 8, and 14. On day 14, the Duyiwei gel group showed comparable effects to the two positive drug groups, indicating that the Duyiwei gel group had a certain effect on alleviating inflammation. Combined with the expression of CD31 and TNF-α, it can be concluded that the Duyiwei gel, fusidic acid cream, and compound phellodendron liquid groups all have the ability to promote angiogenesis and alleviate inflammatory responses, and the gel group had comparable effects to the positive drug groups.

[0071] Table 26 α-SMA positive expression determination results Note: Compared with the control group, a P<0.05; compared with the fusidic acid cream group, b P<0.05; compared with the compound Huangbai liquid group, c P<0.05 α-Smooth muscle actin (α-SMA) is mainly used to mark smooth muscle fibroblasts: as shown in Table 26 and Figure 22 As shown in the results, the expression of α-SMA in the drug-treated groups was higher than that in the control group, that is, more blood vessels were formed in the drug-treated groups, and on the 14th day, there was no significant difference in the expression of α-SMA between the gel group and the fusidic acid cream group, indicating that Du Yiwei gel has the ability to enhance a-SMA expression and promote angiogenesis.

[0072] Table 27 MPO positive expression determination results Note: Compared with the control group, a P<0.05; compared with the fusidic acid cream group, b P<0.05 Myeloperoxidase (MPO) is a marker of systemic inflammation. MPO is mainly used to detect wound inflammation: Analysis results are shown in Table 27 and Figure 23 The results showed that the MPO level of the control group was significantly higher than that of the drug-treated group, and both the Duyiwei gel group and the compound Huangbo liquid group showed lower MPO levels, indicating that the Duyiwei gel group had an anti-inflammatory effect comparable to that of the compound Huangbo liquid group.

[0073] Based on the test results of α-SMA and MPO: the Duyiwei gel group and the positive drug group not only have stronger ability to promote angiogenesis, but also can significantly reduce the inflammatory response.

[0074] The above results show that Du Yiwei gel can promote the healing of infected wounds, and its overall healing effect is better than that of Du Yiwei extract, and is also better than that of fusidic acid cream and compound phellodendron liquid currently commonly used in clinical practice.

[0075] In summary, the Lamiophlomis gel of the present invention has a high drug loading capacity, good stability and fluidity, and its therapeutic effect in repairing acute wounds is comparable to that of the Lamiophlomis extract at the same dose; its therapeutic effect in repairing infectious wounds is significantly better than that of the Lamiophlomis extract at the same dose, and is also better than the fusidic acid cream and compound phellodendron liquid currently commonly used in clinical practice, and has practical value for promotion and application.

Claims

1. A gel preparation comprising: It is an external preparation prepared from the following raw materials and auxiliary materials: 1.25-5 parts by weight of Lamiophlomis lappa extract, 0.5-1 parts by weight of Carbomer 940, 0.01-0.05 parts by weight of ethylparaben, 0.5-1 parts by volume of triethanolamine, 1-2 parts by volume of glycerol, and 1.5-2 parts by volume of 1,2-propylene glycol; The Lamiophlomis lappa extract is a water extract of Lamiophlomis lappa; Each 1g of the water extract is equivalent to 3-5g of Radix Angelicae Pubescentis; The carbomer 940 is dissolved in water to form a solution with a concentration of 3-3.8%; The ethyl hydroxybenzoate is dissolved in anhydrous ethanol to form a solution with a concentration of 3-3.5%.

2. The gel of claim 1, wherein It is an external preparation prepared from the following raw materials and excipients: 2.5 parts by weight of Lamiophlomis lappa extract, 0.875 parts by weight of Carbomer 940, 0.025 parts by weight of ethylparaben, 0.75 parts by volume of triethanolamine, 1.875 parts by volume of glycerol, and 1.875 parts by volume of 1,2-propylene glycol; The carbomer 940 is dissolved in water to form a solution with a concentration of 3.5%; The ethylparaben is dissolved in anhydrous ethanol to form a solution with a concentration of 3.3%.

3. A method for preparing the Lamiophlomis gel according to any one of claims 1 to 2, characterized in that: The steps include: 1) Weigh the raw materials and excipients according to the ratio. Take carbomer 940 and dissolve it in water to obtain a carbomer 940 solution. Take ethyl paraben and dissolve it in ethanol to obtain an ethyl paraben solution. 2) Take the carbomer 940 solution obtained in step 1), place it at 2-8°C to swell and defoam, then add triethanolamine, glycerol, 1,2-propylene glycol and the ethylparaben solution obtained in step 1), mix well, and finally add the Lamiophlomis radiata extract and stir well to obtain the product.

4. The method according to claim 3, wherein: Step 1) The concentration of the Carbomer 940 solution is 3.5%.

5. The method according to claim 3, wherein: In step 1), the ethanol is anhydrous ethanol; and the concentration of the ethyl hydroxybenzoate solution is 3.3%.

6. Use of the Lamiophlomis gel according to claim 1 or 2 in preparing a medicament for repairing wounds.

7. The use according to claim 6, characterized in that: The medicine is a medicine for repairing acute wounds.

8. The use according to claim 6, characterized in that: The medicine is a medicine for repairing infected wounds.

9. The use according to any one of claims 6 to 8, characterized in that: The drug has the effects of anti-inflammation, analgesia, promoting angiogenesis, promoting granulation tissue formation and / or promoting collagen deposition.

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