Use of a garlic bacteriostatic paste in the treatment of diabetic ulcers

Abstract

This invention provides the use of garlic and radish antibacterial ointment in the treatment of diabetic ulcers. Specifically, the garlic and radish antibacterial ointment has the effects of reducing ulcer area, improving wound healing rate, improving wound morphology, inhibiting tissue necrosis / promoting the shedding of necrotic tissue, inhibiting tissue exudation, promoting granulation / collagen tissue formation, and inhibiting inflammation, significantly improving the pathological characteristics of diabetic ulcers and exhibiting excellent therapeutic effects.

Description

Technical Field

[0001] This invention belongs to the technical field, and more specifically, this invention relates to the use of a garlic and scallion antibacterial ointment in the treatment of diabetic ulcers. Background Technology

[0002] Diabetes mellitus, a chronic metabolic disease characterized by hyperglycemia, is experiencing an increasing incidence rate and a trend towards younger onset. According to a survey by the International Diabetes Federation, there were approximately 465 million people with diabetes worldwide in 2020, and this number is projected to increase to 693 million by 2045. About 15% of these patients will develop diabetic ulcers (DU). DU is a difficult-to-heal ulcer caused by infection resulting from long-term hyperglycemia leading to peripheral vascular and / or neuropathy. It is one of the most serious and challenging chronic complications of diabetes, especially diabetic foot ulcers, which are a major cause of disability and death among diabetic patients, resulting in persistently high amputation and mortality rates.

[0003] Currently, treatment methods for diabetic duodenitis (DU) mainly include blood sugar control, anti-infection therapy, topical dressings, negative pressure therapy, surgical debridement, skin grafting, and bioengineered skin. However, these treatments are time-consuming and costly. The enormous medical expenses and limited treatment options severely impact patients' physical and mental health and impose a significant economic burden on society. Furthermore, there are few drugs available for treating DU on the market, making it difficult to meet the huge market demand.

[0004] Therefore, there is an urgent need in this field to develop DU treatment drugs that have clearly defined targets, are easy to use, and combine efficacy and safety. Summary of the Invention

[0005] The purpose of this invention is to provide a drug for treating diabetic ulcers.

[0006] In a first aspect of the invention, there is provided the use of a garlic antibacterial ointment for preparing a medicine, said medicine being used for purposes selected from the group consisting of: 1) Treatment of diabetic ulcers / Improvement of the pathological characteristics of diabetic ulcers; 2) Reduce the ulcer area; 3) Improve wound healing rate; 4) Improve wound morphology; 5) Inhibits tissue necrosis / promotes the shedding of necrotic tissue; 6) Inhibits tissue exudation; 7) Promotes the formation of granulation / collagen tissue; 8) Inhibits inflammation; The garlic antibacterial ointment contains the following ingredients: garlic powder, white petrolatum, glyceryl stearate, liquid paraffin, and peppermint oil.

[0007] In another preferred embodiment, the drug is a topical preparation.

[0008] In another preferred embodiment, the drug is an ointment.

[0009] In another preferred embodiment, the drug is used in the early stage of diabetes, the ulcer stage, and / or the stage complicated by infection.

[0010] In another preferred embodiment, the drug is a pharmaceutical composition comprising: 1) Garlic and garlic antibacterial ointment; and 2) Pharmaceutically acceptable carrier.

[0011] In another preferred embodiment, the inhibition of inflammation includes inhibiting the infiltration of inflammatory cells.

[0012] In another preferred embodiment, the inflammatory cells include macrophages and neutrophils.

[0013] In another preferred embodiment, the inhibition of inflammation includes inhibiting the infiltration of M1 macrophages.

[0014] In another preferred embodiment, the inhibition of inflammation includes increasing M2 macrophage infiltration.

[0015] In another preferred embodiment, the garlic antibacterial ointment comprises the following components (parts by weight): Tube A: Garlic powder 10-30%, white petrolatum 60-80%, light liquid paraffin 5-15%, white beeswax 3-8%, lanolin 0.5-5%, paraffin 0.5-5%, menthol 0.1-0.5%; Tube B: Glycerin 5-15%, Carbomer 0.5-1.5%, Triethanolamine 0.5-1.5%, Sodium Bisulfite 0.02-0.06%, Benzalkonium Bromide 0.01-0.05%.

[0016] In another preferred embodiment, the garlic antibacterial ointment comprises: Tube A: Garlic powder 10-20%, white petrolatum 70-80%, light liquid paraffin 5-10%, white beeswax 3-6%, lanolin 1-3%, paraffin 1-3%, menthol 0.1-0.3%; Tube B: Glycerin 5-10%, Carbomer 0.5-1.0%, Triethanolamine 0.5-1.0%, Sodium Bisulfite 0.02-0.06%, Benzalkonium Bromide 0.01-0.03%.

[0017] In another preferred embodiment, the garlic antibacterial ointment further includes preservatives and water.

[0018] In another preferred embodiment, the method of using the garlic antibacterial ointment is as follows: apply equal amounts of both tube A and tube B evenly to the affected area.

[0019] In a second aspect of the invention, a method for treating diabetic ulcers is provided, the method comprising administering to a subject a therapeutically effective amount of garlic antibacterial ointment as defined in the first aspect of the invention.

[0020] In another preferred embodiment, the effective therapeutic dose is determined based on the condition of the subject.

[0021] In a third aspect of the invention, a kit is provided comprising garlic antibacterial ointment as defined in the first aspect of the invention.

[0022] In another preferred embodiment, the kit also includes instructions for use.

[0023] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0024] Figure 1 The appearance of wound healing in diabetic ulcer rats after intervention with garlic freeze-dried powder is shown.

[0025] Figure 2 The size and healing rate of diabetic ulcers in rats were shown. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. vs. model group. n = 6.

[0026] Figure 3 The scores for diabetic ulcer wounds in rats are shown. A: Wound edge score; B: Necrotic tissue type score; C: Exudate type score; D: Exudate quantity score; E: Granulation tissue score; F: Total wound healing score. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. vs. model group. n = 6.

[0027] Figure 4 HE staining results of diabetic rat ulcer wounds are shown.

[0028] Figure 5 Masson staining results are shown for diabetic rat ulcer wounds.

[0029] Figure 6The following data are presented: (A) Inflammatory infiltration of iNOS M1 macrophages in rat wound skin; (B) Inflammatory infiltration of CD206 M2 macrophages; (C) Inflammatory infiltration of MPO+ neutrophils; (D) Percentage of infiltrated area by iNOS M1 macrophages; (E) Percentage of infiltrated area by CD206 M2 macrophages; (F) Percentage of infiltrated area by MPO+ neutrophils. Detailed Implementation

[0030] Through extensive and in-depth research, the inventors have developed a garlic-based antibacterial ointment for the treatment of diabetic ulcers. This ointment is primarily composed of garlic powder, with added excipients such as white petrolatum, glyceryl stearate, liquid paraffin, and peppermint oil to form a paste. Experiments have demonstrated that this garlic-based antibacterial ointment can reduce ulcer area in diabetic mice, improve wound healing rate, improve wound morphology, inhibit tissue necrosis / promote necrotic tissue shedding, inhibit tissue exudation, promote granulation / collagen tissue formation, and inhibit inflammation, significantly improving the pathological characteristics of diabetic ulcers and exhibiting excellent therapeutic effects. This invention is based on these findings.

[0031] the term To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing this invention, it should be understood that it is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied.

[0032] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0033] diabetic ulcer Diabetic ulcers are a serious chronic complication of diabetes, most commonly occurring in the feet (diabetic foot), but can also occur in other parts of the lower limbs. They are primarily caused by poor long-term blood sugar control in diabetic patients, leading to neuropathy and vascular disease, resulting in wounds that are difficult to heal after skin damage.

[0034] Diabetic ulcers are usually the result of multiple factors working together: Neuropathy: Long-term high blood sugar can damage nerves (especially sensory nerves and autonomic nerves). Loss of sensation: The patient's sensation of pain, temperature, and pressure is reduced or absent. Even if there are foreign objects (such as small stones) in the shoes or the shoes are too tight and rub the feet raw, the patient will not feel pain, causing ulcers to occur without their knowledge. Autonomic neuropathy: Reduced sweating, leading to dry, cracked skin that is more susceptible to bacterial invasion; Vascular lesions: High blood sugar leads to arteriosclerosis, and narrowing or occlusion of blood vessels in the lower limbs; Insufficient blood supply: Reduced blood supply to the lower limbs leads to tissue hypoxia and malnutrition. Once a wound occurs, the lack of sufficient blood to bring the oxygen and nutrients needed for repair makes the wound extremely difficult to heal. Infection: A high blood sugar environment is conducive to bacterial growth. Coupled with a weakened immune system, even minor wounds can easily develop into serious infections that are difficult to control.

[0035] Symptoms of diabetic ulcers vary depending on their severity and pathological type, and can generally be divided into early signs, ulcer stage, and severe complication stage.

[0036] 1) Early signs (symptoms of high-risk foot) Before the skin breaks down, patients may experience the following symptoms: Abnormal sensation: numbness, burning, tingling, or a feeling of walking on cotton (reduced sensation) in the distal extremities (toes, soles of feet).

[0037] Foot deformities: Due to neuropathy leading to muscle atrophy, the arch of the foot may collapse, toes curling into claw-like shapes (claw toes), and metatarsal heads may protrude.

[0038] Dry skin: Skin that is dry and cracked without sweating, and may appear purplish or pale.

[0039] 2) Ulcer stage When the skin is broken, it will exhibit typical ulcer characteristics: It usually occurs at pressure points on the sole of the foot (such as the front of the foot, heel), between the toes, or in areas where shoes rub against the skin. Wound morphology: Surrounded by calluses: Due to abnormal pressure distribution caused by neuropathy, a thick, hard callus often forms around the ulcer. After removing the callus, a deep, pitted ulcer may be revealed underneath.

[0040] Clear boundaries: The boundaries of ulcers are mostly round or irregular in shape.

[0041] Base color: Depending on the blood supply, the base of the wound may appear red (granulation tissue), yellow (tendon, fat) or black (necrotic fascia, bone).

[0042] Discharge: Ulcers often have exudate. If infection occurs, there will be purulent discharge with a foul odor.

[0043] 3) Co-infection (complication) Redness, swelling, heat, and pain: Although patients with neuropathy may not feel pain, in severe infections, the surrounding soft tissues will show obvious redness, swelling, and heat. It is important to note that some patients may not experience significant pain even if the infection is deep due to severe neuropathy, which can easily lead to delays in diagnosis and treatment.

[0044] Systemic symptoms: When the infection spreads severely, patients may experience fever, chills, general weakness, and a sharp rise in blood sugar.

[0045] Gangrene: tissue necrosis caused by blockage of blood vessels.

[0046] Dry gangrene: often seen in arterial occlusion, where the toes or feet become dry and black, resembling a mummy.

[0047] Wet gangrene: It is often seen in infections complicated by vascular lesions. It presents with local swelling, ulceration, foul odor, and a large amount of pus. The condition progresses rapidly and is life-threatening.

[0048] Due to the complexity of the condition, treatment usually requires multiple collaborations: such as controlling blood sugar, debridement, anti-infection, improving blood supply, decompression, and wound dressing.

[0049] This invention demonstrates, through an in vivo animal model of diabetic ulcer rats, that the garlic antibacterial ointment has a significant effect on accelerating the healing of chronic diabetic ulcer wounds.

[0050] Garlic Antibacterial Paste The garlic antibacterial agent of this invention includes tube A and tube B. Equal amounts of tube A and tube B are mixed and then applied to the affected area.

[0051] The main components of garlic powder are alliin and alliinase. In the presence of water, alliinase rapidly catalyzes the cleavage of alliin to produce the active ingredient allicin. Allicin is unstable due to the presence of allyl and sulfoxide groups in its structure. Based on the unique properties of garlic powder, this product is designed as two different dosage forms for combined use. Tube A is an ointment made from garlic powder and an oily matrix. Tube A is anhydrous, allowing the garlic powder to exist in its original form and remain stable. The other tube, Tube B, contains no active ingredient and has a water content as high as 90%. When used together, the gelling agent provides sufficient moisture for the reaction of alliin and alliinase in the garlic powder, enabling the in-situ production of allicin and its active ingredient.

[0052] Tube A: An ointment containing garlic powder, composed of the following components by weight percentage: Garlic powder 10-30%, white petrolatum 60-80%, light liquid paraffin 5-15%, white beeswax 3-8%, lanolin 0.5-5%, paraffin 0.5-5%, menthol 0.1-0.5%, and appropriate amount of preservatives.

[0053] Preferably, the ingredients are 10-20% garlic powder, 70-80% white petrolatum, 5-10% light liquid paraffin, 3-6% white beeswax, 1-3% lanolin, 1-3% paraffin, 0.1-0.3% menthol, and an appropriate amount of preservatives.

[0054] Preferably, the ingredients are 10% garlic powder, 73% white petrolatum, 7.0% light liquid paraffin, 5.5% white beeswax, 2.0% lanolin, 2.0% paraffin, 0.2% menthol, and an appropriate amount of preservatives.

[0055] Garlic powder 11%, white petrolatum 72%, light liquid paraffin 7.0%, white beeswax 5.5%, lanolin 2.0%, paraffin 2.0%, menthol 0.2%, and appropriate amount of preservatives.

[0056] Garlic powder 12%, white petrolatum 72%, light liquid paraffin 6.0%, white beeswax 5.5%, lanolin 2.0%, paraffin 2.0%, menthol 0.2%, and appropriate amount of preservatives.

[0057] Garlic powder 13%, white petrolatum 71%, light liquid paraffin 6.0%, white beeswax 5.5%, lanolin 2.0%, paraffin 2.0%, menthol 0.2%, and appropriate amount of preservatives.

[0058] Garlic powder 14%, white petrolatum 71%, light liquid paraffin 6.0%, white beeswax 4.5%, lanolin 2.0%, paraffin 2.0%, menthol 0.2%, and appropriate amount of preservatives.

[0059] Preferably, the ingredients are 15% garlic powder, 70% white petrolatum, 6% light liquid paraffin, 4% white beeswax, 2.0% lanolin, 2.0% paraffin, and an appropriate amount of preservative.

[0060] B tube: An additional solvent, composed of the following components by mass percentage: The ingredients are 5-15% glycerol, 0.5-1.5% carbomer, 0.5-1.5% triethanolamine, 0.02-0.06% sodium bisulfite, 0.01-0.05% benzalkonium bromide, and the balance is purified water.

[0061] Preferably, the composition is 5-10% glycerol, 0.5-1.0% carbomer, 0.5-1.0% triethanolamine, 0.02-0.06% sodium bisulfite, 0.01-0.03% benzalkonium bromide, and the balance being purified water.

[0062] Preferably, the composition is 7.0% glycerol, 0.8% carbomer, 0.8% triethanolamine, 0.02% sodium bisulfite, 0.02% benzalkonium bromide, and the balance being purified water.

[0063] Preferably, the composition is 8.0% glycerol, 0.8% carbomer, 0.8% triethanolamine, 0.02% sodium bisulfite, 0.02% benzalkonium bromide, and the balance being purified water.

[0064] Preferably, the composition is 9.0% glycerol, 0.9% carbomer, 0.8% triethanolamine, 0.03% sodium bisulfite, 0.02% benzalkonium bromide, and the remainder is purified water.

[0065] Preferably, the composition is 10.0% glycerol, 0.9% carbomer, 0.8% triethanolamine, 0.04% sodium bisulfite, 0.02% benzalkonium bromide, and the balance being purified water.

[0066] Preferably, the composition is 10.0% glycerol, 1.0% carbomer, 0.9% triethanolamine, 0.04% sodium bisulfite, 0.02% benzalkonium bromide, and the remainder is purified water.

[0067] In this invention, a suitable formula is selected for administration based on the symptoms of the disease.

[0068] Pharmaceutical Composition The present invention also provides a pharmaceutical composition. In a preferred embodiment, the composition comprises a garlic antibacterial ointment and a pharmaceutically acceptable carrier.

[0069] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to) skin or topical administration. Preferably, the pharmaceutical composition is applied topically to the skin and used in combination in tubes A and B. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably an ointment, tablet, capsule, granule, injection, or infusion. Preferably, the garlic antibacterial ointment of the present invention is a topical preparation.

[0070] Pharmaceutically acceptable carriers include, but are not limited to, saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. Preferably, the pharmaceutical composition of the present invention further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents.

[0071] Preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by employing experiments not exceeding the norm. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are within the scope of a skilled physician's expertise.

[0072] Compared with the prior art, the main advantages of the present invention include: 1. The garlic antibacterial ointment of the present invention significantly improves the pathological characteristics of diabetic ulcers, such as reducing ulcer area, increasing wound healing rate, improving wound morphology, inhibiting tissue necrosis / promoting necrotic tissue shedding, inhibiting tissue exudation, promoting granulation / collagen tissue formation, and inhibiting inflammation.

[0073] 2. The garlic antibacterial ointment of the present invention is simple to prepare, convenient to use, and inexpensive, making it suitable for long-term treatment.

[0074] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0075] Experimental materials Streptozotocin (STZ), sodium citrate buffer, sodium pentobarbital, physiological saline, freeze-dried garlic powder, SD rats Experimental methods 1. Establishment of a rat model of diabetic ulcers Seventy rats were randomly divided into a normal control group (n=10) and a diabetic group (n=60). After one week of acclimatization, the rats were fasted but allowed free access to water for 12 hours. On the day of model establishment, the rats were weighed and their blood glucose levels were measured via tail vein. Then, they were injected intraperitoneally with STZ buffer at a dose of 50 mg / kg. On days 3, 7, and 14 after the intraperitoneal injection, random blood glucose levels were measured via tail vein. A blood glucose level greater than 16.7 mol / L on three separate occasions was considered a successful establishment of the diabetic rat model. The rats continued to be fed, with the normal control group fed a standard diet and the diabetic ulcer model group and the drug-treated group fed a high-sugar, high-fat diet. Fourteen days later, all rats were anesthetized by intraperitoneal injection of sodium pentobarbital at 45 mg / kg. After hair removal, the posterior cervical spinal region was fully exposed. A 2 cm diameter circular biopsy punch was used to create a uniform ulcer model area, reaching the subcutaneous fascia, thus establishing an open, full-thickness skin ulcer rat model.

[0076] 2. Grouping and Dosing The ointment is made primarily from garlic powder, with the addition of excipients such as white petrolatum, glyceryl stearate, liquid paraffin, and peppermint oil. The low-dose ointment contains 1.91 mg / g of freeze-dried garlic powder (based on alliin content, 1g of freeze-dried powder contains 0.3% alliin); the medium-dose group contains 3.4 mg / g of freeze-dried garlic powder (based on alliin content); and the high-dose group contains 4.75 mg / g of freeze-dried garlic powder (based on alliin content).

[0077] Experimental mice were randomly divided into four groups: a blank control group (Control), a model group (Model), a positive control group (F-Fespixon), a low-dose administration group (L-Allicin), a medium-dose administration group (M-Allicin), a high-dose administration group (H-Allicin), and a matrix group (Vehicle), with six rats in each group. Wound dressings were changed starting on the second day after dorsal skin modeling, and the wounds in all groups were rinsed with physiological saline.

[0078] The positive control group received Xiangleitang Foot Cream, a topical product used to help improve diabetic foot skin problems. It mainly helps prevent infection and repair damaged skin through antibacterial, healing-promoting, and inflammation-relieving effects. It is China's first new drug for diabetic foot wounds and ulcers.

[0079] The matrix group is formulated into ointments (oil solutions, emulsions, ointments / oil pastes, liposomes, microspheres, solid lipid nanoparticles, nanostructured lipid carriers, microemulsions / nanoemulsions, self-emulsions, micelles) using excipients such as white petrolatum without garlic freeze-dried powder, glyceryl stearate, liquid paraffin, and peppermint oil.

[0080] The low-dose group received topical application of 1.91 mg / g of lyophilized garlic powder (based on alliin content, 1g of lyophilized powder contains 0.3% alliin); the medium-dose group received topical application of 3.4 mg / g of lyophilized garlic powder; and the high-dose group received topical application of 4.75 mg / g of lyophilized garlic powder. The normal control group and the model group received no medication. All groups were bandaged with sterile gauze and secured with medical tape. The dressings were changed once daily.

[0081] 3. Clinical assessment (1) Starting from D1, take photos of the wounds of diabetic ulcer rats every day. When taking photos, place a ruler on the edge of the ulcer for comparison.

[0082] (2) Each photo was analyzed using image analysis software (ImageJ) to quantitatively measure the ulcer area. The degree of ulcer healing was calculated as follows: Ulcer healing degree = (A1-An) / A1*100%, where A1 is the original ulcer area on day D1 and An is the unhealed ulcer area on each day after day Dn.

[0083] 4. Grading standards for the severity and healing status of diabetic ulcers Table 1. Scoring criteria for wound healing 5. Paraffin-embedded sections of skin tissue Rat skin tissue was fixed in 4% paraformaldehyde for at least 24 hours. The tissue was then removed from the fixative and sequentially dehydrated in 70%, 80%, 90%, 95%, anhydrous ethanol I, and anhydrous ethanol II. Afterward, the tissue was cleared in xylene. The cleared tissue was immediately placed in pre-melted paraffin wax and embedded in a groove, where the wax solidified into a rectangular block. Sections were then prepared using a microtome, typically to a thickness of 5-7 μm. The slides were set to a suitable temperature and floated on warm water to flatten the tissue. The slides were then lifted using a glass slide and placed in a 60°C oven to bake until the water dried and the wax melted. Finally, they were placed in a 37°C incubator for staining.

[0084] 6. Hematoxylin-eosin (H&E) staining Paraffin-embedded tissue sections were sequentially dewaxed and hydrated using xylene I, xylene II, anhydrous ethanol I, anhydrous ethanol II, 95% ethanol, 90% ethanol, 80% ethanol, 70% ethanol, and distilled water. After staining with hematoxylin, the sections were washed with water to remove excess dye, then immersed in hydrochloric acid-alcohol differentiation solution until the sections turned pale pink. They were immediately rinsed with running water and then immersed in ammonia water for inversion until the sections turned blue. Finally, they were stained with eosin. The stained sections were then sequentially dehydrated and cleared using 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, anhydrous ethanol I, anhydrous ethanol II, xylene I, and xylene II. Finally, they were mounted with neutral resin, avoiding air bubbles. The stained sections were then observed under a microscope for histopathological changes in each group of rats.

[0085] 7. Masson's trichrome staining Fixed tissue specimens were routinely dehydrated, cleared, and embedded in paraffin. Serial sections (approximately 4 μm thick) were prepared using a rotary microtome and dried at 60°C for 1-2 hours. Sections were dewaxed sequentially with xylene (twice, 5-10 min each time), rehydrated with a gradient of ethanol (100%, 95%, 85%, 75%), and then rinsed with distilled water. Nuclear staining was then performed (Weigert iron hematoxylin, approximately 5-10 min), followed by thorough rinsing with running water and appropriate differentiation with a weakly acidic solution, then rinsing again. Next, cytoplasm and muscle fibers were stained with acid fuchsin / Pounosulfan (approximately 5-10 min), followed by rinsing with distilled water. The cells were then differentiated in phosphomolybdic acid / phosphotungstic acid solution (approximately 5-10 min) to improve the contrast between collagen and muscle fibers. Finally, collagen fibers were counterstained with aniline blue / brilliant green (depending on the reagent system) (approximately 3-10 min), followed by brief differentiation with a weakly acidic solution (approximately 0.5-2 min), and rinsed with distilled water. Finally, the slides were rapidly dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin. Microscopic observation and image acquisition revealed that collagen fibers appeared blue (or green, depending on the counterstaining system), myofibrils / cytoplasm appeared red, and cell nuclei appeared bluish-black. For quantitative analysis, ImageJ was used to measure the collagen area percentage, and statistical analysis was performed under the same magnification and exposure conditions.

[0086] 8. Immunohistochemistry (IHC) After dewaxing and hydration, paraffin tissue sections were placed in boiling 10 mmol / L Tris-EDTA (pH 9.0) antigen retrieval solution and boiled for 20 minutes. After natural cooling, the sections were washed twice with PBST. The sections were then soaked in 3% H2O2 at room temperature in the dark to remove endogenous peroxidase. After wiping away moisture around the tissue with filter paper, the tissue was circled with an immunohistochemical pen (the circle should be closed at both ends and not drawn on the tissue itself). Blocking solution was carefully added to cover the tissue, and the reaction was allowed to proceed at room temperature for 30 minutes. The blocking solution was discarded, and the sections were incubated with primary antibody at 4°C overnight. After washing, the sections were incubated with secondary antibody at room temperature for 30 minutes. Fresh DAB chromogenic solution was added in the dark. When a yellow-brown positive area appeared under the microscope, the sections were rinsed with running water. The sections were counterstained sequentially with hematoxylin, differentiation solution, and blue solution. Dehydration and clearing were performed using graded ethanol and xylene. The sections were mounted with neutral resin, avoiding air bubbles. The sections were observed under an optical microscope, photographed, and the positive rate was calculated.

[0087] Example 1. Effect of garlic and scallion antibacterial ointment on ulcer area in diabetic rats. like Figure 1 As shown, the changes in wound area at five time points (days 1, 3, 6, 9, and 12) for different treatment groups are illustrated. Each group consisted of a prototype skin defect of the same size. Over time, the wound area, color, moisture, and tissue structure changed to varying degrees. Wound area (Table 2) and healing rate (Table 3) were calculated and plotted as line graphs. Figure 2 ).

[0088] In the blank control group, without modeling treatment, wound healing was the fastest, with neat wound edges and mild inflammation, reaching near-complete healing by day 12. The model group recovered the slowest, characterized by persistent necrotic tissue and exudate, resulting in slow healing. The low-dose and medium-dose groups initially showed some inflammation and exudate, but the wounds gradually shrank over time, with significantly improved healing compared to the model group, although the overall efficacy was relatively weaker. The high-dose group exhibited a significant coarse healing effect, with the wound rapidly drying and shrinking after day six, nearing healing by day 12, with treatment efficacy comparable to the positive control group.

[0089] Table 2. Wound area of ​​diabetic ulcer rats Table 3. Wound healing rate in diabetic ulcer rats Example 2. Wound appearance scoring in diabetic rats with lyophilized garlic powder. The wound edge morphology, necrotic tissue type, exudate type, and exudate quantity of each group of rats were assessed according to the wound healing scoring criteria, and line graphs were plotted. Figure 3 AF).

[0090] The scoring of wound edge morphology (Table 4) showed that the model group generally maintained a score above 4 in the first 3 days, characterized by significantly curled and thickened wound edges, a firm texture, and a resilient feel, indicating significant inflammatory hyperplasia at the wound edges. In contrast, the scores of the medium-dose and high-dose garlic freeze-dried powder treatment groups were significantly lower than those of the model group, with good wound edge adhesion, flush with the wound base or with relatively smooth edges, suggesting that garlic freeze-dried powder intervention has a positive effect on the repair of wound edge tissue.

[0091] The scoring of necrotic tissue types (Table 5) showed that the model group's scores remained mostly in the range of 3-4 points from day 1 to day 5. The main characteristic was the presence of yellow necrotic tissue or attached black eschar on the wound surface, indicating significant local tissue necrosis, possibly accompanied by an inflammatory response. In contrast, the scores of the positive control group and each dose group of garlic freeze-dried powder decreased more rapidly, especially the high-dose group, whose score dropped below 2 points after day 6, indicating that it had a stronger ability to promote the shedding of necrotic tissue and the growth of new tissue.

[0092] The evaluation of exudate type (Table 6) showed that the model group had a higher score at the beginning of the experiment, with exudate that was viscous, purulent, or mixed with necrotic tissue debris, and some of it was green or had an odor, reflecting a more severe wound infection. In the treatment group, the exudate type scores of the medium-dose and high-dose groups decreased rapidly, approaching the level of 2-3 points after day 5, and the exudate gradually became clear and watery, indicating good local infection control and reduced inflammatory response.

[0093] The scoring of exudate volume (Table 7) showed that the model group wound maintained an exudate volume score above 4 points for the first 5 days, indicating that the wound was in a state of high exudation and significant tissue edema. However, as the treatment progressed, the scores of the high-dose garlic freeze-dried powder group and the positive control group significantly decreased to 2-3 points, reflecting a reduction in exudate volume and moderate wound moisture, which is conducive to the formation of granulation tissue and wound healing.

[0094] Table 8 shows that the scores of granulation tissue in rat wounds gradually decreased over time in all groups. Compared with the normal control group, the score in the model group decreased more slowly, indicating impaired granulation tissue repair in diabetic wounds. Compared with the model group, the granulation tissue scores in each dose group of the garlic antibacterial ointment gradually decreased after administration, with the high-dose group showing the largest decrease, followed by the medium-dose and low-dose groups. This suggests that the drug can promote the formation and repair of granulation tissue in diabetic wounds and has a certain dose-dependent effect.

[0095] In summary, the overall score of the model group was statistically different from that of the blank control group, indicating that the diabetic ulcer model was successfully established. The scores and wound healing rates of each dose group of the garlic freeze-dried powder were statistically different from those of the model group, indicating that the garlic freeze-dried powder has a significant therapeutic effect in promoting wound healing and exhibits a clear dose-dependent effect. The high-dose group was superior to the medium- and low-dose groups in improving wound edge morphology, reducing necrotic tissue, controlling exudation, and promoting granulation tissue formation, suggesting its promising application in wound repair.

[0096] Table 4. Wound edge scoring in rats Table 5. Scoring of Necrotic Tissue Type in Rat Wounds Table 6. Scoring of wound exudate type in rats Table 7. Scoring of wound exudate quantity in rats Table 8. Rat Wound Granulation Tissue Scoring Example 3. Garlic freeze-dried powder intervention reduces inflammatory response in diabetic ulcer rats. HE staining analysis of skin tissue from rat lesions ( Figure 4 In the control group, the skin tissue structure was intact, the epidermis was continuous, the stratum corneum was intact, and the dermal collagen fibers were densely and orderly arranged, with no obvious inflammatory cell infiltration. Compared with the control group, the epidermis at the wound edge of the model group was obviously interrupted, the wound surface was covered with a thicker scab and necrotic tissue, and there was a large area of ​​tissue loss in the dermis and subcutaneous tissue. The growth of new epithelium at the wound edge towards the center of the wound was limited, and the repair of the ulcer wound was significantly delayed.

[0097] Compared with the model group, the garlic freeze-dried powder intervention group showed a significant reduction in necrotic tissue and inflammatory cell infiltration, with relatively continuous new epithelial growth covering the wound edges. The high-dose garlic freeze-dried powder group showed the most significant improvement, with only a small number of scattered inflammatory cells remaining.

[0098] In summary, the results show that treatment with freeze-dried garlic powder, especially at high doses, can significantly reduce the local inflammatory response in ulcerated lesions of diabetic rats.

[0099] Example 4. Garlic freeze-dried powder intervention increases collagen tissue formation in diabetic ulcer rat wounds. Masson staining of rat ulcer wounds ( Figure 5In the control group, collagen deposition and tissue structure remained normal, and collagen production and deposition were at a natural healing level. Compared with the normal control group, the collagen volume fraction in the model group was significantly reduced. Compared with the model group, the collagen volume fraction in the garlic freeze-dried powder group showed a significantly higher change; especially the high-dose garlic freeze-dried powder group showed a significantly increased collagen volume fraction compared with the model group, indicating a significant increase in collagen production.

[0100] Example 5. Garlic freeze-dried powder intervention improves the degree of immune cell infiltration in the wound of diabetic ulcer rats. The extensive infiltration of immune cells, such as macrophages and neutrophils, into skin tissue is a hallmark of the inflammatory phase of a wound. Immunohistochemistry was used to detect neutrophil surface markers MPO, M1 macrophage surface marker iNOS, and M2 macrophage surface marker CD206 to assess the extent of immune cell infiltration. Figure 6 (AF). In the model group, neutrophil infiltration was significant, macrophages showed a predominantly pro-inflammatory phenotype and insufficient reparative phenotype, and the wound was in a state of "high inflammation". Compared with the model group, the garlic freeze-dried powder group significantly inhibited inflammatory infiltration and macrophage pro-inflammatory polarization, while enhancing the reparative immune response.

[0101] in conclusion This study confirms that treatment of diabetic ulcer wounds with freeze-dried garlic powder, especially at high doses, can significantly improve the healing rate of diabetic ulcers, reduce the ulcer area, alleviate local inflammation, and promote collagen production, similar to the effects of the positive control drug Xiangleitang Foot Ointment, demonstrating the potential pharmacological value of freeze-dried garlic powder in the treatment of diabetic ulcers.

[0102] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. The use of a garlic antibacterial ointment, characterized in that, For the preparation of a drug, said drug being used for purposes selected from the group consisting of: 1) Treatment of diabetic ulcers / Improvement of the pathological characteristics of diabetic ulcers; 2) Reduce the ulcer area; 3) Improve wound healing rate; 4) Improve wound morphology; 5) Inhibits tissue necrosis / promotes the shedding of necrotic tissue; 6) Inhibits tissue exudation; 7) Promotes the formation of granulation / collagen tissue; 8) Inhibits inflammation; The garlic antibacterial ointment contains the following ingredients: garlic powder, white petrolatum, glyceryl stearate, liquid paraffin, and peppermint oil.

2. The use as described in claim 1, characterized in that, The drug is a topical preparation.

3. The use as described in claim 1, characterized in that, The inhibition of inflammation includes inhibiting the infiltration of inflammatory cells.

4. The use as described in claim 1, characterized in that, The inflammatory cells include macrophages and neutrophils.

5. The use as described in claim 1, characterized in that, The inhibition of inflammation includes inhibiting the infiltration of M1 macrophages.

6. The use as described in claim 1, characterized in that, The inflammation suppression includes increasing M2 macrophage infiltration.

7. The use as described in claim 1, characterized in that, The garlic antibacterial ointment comprises the following components (parts by weight): Tube A: Garlic powder 10-30%, white petrolatum 60-80%, light liquid paraffin 5-15%, white beeswax 3-8%, lanolin 0.5-5%, paraffin 0.5-5%, menthol 0.1-0.5%; and Tube B: Glycerin 5-15%, Carbomer 0.5-1.5%, Triethanolamine 0.5-1.5%, Sodium Bisulfite 0.02-0.06%, Benzalkonium Bromide 0.01-0.05%.

8. The use as described in claim 1, characterized in that, The garlic antibacterial paste comprises the following components (parts by weight): Tube A: Garlic powder 10-20%, white petrolatum 70-80%, light liquid paraffin 5-10%, white beeswax 3-6%, lanolin 1-3%, paraffin 1-3%, menthol 0.1-0.3%; and Tube B: Glycerin 5-10%, Carbomer 0.5-1.0%, Triethanolamine 0.5-1.0%, Sodium Bisulfite 0.02-0.06%, Benzalkonium Bromide 0.01-0.03%.

9. The use as described in claim 7 or 8, characterized in that, The A tube contains anhydrous formulation, while the B tube contains aqueous formulation.

10. A reagent kit, characterized in that, The kit includes the garlic antibacterial ointment as defined in claim 1.

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