Duloxetine hydrochloride and its use

Abstract

The present application belongs to the field of pharmaceutical preparations, and particularly relates to a gel of Duiyiwai and application thereof. The gel of Duiyiwai is an external preparation prepared from raw and auxiliary materials including the following parts: Duiyiwai extract 1.25-5 parts by weight, carbomer 940 0.5-1 part by weight, hydroxyphenyl ethyl ester 0.01-0.05 part by weight, triethanolamine 0.5-1 volume part, glycerol 1-2 volume parts, 1,2-propylene glycol 1.5-2 volume parts. The gel of Duiyiwai has the same curative effect as the Duiyiwai extract of the same dosage in repairing acute wounds; but has a significantly better curative effect than the Duiyiwai extract of the same dosage in repairing infected wounds, and is also better than the commonly used fuscide acid cream and compound Huangbai liquid in the clinic, and has practical popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine preparations, specifically relating to a unique herbal gel and its application. Background Technology

[0002] *Lamiophlomis rotata* (Benth.) Kudo, also known as Dubutong or "Daba" or "Dabuba" in Tibetan, is the only plant in the genus *Lamiophlomis* of the Lamiaceae family. It is a commonly used Tibetan medicine with analgesic, hemostatic, and anti-inflammatory effects, characterized by significant efficacy, few side effects, and a wide range of clinical applications. Tablets, capsules, and granules made from *Lamiophlomis rotata* are available, and it has been reported to be used for various pain treatments in gynecology, surgery, internal medicine, dermatology, oncology, and ENT.

[0003] Pharmaceutical gels refer to viscous liquid or semi-solid preparations made from drugs and excipients capable of forming gels, in the form of solutions, suspensions, or emulsions. As a novel dosage form, pharmaceutical gels have become a research hotspot in the field of pharmaceutical formulations in recent years, and their advantages are gradually becoming apparent in clinical use. Patent CN 1739609A discloses a *Ligusticum striatum* gel with significant analgesic effects; however, this gel is an oral preparation, metabolized by the liver, resulting in drug conversion in the liver and thus reducing its bioavailability. Topical preparations are absorbed directly through the skin or mucous membranes, avoiding the first-pass effect of oral drugs through the gastrointestinal tract and liver, thereby improving drug bioavailability. Due to the lower amount of drug absorbed by topical preparations, the systemic exposure is lower, and the incidence of adverse reactions is also lower. However, there are currently no reports on gels made with *Ligusticum striatum* as the sole active ingredient that significantly improve skin pathological conditions when applied topically to the skin. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a unique gel, which is a topical preparation made from raw and auxiliary materials comprising the following proportions:

[0005] The following ingredients are present: 1.25-5 parts by weight of *Lysimachia christinae* extract, 0.5-1 part by weight of carbomer 940, 0.01-0.05 parts by weight of ethylparaben, 0.5-1 part by volume of triethanolamine, 1-2 parts by volume of glycerol, and 1.5-2 parts by volume of 1,2-propanediol.

[0006] The extract of *Duyiwei* is an aqueous extract of *Duyiwei*.

[0007] Each 1g of the water extract is equivalent to 3-5g of *Duyiwei*.

[0008] The carbomer 940 is dissolved in water to form a solution with a concentration of 3-3.8%;

[0009] The ethylparaben was dissolved in anhydrous ethanol to form a solution with a concentration of 3-3.5%.

[0010] Furthermore, it is a topical preparation made from raw materials and excipients in the following proportions:

[0011] 2.5 parts by weight of *Lysimachia christinae* 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-propanediol.

[0012] The carbomer 940 was dissolved in water to form a 3.5% solution;

[0013] The ethylparaben was dissolved in anhydrous ethanol to prepare a 3.3% solution;

[0014] Each 1g of the water extract is equivalent to 4.8g of *Duyiwei*.

[0015] The present invention also provides a method for preparing the aforementioned unique flavor gelling agent, comprising the following steps:

[0016] 1) Weigh the raw and auxiliary materials according to the ratio, take carbomer 940, add water to dissolve it to obtain carbomer 940 solution; take ethylparaben, add ethanol to dissolve it to obtain ethylparaben solution;

[0017] 2) Take the carbomer 940 solution obtained in step 1), place it at 2~8℃ to swell and defoam, then add triethanolamine, glycerol, 1,2-propanediol and the ethylparaben solution obtained in step 1), mix well, and finally add the extract of *Lysimachia christinae*, stir evenly, and the product is obtained.

[0018] Further, the concentration of the carbomer 940 solution in step 1) is 3.5%.

[0019] Furthermore, the ethanol in step 1) is anhydrous ethanol; the concentration of the ethylparaben solution is 3.3%.

[0020] The present invention also provides the use of the aforementioned unique flavor gel in the preparation of a medicine for repairing wounds.

[0021] Furthermore, the drug is a drug for repairing acute wounds.

[0022] Furthermore, the drug is a drug for repairing infected wounds.

[0023] Furthermore, the drug has anti-inflammatory, analgesic, angiogenesis-promoting, granulation tissue-promoting, and / or collagen-deposition-promoting effects.

[0024] This invention relates to a gel containing *Duyiwei* (a type of medicinal herb), which, by combining *Duyiwei* extract with specific excipients, not only achieves a high drug loading capacity for the *Duyiwei* extract but also ensures excellent stability and flowability of the gel itself. Animal experiments have demonstrated that the efficacy of this *Duyiwei* gel in repairing acute wounds is comparable to that of *Duyiwei* extract at the same dosage; in repairing infected wounds, its efficacy is significantly superior to that of *Duyiwei* extract at the same dosage, and also superior to fusidic acid cream and compound berberine solution commonly used in clinical practice, thus possessing practical application value.

[0025] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0026] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0027] Figure 1 Cumulative pain score;

[0028] Figure 2 Initial screening of the concentration of Duyiwei gel to promote acute wound healing - wound healing;

[0029] Figure 3 Comparison of the effects of Du Yi Wei gel and Du Yi Wei extract on promoting acute wound healing - Wound healing;

[0030] Figure 4 Comparison of the effects of Du Yi Wei gel and Du Yi Wei extract on promoting acute wound healing - HE staining;

[0031] Figure 5 Comparison of the effects of Du Yi Wei gel and Du Yi Wei extract on promoting acute wound healing - Masson staining;

[0032] Figure 6 Study on the activity of Duyiwei gel in promoting acute wound healing - wound healing;

[0033] Figure 7 Study on the activity of Du Yi Wei gel in promoting acute wound healing - HE staining;

[0034] Figure 8 Study on the activity of Du Yi Wei gel in promoting acute wound healing - Masson staining;

[0035] Figure 9 Study on the activity of Duyiwei gel in promoting acute wound healing - CD31 immunohistochemistry;

[0036] Figure 10 Study on the activity of Duyiwei gel in promoting acute wound healing - TNF-α immunohistochemistry;

[0037] Figure 11 Study on the activity of Duyiwei gel in promoting acute wound healing - α-SMA immunofluorescence;

[0038] Figure 12 Study on the activity of Duyiwei gel in promoting acute wound healing - MPO immunofluorescence;

[0039] Figure 13 Initial screening of the concentration of Duyiwei gel to promote infection-inducing wound healing - wound healing;

[0040] Figure 14 Comparison of the effects of Du Yi Wei gel and Du Yi Wei extract on promoting the healing of infected wounds - Wound healing;

[0041] Figure 15 Comparison of the effects of Du Yi Wei gel and Du Yi Wei extract on promoting the healing of infected wounds - HE staining;

[0042] Figure 16 Comparison of the effects of Du Yi Wei gel and Du Yi Wei extract on promoting the healing of infected wounds - Masson staining;

[0043] Figure 17 Study on the activity of Duyiwei gel in promoting the healing of infected wounds - Wound healing;

[0044] Figure 18 Study on the healing activity of Duyiwei gel in promoting infected wounds - HE staining;

[0045] Figure 19 Study on the healing activity of Duyiwei gel in promoting infected wounds - Masson staining;

[0046] Figure 20 Study on the healing activity of Duyiwei gel in promoting infected wounds - CD31 immunohistochemistry;

[0047] Figure 21 Study on the healing activity of Duyiwei gel in promoting infected wounds - TNF-α immunohistochemistry;

[0048] Figure 22 Study on the activity of Duyiwei gel in promoting acute wound healing - α-SMA immunofluorescence;

[0049] Figure 23 Study on the activity of Duyiwei gel in promoting acute wound healing - MPO immunofluorescence. Detailed Implementation

[0050] The raw materials, reagents and equipment used in the specific embodiments of this invention were all purchased commercially. Among them, the Duyiwei extract and Duyiwei capsules were provided by Kangxian Duyiwei Biopharmaceutical Co., Ltd. The preparation method of Duyiwei extract is as follows: Duyiwei slices are crushed, decocted with water three times, one hour each time, the decoctions are combined, filtered, the filtrate is concentrated and dried below 80°C, crushed, and Duyiwei extract is obtained; each 1g of Duyiwei extract is equivalent to 4.8g of slices.

[0051] Example 1: Preparation of the unique flavor gelling agent of the present invention

[0052] Formula: 2.5g of *Lysimachia christinae* extract, 0.875g of carbomer 940, 0.025g of ethylparaben, 0.75ml of triethanolamine, 1.875ml of glycerol, and 1.875ml of 1,2-propanediol;

[0053] Preparation method:

[0054] 1) Dissolve carbomer 940 in 25 ml of water to prepare a 3.5% g / ml carbomer 940 solution; dissolve ethylparaben in 0.75 ml of anhydrous ethanol to prepare a 3.3% g / ml ethylparaben solution.

[0055] 2) Take the carbomer 940 solution obtained in step 1), swell and defoam at 4°C, then add triethanolamine, glycerol, 1,2-propanediol and the ethylparaben solution obtained in step 1) at room temperature and mix well. Then add the extract of *Duyiwei* and stir evenly to obtain the final product.

[0056] The beneficial effects of the present invention are illustrated below through experimental examples:

[0057] Experimental Example 1: Study on Unique Flavor Gel

[0058] I. Research on the formulation screening and preparation process of Duyiwei gel

[0059] 1. Experimental Objective

[0060] To prepare a Du Yi Wei gel with excellent stability and excipient properties, so as to improve the efficacy of Du Yi Wei.

[0061] 2. Prescription Design

[0062] There are many types of excipients for gelling agents. The composition and ratio of these excipients not only affect the gel's formation but also the efficacy of the active ingredients. Based on previous experimental results, from excipients including carbomer, methylcellulose, hydroxypropyl methylcellulose, sodium phosphomethylcellulose, alginate, tragacanth gum, gelatin, starch, lauryl azone, menthol, pH adjusters, humectants, thickeners, transdermal penetration enhancers, and preservatives, we determined that using carbomer 940 as the main matrix, with the addition of appropriate amounts of glycerol, 1,2-propanediol, triethanolamine, ethylparaben (EPF), and *Duyiwei* extract powder, and mixing thoroughly, *Duyiwei* gel showed good gelling results and is expected to maximize the efficacy of *Duyiwei* extract.

[0063] Meanwhile, regarding the dosage of Carbomer 940, referring to the recommended dosage concentration of 0.5%~2% in the "Handbook of Pharmaceutical Excipients", it has been impossible to obtain a gel with good excipient effect. Therefore, based on the characteristics of the water extract of Duyiwei, the dosage of Carbomer 940 was further adjusted in order to make the gel have ideal rheological properties, improve the patient's user experience, and fully exert the efficacy of Duyiwei.

[0064] 3. Prescription Examination

[0065] The optimal prescription was screened by combining different concentrations of *Dioscorea opposita* extract with different matrix ratios.

[0066] 4. Stability of traits

[0067] The properties and stability of Du Yi Wei gel with different drug loading were observed.

[0068] 5. Results

[0069] 5.1 Investigation of blank gel formulation

[0070] Gel matrices with different carbomer concentrations were prepared according to Table 1. 2 mL of each gel matrix was added to triethanolamine, glycerol, and 1,2-propanediol to obtain blank gels. Observation of the gel morphology revealed that the addition of glycerol, 1,2-propanediol, and ethylparaben did not affect the gel properties; the resulting gel was a colorless, transparent semi-solid that maintained a certain shape.

[0071] Table 1 Blank Gel Prescriptions

[0072]

[0073] 5.2 Investigation of the formula for Duyiwei gel

[0074] Blank gels were prepared according to section 5.1. Different masses of *Ligusticum striatum* extract powder were added to 2 mL of each blank gel, and the properties of *Ligusticum striatum* gels with different drug loadings (0, 5, 8, 10, and 15%) were examined (Table 2). Table 2 shows that *Ligusticum striatum* extract has a significant impact on the properties of the gel. When the carbomer concentration is 4% and the *Ligusticum striatum* concentration is 8-10%, the excipient effect is better, and the drug loading is also higher.

[0075] Table 2 Screening of Du Yi Wei gel formulations

[0076]

[0077] 5.3 Stability Study of the Gel Properties of Du Yi Wei

[0078] To meet the stability requirements of the gel, after formulation investigation and numerous trials, the relevant parameters were further adjusted based on the results. Specifically, 25 mL of *Duyiwei* gels with different drug loadings were prepared according to Table 3. First, a (3.5%) CP940 solution was prepared and, after complete dissolution, placed in a 4°C refrigerator to swell and defoam. Then, at room temperature, triethanolamine, glycerol, 1,2-propanediol, and anhydrous ethanol solution of ethylparaben were added sequentially according to the table below, and stirred thoroughly to obtain a blank gel matrix. Appropriate amounts of *Duyiwei* extract powder were added to the blank gels, and stirred thoroughly to obtain *Duyiwei* gels with different drug loadings, and their properties were preliminarily investigated. Finally, the *Duyiwei* gels were transferred to squeezeable plastic bottles and allowed to stand for stability observation.

[0079] Table 3 Optimization of the Du Yi Wei gel prescription

[0080]

[0081] The prepared Du Yi Wei gels with different drug loading were transferred to squeezeable plastic bottles and placed at 4°C to observe their stability. The results are shown in Table 4.

[0082] Table 4. Stability Study of Appearance and Properties of Du Yi Wei Gel

[0083]

[0084] As shown in Table 4, when the drug loading is 10%, the gel exhibits the best color, odor, excipient properties, extrusion from the soft bottle, and flowability within the soft bottle, while also demonstrating high drug loading and good stability. Specifically, the preparation method for the gel in Group 2 with a drug loading of 10% is as follows:

[0085] Dissolve 0.875g of carbomer 940 in 25ml of water to prepare a 3.5% g / ml carbomer 940 solution; dissolve 0.025g of ethylparaben in 0.75ml of anhydrous ethanol to prepare a 3.3% g / ml ethylparaben solution; take 25ml of the prepared carbomer 940 solution, place it in a 4℃ refrigerator to swell and defoam, then add 0.75ml of triethanolamine, 1.875ml of glycerol, 1.875ml of 1,2-propanediol and the prepared ethylparaben solution in sequence and mix well; finally add 2.5g of *Lysimachia christinae* extract and stir well to obtain the final product.

[0086] II. Evaluation of Pharmacodynamic Effects

[0087] (1) Evaluation of postoperative analgesic effect in rats

[0088] 1. Experimental Objective

[0089] A rat paw incision pain model was established to evaluate the analgesic effect of topical Duyiwei gel (Duyiwei gels prepared according to the optimized process of the prescription in Table 3 with concentrations of 10%, 15%, and 20%), and to compare it with compound polymyxin B ointment and oral Duyiwei capsules.

[0090] 2 Experimental Methods

[0091] At least 90 adult male SD rats (8-10 weeks old, weighing 200-220 g) were selected and fed 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 four groups: a control group, an acute pain model group, a Duyiwei capsule group (divided into high, medium, and low doses), a compound polymyxin B ointment group, and a Duyiwei gel group (divided into high, medium, and low doses), with 10 rats in each group. The rats were placed in observation cages for two days to acclimatize before the experiment.

[0092] Except for the control group, all rats in the experimental groups underwent left plantar laceration surgery (the most common postoperative pain model). Rats other than those in the control group were placed in a transparent induction chamber and induced to sleep with 3% isoflurane inhalation. After falling asleep, they 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 rat's left paw was disinfected with alcohol. A 1 cm longitudinal incision was made anteriorly from 0.5 cm at the heel edge, penetrating the skin, fascia, and plantar muscles. The skin was then sutured with two stitches using 5-0 antibacterial absorbable sutures. Hemostasis was achieved by pressure, and the wound was cleaned and disinfected to establish an acute rat model of plantar laceration pain.

[0093] After the foot was cut open, the model group was given physiological saline by gavage (1 mL / 200 g). The Duyiwei capsule group was given the capsule contents solution by gavage (dissolved in physiological saline at concentrations of 3%, 6%, and 9%, respectively, at a dose of 1 mL / 200 g). The compound polymyxin B ointment group was given compound polymyxin B ointment (0.04 mL / 200 g) applied to the incision site. The Duyiwei gel group was given Duyiwei gel applied to the incision site at a dose of 0.04 mL / 200 g.

[0094] One hour after establishing the pain model, all rats were placed in glass boxes (40 cm × 40 cm × 40 cm) and the "cumulative pain scoring method" was used to observe the hind paw's ground contact and weight-bearing on the surgical side. A score of 2 was given if the hind paw on the surgical side was raised and not on the ground due to pain; 1 point was given if the hind paw on the surgical side was on the ground but not bearing weight (i.e., the hind paw did not turn white); and 0 points were given if the hind paw on the surgical side was on the ground and bearing weight. The score for the non-surgical side was the same as the surgical side. Observations were conducted every 5 minutes for 1 minute starting 1 hour post-surgery. The most frequently adopted standing posture of the rat within 1 minute was used as the standard. Observations were continued for a total of 1 hour, and the cumulative pain score (0~24 points) was calculated using the following formula:

[0095] Cumulative pain score = Pain score on the surgical side - Pain score on the non-surgical side

[0096] 3. Results

[0097] The lower the cumulative pain score, the better the analgesic effect. (See Table 5 and...) Figure 1 As shown, the 6% and 9% Duyiwei capsule groups had the lowest cumulative pain scores, at 5.00 and 5.33 respectively; followed by the 20% Duyiwei gel group, which had a relatively low cumulative pain score of 6.33. Although there was no statistically significant difference between the gel group and the model group, the cumulative pain score results indicated that the gel group showed a trend towards analgesic effects and was superior to the compound polymyxin B group. Compound polymyxin B contains lidocaine hydrochloride, which has analgesic effects, indicating that Duyiwei gel also has a pain-relieving effect, and its analgesic effect is superior to that of compound polymyxin B.

[0098] Table 5 Pain Cumulative Score Table

[0099]

[0100] 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

[0101] (2) Pharmacodynamic evaluation of Duyiwei gel in promoting acute wound healing

[0102] 1. Experimental Objective

[0103] An acute wound model was established in rats. The wound repair activity of Du Yi Wei gel (Du Yi Wei gels prepared according to the optimized process of the prescription in Table 3 with concentrations of 10%, 15%, and 20%) was evaluated by pharmacodynamic experiments and compared with Du Yi Wei extract, Kangfuxin liquid, and recombinant human epidermal growth factor gel.

[0104] 2 Experimental Methods

[0105] 2.1 Initial screening of the concentration of Duyiwei gel to promote acute wound healing

[0106] Six rats were randomly selected and anesthetized with 2% sodium pentobarbital (60 mg / kg). The hair on the back was then removed using an electric animal razor, and a safe, non-irritating depilatory cream was used to further remove the hair at the root, fully exposing 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 drape. Four circular, full-thickness skin defects, each 8 mm in diameter, were created on both sides of the midline of the back in each rat using a skin biopsy punch, continuing until the depth reached the fascia.

[0107] Four wound treatments were performed on each rat as follows: 1) Untreated blank control group; 2-4) 10%, 15%, and 20% Duyiwei gel groups. During the initial screening of gel concentration, the gel's air permeability and exudate absorption performance were simultaneously evaluated. For 14 consecutive days, the healing of the rat's dorsal wounds was observed and photographed daily to preliminarily determine the optimal concentration of Duyiwei gel for promoting wound healing.

[0108] 2.2 Comparison of the effects of Du Yi Wei gel and extract on promoting acute wound healing

[0109] Six rats were randomly selected and anesthetized with 2% sodium pentobarbital (60 mg / kg). The hair on the back was then removed using an electric animal razor, followed by further removal of the hair at the root using a safe and non-irritating depilatory cream to fully 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 drape. Four circular full-thickness skin defects, each 8 mm in diameter, were created on the back of each rat using a skin biopsy punch, extending to the fascia, and evenly distributed in a triangular pattern on the rat's back. The four wounds on each rat were treated as follows: 1) Untreated blank control group; 2-3) 10% *Duyiwei* gel group; 4) 10% *Duyiwei* extract group.

[0110] The healing of the wounds on the backs of rats was observed and photographed daily for 14 consecutive days, and the wound healing status was calculated on days 3, 8, and 14. Rats were sacrificed on days 3, 8, and 14, and skin samples from the wounds were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm. The samples were stained with hematoxylin-eosin (H&E) and Masson's trichrome staining, respectively. Microscopic photographs were taken, and the distribution of epidermal regeneration and collagen deposition around the wounds was observed through the photographed tissue sections.

[0111] 2.3 Study on the activity of Duyiwei gel in promoting acute wound healing

[0112] After determining the optimal concentration of Duyiwei gel for promoting healing, 10 rats were anesthetized with 2% sodium pentobarbital (60 mg / kg). The hair on their backs was then removed using an electric animal razor, and a safe, non-irritating depilatory cream was used to further remove the hair at the roots, fully exposing 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 disposable sterile surgical drapes. Four circular, full-thickness skin defects with a diameter of 8 mm were created on both sides of the midline of the back in each rat using a skin biopsy punch, extending to the fascia. The four wounds in each rat were treated as follows: 1) Untreated blank control group; 2) Duyiwei gel optimal dose group; 3) Kangfuxin liquid group; 4) Recombinant human epidermal growth factor gel group.

[0113] Wound healing was observed at 0, 3, 8, and 14 days. Wound photographs were taken, and the wound area was measured using ImageJ software. The wound healing rate was calculated using the following formula:

[0114] Wound shrinkage (%) = (A0 − At) / A0 × 100%

[0115] Where A0 represents the wound area on day 0, and At represents the wound area at different times.

[0116] This study evaluated the effects of Duyiwei gel on epidermal regeneration, inflammation, angiogenesis, and oxidative stress in the wound area. Rats were sacrificed on days 3, 8, and 14, and skin samples from the wound were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm. The sections were stained with hematoxylin-eosin (H&E) and Masson's trichrome, respectively. All sections were analyzed, and microscopic photographs were taken to observe the distribution of epidermal regeneration and collagen deposition around the wound.

[0117] Immunohistochemistry was used to analyze the expression levels of regenerative skin cytokines CD31 and TNF-α in the wound site. In addition, skin tissue sections on days 3, 8, and 14 were subjected to immunofluorescence staining with α-SMA (smooth muscle actin) and MPO (myeloperoxidase), with cell nuclei further stained with a fixative containing DAPI. The slides were then observed under an inverted fluorescence microscope.

[0118] 3. Results

[0119] 3.1 Initial screening results of the concentration of Duyiwei gel in promoting acute wound healing

[0120] Table 6 Results of wound appearance healing rate determination during initial screening at high concentrations

[0121]

[0122] Based on the observations of wound healing progress during the experiment, from Table 6 and Figure 2 It is evident that no significant differences were observed among the gel concentration groups in promoting wound healing. However, after comprehensively considering the preparation characteristics, stability assessment, and deposition behavior of the gel after application to the wound, it was found that the 15% and 20% concentrations of *Duyiwei* gel exhibited excessive viscosity, leading to significant adhesion at the wound site. Therefore, a 10% concentration of *Duyiwei* gel was selected for subsequent wound healing promotion experiments.

[0123] 3.2 Comparison of the effects of Du Yi Wei gel and extract on promoting acute wound healing

[0124] Table 7 Results of wound appearance healing rate determination compared with extract.

[0125]

[0126] Based on the analysis of the wound surface recovery, from Table 7 and Figure 3 It is evident that, compared with the control group, the wound healing rates of the Duyiwei gel group and the Duyiwei extract group were higher on days 3 and 14; the healing rate of the Duyiwei extract group was higher than that of the Duyiwei gel group on days 3 and 8, while the healing rates of the Duyiwei extract group and the Duyiwei gel group were comparable on day 14. This indicates that the Duyiwei gel and Duyiwei extract are equally efficient in repairing acute wounds.

[0127] Table 8 Results of Granulation Tissue Interstitial Space Measurement

[0128]

[0129] Note: Compared with the control group, a P < 0.001; compared with the extract group, b P < 0.01

[0130] The wound refilling, granulation tissue formation, and epithelial reconstruction were assessed based on the HE staining results, as shown in Table 8 and... Figure 4 It can be seen that during the treatment, the granulation tissue gaps in each group gradually decreased, indicating that the wound was healing. However, the drug-treated group had smaller granulation tissue gaps and a large number of new granulation tissues, indicating better tissue healing quality. By day 14, 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. There was no significant difference between the Duyiwei gel group and the Duyiwei extract group.

[0131] Table 9 Results of Collagen Deposition Measurement

[0132]

[0133] Note: Compared with the control group, a P < 0.001; compared with the extract group, b P < 0.01

[0134] Masson staining was used to assess collagen deposition and tissue healing, as shown in Table 9 and Figure 5 As observed, on days 8 and 14 of treatment, the density and thickness of collagen fibers in the *Duyiwei* gel group and the extract group were significantly greater than those in the control group, indicating that both *Duyiwei* gel and extract can promote collagen deposition at a faster rate. Furthermore, the treated groups also exhibited more abundant and structurally intact identifiable angiogenesis. This further demonstrates the significant effects of *Duyiwei* gel and extract in promoting tissue healing and angiogenesis.

[0135] 3.3 Study on the activity of Duyiwei gel in promoting acute wound healing

[0136] Table 10 Results of wound appearance healing rate compared with positive control drug

[0137]

[0138] Note: Compared with the control group, a P < 0.001; compared with the gel group, b P < 0.01

[0139] Based on the analysis of the wound surface recovery, from Table 10 and Figure 6 It is evident that the wound healing rate was higher in the early stage of the Kangfuxin liquid group, which may be related to the characteristics of the liquid formulation of Kangfuxin liquid. The rate was slightly lower in the mid-stage of the Duyiwei gel group, and there was no significant difference in the healing rate among the groups in the later stage. Overall, there was no significant difference in the wound healing rate between the Duyiwei gel group, the recombinant human epidermal growth factor group, and the control group.

[0140] Table 11 Results of Granulation Tissue Interstitial Space Measurement

[0141]

[0142] Note: Compared with the control group, a P < 0.001; compared with the gel group, b P < 0.05

[0143] The wound refilling, granulation tissue formation, and epithelial reconstitution were assessed based on the HE staining results, as shown in Table 11 and... Figure 7 It is evident that each treatment group exhibited good wound healing performance, with smaller granulation tissue gaps than the control group, stronger tissue adhesion, and more angiogenesis. The wound tissue granulation recovery in the treatment groups was better, but the healing effect of Duyiwei gel was not as good as that of Kangfuxin liquid.

[0144] Table 12 Results of Collagen Deposition Measurement

[0145]

[0146] Note: Compared with the control group, a P < 0.001; compared with the gel group, b P < 0.001

[0147] Masson section staining was used to assess collagen deposition and tissue healing, as shown in Table 12 and Figure 8 It is evident that the drug-treated group showed greater collagen fiber deposition than the control group, with a faster collagen deposition rate, and also exhibited more complete and clearly identifiable angiogenesis. The gel's effect was initially inferior to the two positive control groups. Considering both HE and Masson results, all drug-treated groups effectively promoted the recovery of skin appendages, but the gel group's effect was slightly lower than that of the Kangfuxin liquid and recombinant human epidermal growth factor groups.

[0148] Table 13 Results of CD31 positive expression assay

[0149]

[0150] Note: Compared with the control group, a P < 0.001; compared with the gel group, b P < 0.001

[0151] CD31 is an intercellular adhesion molecule found on endothelial cells and is used to assess angiogenesis and stability. (From Table 13 and...) Figure 9 It is evident that CD31 expression gradually increases from the early to the middle stages of wound healing, and then decreases in the later stages. Compared with the control group, the drug-treated 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 is close to that of Kangfuxin liquid and superior to recombinant human epidermal growth factor.

[0152] Table 14 Results of TNF-α positive expression assay

[0153]

[0154] Note: Compared with the control group, a P < 0.05

[0155] Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine secreted by M1 macrophages and is used to assess inflammation during wound healing. (See Table 14 and...) Figure 10 It was observed that on day 3, the expression level of TNF-α in the control group was significantly higher than that in other treatment groups. On days 8 and 14, the expression level of TNF-α in the control group decreased slightly but remained higher than that in the treatment groups, indicating that the inflammatory response in the control group was more severe during the treatment process. Duyiwei gel and the positive control drug had comparable anti-inflammatory effects. Combining the expression levels of CD31 and TNF-α, it can be seen that Duyiwei gel, Kangfuxin liquid, and recombinant human epidermal growth factor groups had stronger angiogenesis-promoting abilities and lower inflammatory responses, but the gel group was less effective than the positive control drug group.

[0156] Table 15 Results of α-SMA Positive Expression Assay

[0157]

[0158] Note: Compared with the control group, a P < 0.001

[0159] The detection of α-smooth muscle actin (α-SMA) is mainly used to label smooth muscle myofibroblasts, as shown in Table 15. Figure 11 The results showed that the control group had lower α-SMA positive staining, indicating minimal angiogenesis. 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 group had the ability to enhance α-SMA expression and promote angiogenesis, which is beneficial to wound tissue healing. Moreover, the effects of Duyiwei gel and the positive drug were similar.

[0160] Table 16 Results of MPO Positive Expression Assay

[0161]

[0162] Note: Compared with the control group, a P < 0.001

[0163] Myeloperoxidase (MPO) is a marker of systemic inflammation. MPO is primarily used to detect wound inflammation. (Table 16 and...) Figure 12The results showed that MPO expression in the control group was significantly higher than in other treatment groups. This indicates that the inflammatory response was more severe in the control group during the wound healing phase, while the Duyiwei gel group and the positive control group effectively reduced the inflammatory response at the wound site, thus ensuring the normal repair of wound tissue. Furthermore, the effects of Duyiwei gel and the positive control group were comparable. Based on the combined results of α-SMA and MPO detection, it can be concluded that the treatment groups not only possess stronger angiogenesis-promoting capabilities but also significantly reduce the inflammatory response.

[0164] The above results indicate that Duyiwei gel can promote the healing of acute wounds, and its overall healing effect is comparable to that of Duyiwei extract, but not as good as Kangfuxin liquid and recombinant human epidermal growth factor, which are currently widely used in clinical practice.

[0165] (3) Study on the effect of Duyiwei gel on promoting the healing of infected wounds

[0166] 1. Experimental Objective

[0167] A rat model of bacterial infection was established to evaluate the antibacterial and wound-healing activity of Du Yi Wei gel (gels prepared according to the optimized process of the prescription in Table 3 with Du Yi Wei concentrations of 10%, 15%, and 20%), and to compare its antibacterial and wound-healing effects with those of compound Phellodendron amurense liquid and fusidic acid cream.

[0168] 2. Experimental Methods

[0169] 2.1 Initial Screening of the Concentration of Duyiwei Gel for Promoting Infection-Promoting Wound Healing

[0170] Six rats were randomly selected and anesthetized with 2% sodium pentobarbital (60 mg / kg). The hair on the back was then removed using an animal electric shaver, followed by further removal of the hair at the root using a safe and non-irritating depilatory cream to fully 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 drape. Four circular full-thickness skin defects, each 8 mm in diameter, were created on both sides of the midline of the back in each rat using a skin biopsy punch, extending to the fascia. 50 μL of bacterial suspension (Staphylococcus aureus, 10...) was added to the wounds. 8 (CFU / mL), and the wound was covered with a sterile PU film for 24 h to create a bacterial infection wound model.

[0171] Four wound treatments were performed on each rat as follows: 1) Untreated blank control group; 2-4) 10%, 15%, and 20% Duyiwei gel groups. During the initial screening of gel concentration, the air permeability of the sterile PU membrane, the air permeability of the gel, and its ability to absorb exudate were simultaneously evaluated. For 15 consecutive days, the healing of the rat's dorsal wounds was observed and photographed daily to preliminarily screen the optimal concentration of Duyiwei gel for promoting the healing of bacterial wounds.

[0172] 2.2 Comparison of the effects of Du Yi Wei gel and extract on promoting the healing of infected wounds

[0173] Ten rats were randomly selected and anesthetized with 2% sodium pentobarbital (60 mg / kg). The hair on the back was then removed using an animal electric shaver, followed by further removal of the hair at the root using a safe and non-irritating depilatory cream to fully 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 drape. Four circular full-thickness skin defects, each 8 mm in diameter, were created on the back of each rat using a skin biopsy punch, extending to the fascia, and evenly distributed in a triangular pattern on the rat's back. 50 μL of bacterial suspension (Staphylococcus aureus, 10...) was added to each wound. 8 (CFU / ml), and the wound was covered with a sterile PU film for 24 h to create a bacterial infection wound model.

[0174] The four wounds of each rat were treated as follows: 1) Untreated blank control group; 2-3) 10% Du Yi Wei gel group; 4) 10% Du Yi Wei extract group.

[0175] For 15 consecutive days, the healing of the wounds on the rats' backs was observed and photographed daily. Wound healing was recorded on days 3, 9, and 15. Rats were sacrificed on days 3, 8, and 14 after drug administration. Skin samples from the wounds were collected, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a thickness of 4 μm. The sections were stained with hematoxylin-eosin (H&E) and Masson's trichrome staining, respectively. Microscopic photographs were taken to observe the formation of granulation tissue and the distribution of collagen deposition in the wound sections.

[0176] 2.3 Study on the healing activity of Duyiwei gel in promoting infected wounds

[0177] After determining the optimal concentration of the unique gel for promoting wound healing in infected areas, ten rats were anesthetized with 2% sodium pentobarbital (60 mg / kg). The hair on their backs was then removed using an electric shaver, followed by further removal of the hair roots with a safe and non-irritating depilatory cream to fully expose the areas on either side of the midline of the back. The areas on either side of the midline of the back were 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 midline of the back in each rat using a skin biopsy punch, extending to the fascia. 50 μl of bacterial suspension (Staphylococcus aureus, 10...) was added to the wound. 8 (CFU / ml), and the wound was covered with a sterile PU film for 24 h to create a bacterial infection wound model.

[0178] Four wound treatments were administered to each rat as follows: 1) untreated control; 2) 10% Duyiwei gel group; 3) Fusidic acid cream group; 4) Compound Phellodendron amurense liquid group. Wound healing was observed on days 3, 9, and 15, and wound photographs were taken. The wound area was measured using ImageJ software, and the wound healing rate was calculated. Rats were sacrificed on days 3, 8, and 14 after drug application. Corresponding regenerated skin tissue samples were collected, fixed with 4% paraformaldehyde, dehydrated with graded alcohols, and embedded in paraffin. The tissue was transversely sectioned into 3 μm thick slices. Hematoxylin-eosin (H&E) and Masson's trichrome were used as histological evaluation indicators, platelet-endothelial cell adhesion molecule (CD31) and tumor necrosis factor-α (TNF-α) were used as immunohistochemical evaluation indicators, and α-smooth muscle actin (α-SMA) and myeloperoxidase (MPO) were used as immunofluorescence evaluation indicators. Cell nuclei were stained with DAPI fixative to comprehensively evaluate granulation tissue formation, collagen deposition, tissue angiogenesis, and tissue inflammation in the infected wound.

[0179] 3. Results

[0180] 3.1 Initial screening results of the concentration of Duyiwei gel in promoting infection healing

[0181] Table 17 Results of wound healing rate determination based on initial concentration screening

[0182]

[0183] Based on the observation results of wound healing during the experiment (Table 17 and...), Figure 13 No significant differences were observed among the gel concentration groups in promoting wound healing. Considering the properties and stability examined during the initial preparation of the gel, as well as the deposition of the gel on the wound during the experiment, the 10% Duyiwei gel group was ultimately selected for subsequent experiments promoting infection-induced wound healing.

[0184] 3.2 Comparison of the effects of Du Yi Wei gel and extract on promoting the healing of infected wounds

[0185] Table 18 Results of wound appearance healing rate comparison with extract.

[0186]

[0187] The wound healing rate was calculated and analyzed based on the experimental record charts. The results are shown in Table 18 and... Figure 14As shown, compared with the control group, the overall wound healing rate of the Duyiwei gel group was higher during the observation period, while the Duyiwei extract group only showed a higher healing rate on day 15. Compared with the Duyiwei extract group, the Duyiwei gel group had a higher healing rate on days 3 and 8, while the healing rate was comparable on day 15. This indicates that Duyiwei gel is more effective than Duyiwei extract in repairing infected wounds.

[0188] Table 19 Results of Granulation Tissue Interstitial Measurement

[0189]

[0190] Note: Compared with the control group, a P < 0.001; compared with the extract group, b P < 0.001

[0191] The wound refilling, granulation tissue formation, and epithelial reconstruction were assessed based on the HE staining results, as shown in Table 19 and... Figure 15 As shown in the analysis figure, there were significant differences between the Duyiwei gel group and the extract group and the control group during the healing process. On day 14, the gel group showed the smallest tissue gap, which was significantly different from the Duyiwei extract group. This phenomenon indicates that the Duyiwei gel group has a better effect on promoting wound healing than the control group and the extract group.

[0192] Table 20 Results of Collagen Deposition Measurement

[0193]

[0194] Note: Compared with the control group, a P < 0.05; compared with the extract group, b P < 0.001

[0195] Masson section staining was used to assess collagen deposition and tissue healing, as shown in Table 20 and... Figure 16 It is evident that, compared to the control group, both the Duyiwei gel group and the extract group exhibited higher collagen deposition capacity during the healing process, with the gel group showing significantly superior collagen deposition capacity compared to the extract group. This indicates that the Duyiwei gel group can significantly accelerate collagen deposition at the wound site.

[0196] 3.3 Study on the healing activity of Duyiwei gel in promoting infected wounds

[0197] Table 21 Results of wound appearance healing rate compared with positive control drug

[0198]

[0199] The wound healing rate was calculated and analyzed based on the experimental record charts. The results are shown in Table 21 and... Figure 17As shown, on days 3 and 15, compared with the control group, the wound healing rate of the treatment group was higher than that of the control group, and the healing-promoting effect of the treatment group was comparable on day 15.

[0200] Table 22 Results of Granulation Tissue Interstitial Space Measurement

[0201]

[0202] 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 Phellodendron amurense liquid group, c P < 0.05

[0203] The wound refilling, granulation tissue formation, and epithelial reconstitution were assessed based on the HE staining results, as shown in Table 22 and... Figure 18 It can be seen that there were significant differences between the treatment group and the control group on days 8 and 14. On day 14, the Duyiwei gel group showed the smallest interstitial space, which was better than the positive drug group.

[0204] Table 23 Results of Collagen Deposition Measurement

[0205]

[0206] 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 Phellodendron amurense liquid group, c P < 0.05

[0207] Masson staining was used to assess collagen deposition and tissue healing, according to Table 23 and Figure 19 The analysis showed that there were significant differences between the Duyiwei gel group, the fusidic acid cream group, and the compound berberine liquid group during the healing process, indicating that the Duyiwei gel group had a better ability to deposit collagen than the positive drug group.

[0208] Table 24 Results of CD31 Positive Expression Assay

[0209]

[0210] 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 Phellodendron amurense liquid group, c P < 0.05

[0211] CD31 is an intercellular adhesion molecule found on endothelial cells, used to assess angiogenesis and stability (Table 24 and...). Figure 20The results showed that, compared with the control group, the expression of CD31 in the healed skin of the treatment group was significantly increased, and the expression of CD31 in the Duyiwei gel group was comparable to that in the compound Huangbai liquid group. This phenomenon indicates that Duyiwei gel can effectively enhance the early vascularization of infected wounds.

[0212] Table 25 Results of TNF-α positive expression assay

[0213]

[0214] 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 Phellodendron amurense liquid group, c P < 0.05

[0215] Tumor necrosis factor-α (TNF-α) is a pro-inflammatory cytokine secreted by M1 macrophages and is used to assess inflammation during wound healing. (See Table 25 and...) Figure 21 It was observed that, compared with the control group, the drug-treated groups showed varying degrees of reduction in TNF-α on days 3, 8, and 14. Furthermore, on day 14, the effect of the Duyiwei gel group was comparable to that of the two positive control groups, indicating that the Duyiwei gel group has a certain effect on reducing inflammation. Combined with the expression levels of CD31 and TNF-α, it can be concluded that the Duyiwei gel, fusidic acid cream, and compound berberine extract all possess the ability to promote angiogenesis and reduce inflammatory responses, and the gel group showed comparable effects to the positive control group.

[0216] Table 26 Results of α-SMA Positive Expression Assay

[0217]

[0218] 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 Phellodendron amurense liquid group, c P < 0.05

[0219] The detection of α-smooth muscle actin (α-SMA) is mainly used to label smooth muscle myofibroblasts: as shown in Table 26 and Figure 22 As shown, the expression of α-SMA in the treatment group was higher than that in the control group, indicating that there was more angiogenesis in the treatment group. Furthermore, on day 14, there was no significant difference in α-SMA expression between the gel group and the fusidic acid cream group, indicating that Duyiwei gel has the ability to enhance α-SMA expression and promote angiogenesis.

[0220] Table 27 Results of MPO Positive Expression Assay

[0221]

[0222] Note: Compared with the control group, a P < 0.05; compared with the fusidic acid cream group, b P < 0.05

[0223] Myeloperoxidase (MPO) is a marker of systemic inflammation, and MPO is mainly used to detect wound inflammation: Analysis results are shown in Table 27. Figure 23 The results showed that the MPO level in the control group was significantly higher than that in the treatment group, while both the Duyiwei gel group and the compound Huangbai liquid group showed lower MPO levels, indicating that the Duyiwei gel group had an anti-inflammatory effect comparable to that of the compound Huangbai liquid group.

[0224] Based on the combined results of α-SMA and MPO tests, the Duyiwei gel group and the positive drug group not only have stronger angiogenesis-promoting capabilities, but also significantly reduce inflammatory responses.

[0225] The above results indicate that Duyiwei gel can promote the healing of infected wounds, and its overall healing effect is better than that of Duyiwei extract, as well as better than fusidic acid cream and compound berberine liquid, which are currently widely used in clinical practice.

[0226] In summary, the *Duyiwei* gel of this invention has a high drug loading capacity, good stability and fluidity. Its efficacy in repairing acute wounds is comparable to that of *Duyiwei* extract at the same dosage. In repairing infected wounds, its efficacy is significantly better than that of *Duyiwei* extract at the same dosage, and it is also superior to fusidic acid cream and compound berberine liquid commonly used in clinical practice. It has practical application value.

Claims

1. A DUEXIS® gel for repairing an infected wound, characterized in that, It is prepared by using the following ingredients: 2.5 parts by weight of extract of Dui Yiyi, 0.875 parts by weight of carbomer 940, 0.025 parts by weight of hydroxyethyl benzoate, 0.75 parts by volume of triethanolamine, 1.875 parts by volume of glycerol, and 1.875 parts by volume of 1,2-propanediol; The preparation method of the extract of Dui Yiyi is as follows: the Dui Yiyi decoction pieces are crushed, and then boiled with water for three times, each time for 1 hour. The decoction is combined, filtered, concentrated, dried below 80℃, and then crushed to obtain the extract. 1g of the extract of Dui Yiyi is equivalent to 3-5g of Dui Yiyi; The preparation method of the gel of Dui Yiyi includes the following steps: 1) Weigh the ingredients according to the ratio, dissolve carbomer 940 in water to obtain a 3.5% carbomer 940 solution, and dissolve hydroxyethyl benzoate in ethanol to obtain a 3.3% hydroxyethyl benzoate solution; 2) Take the carbomer 940 solution obtained in step 1), swell and defoam at 2-8℃, then mix triethanolamine, glycerol, 1,2-propanediol, and the hydroxyethyl benzoate solution obtained in step 1), and finally add the extract of Dui Yiyi and stir until uniform.

2. The use of the gel of Dui Yiyi in claim 1 in the preparation of a medicine for repairing infected wounds.

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