Callicarpa nudiflora effective part for treating diabetic wound ulcer

Abstract

The invention provides an effective part of callicarpa nudiflora. Specifically, the invention provides an effective part of callicarpa nudiflora and a preparation method and application thereof. The effective part of callicarpa nudiflora and verbascoside are found to have excellent biological activity for the first time, the effective part of callicarpa nudiflora and verbascoside can be used as an external preparation to effectively treat diabetic wound ulcer, the preparation method is simple and low in cost, the whole process is carried out at low temperature, active ingredients are not damaged, and the extraction efficiency of the active ingredients is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to an effective part of Callicarpa nudiflora for treating diabetic wound ulcer. BACKGROUND

[0002] Diabetic wound ulcer mainly includes diabetic foot and diabetic skin ulcer, which is a common complication of diabetic patients, mainly manifested as delayed wound healing, increased risk of infection, and even amputation in severe cases. Its mechanism is closely related to microvascular lesions, neuropathy and decreased immune function caused by high blood sugar. High blood sugar damages vascular endothelial cells, leading to blood circulation disorders and affecting wound repair. At the same time, neuropathy makes patients less sensitive to pain, and they are more likely to ignore early damage.

[0003] The current treatment drugs for diabetic wound ulcer include antibiotics, growth factors, antioxidants, hypoglycemic drugs and topical drugs, but they have limited efficacy, high drug resistance, lack of specificity, high cost and unclear long-term effects. Traditional Chinese medicine has the advantages of multi-target and overall regulation, which can improve blood glucose control and repair tissue damage, and has broad prospects in the development of diabetic treatment drugs. At present, only "Sobi one" derived from active ingredients of traditional Chinese medicine is used for the treatment of diabetic wound, and the price is very high, so it is urgent to develop more active substances derived from traditional Chinese medicine and promote the application of Chinese patent medicines in diabetic wound ulcer.

[0004] At present, there is no report on the use of effective part of Callicarpa nudiflora or verbascoside for the treatment of diabetic wound ulcer (diabetic foot). SUMMARY

[0005] The purpose of the present application is to provide a medicine for preventing and / or treating diabetic wound ulcer, and more specifically to provide an effective part of Callicarpa nudiflora, a preparation method thereof and the use thereof for preventing and / or treating diabetic wound ulcer.

[0006] In the first aspect of the present application, an effective part of Callicarpa nudiflora is provided, wherein the effective part contains 50 wt%-90 wt% verbascoside.

[0007] In another preferred embodiment, the effective part contains forsythoside B, verbascoside, luteoloside and isoverbascoside, wherein the content of verbascoside is 50 wt%-80 wt%.

[0008] In another preferred embodiment, the sum of the contents of forsythoside B, luteoloside and isoverbascoside in the effective part is not more than 20 wt%-40 wt%, for example 30 wt%.

[0009] In another preferred embodiment, the effective part contains the following ingredients: Foragenoside B2 4 wt% - 10 wt% content Forasinoside 50 wt% - 80 wt% content Vicenin-2 2 wt% - 4 wt% content, and Isoforasinoside 6 wt% - 10 wt% content.

[0010] In another preferred embodiment, the effective part of S. sinensis is obtained by extracting S. sinensis raw material selected from the group consisting of leaf, root, stem, or a combination thereof; preferably, S. sinensis leaf.

[0011] In another preferred embodiment, the extracting solvent used in the effective part is C1-C5 alcohol selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, t-butanol, or a combination thereof.

[0012] In another preferred embodiment, the extracting solvent used in the effective part is 90-10 vol% ethanol solution, preferably 80-10 vol% ethanol solution, and more preferably 75-20 vol% ethanol solution.

[0013] In another preferred embodiment, the ethanol solution is a solution of ethanol and water.

[0014] In another preferred embodiment, the effective part is obtained by column purification of S. sinensis crude extract using a macroporous resin.

[0015] In another preferred embodiment, the effective part is prepared by the method of the second aspect of the present application.

[0016] In the second aspect of the present application, a method for preparing an effective part of S. sinensis is provided, the method comprising the following steps: (1) taking S. sinensis raw material, adding alcohol solution, and soaking to obtain S. sinensis crude extract; (2) sequentially using a first eluent, a second eluent, and a third eluent to separate and purify the S. sinensis crude extract using a macroporous resin to obtain the effective part of S. sinensis.

[0017] In another preferred embodiment, the S. sinensis raw material in step (1) is S. sinensis leaf. In another preferred embodiment, the alcohol solution in step (1) is C1-C5 alcohol solution selected from the group consisting of methanol solution, ethanol solution, n-propanol solution, isopropanol solution, n-butanol solution, isobutanol solution, or t-butanol solution, preferably ethanol solution.

[0018] In another preferred embodiment, the alcohol solution is 90-20 vol% ethanol solution, preferably 80%-50 vol% ethanol solution, and more preferably 75%±2% vol ethanol solution.

[0019] In another preferred embodiment, the mass-volume ratio of the raw material of naked flower of Callicarpa in step (1) to the alcohol solution is 1 g:10-100 mL. In another preferred embodiment, the soaking time in step (1) is 6h-24h.

[0020] In another preferred embodiment, the soaking times in step (1) are 1-6 times.

[0021] In another preferred embodiment, step (1) comprises: (1-1) taking naked flower of Callicarpa leaves, adding 50-90vol % ethanol, soaking 2-5 times, each time for 8-16h, filtering, and taking the filtrate; (1-2) after concentrating and treating the filtrate obtained in step (1-1), obtaining the crude extract of naked flower of Callicarpa.

[0022] In another preferred embodiment, the concentration is by rotary evaporation, and preferably, the parameters of rotary evaporation are: pressure of 250-80 hPa, temperature of 50-55℃, and rotation speed of 40-80 r.

[0023] In another preferred embodiment, the treatment is drying the concentrated filtrate.

[0024] In another preferred embodiment, the drying is in a vacuum drying oven, and preferably, the parameters of the vacuum drying oven are: temperature of 45℃-65℃, and pressure of less than 0.05 hPa.

[0025] In another preferred embodiment, the first eluent in step (2) is water.

[0026] In another preferred embodiment, the second eluent in step (2) is selected from the group consisting of C1-C5 alcohol solution, preferably ethanol solution.

[0027] In another preferred embodiment, the second eluent in step (2) is 30-10 vol % ethanol solution, preferably 15±2 vol % ethanol solution.

[0028] In another preferred embodiment, the third eluent in step (2) is selected from the group consisting of C1-C5 alcohol solution, preferably ethanol solution.

[0029] In another preferred embodiment, the second eluent in step (2) is 60-30 vol % ethanol solution, preferably 30±2% ethanol solution.

[0030] In another preferred embodiment, the mass-volume ratio of the crude extract of naked flower of Callicarpa to the first eluent is 1 g:0.5-6 L, preferably 1 g:0.5-3 L.

[0031] In another preferred embodiment, the mass-volume ratio of the crude extract of S. nudiflora to the second eluent is 1 g:0.2-6 L, preferably 1 g:0.5-3 L.

[0032] In another preferred embodiment, the mass-volume ratio of the crude extract of S. nudiflora to the third eluent is 1 g:0.5-6 L, preferably 1 g:1-3 L.

[0033] In another preferred embodiment, the effective part of S. nudiflora is obtained after elution by the third eluent.

[0034] In another preferred embodiment, the step (2) further comprises the following pretreatment method: (2-0) Take the macroporous resin, soak it in ethanol, then add it to the washed adsorption column, add ethanol in the adsorption column, and stand to obtain a separation column containing macroporous resin.

[0035] In another preferred embodiment, the ethanol is anhydrous ethanol.

[0036] In another preferred embodiment, the volume-mass ratio of the adsorption column to the macroporous resin is 1 L:100 g-200 g, preferably 1 L:150 g-170 g.

[0037] In another preferred embodiment, after adding ethanol in the adsorption column, the height of the ethanol exceeds the macroporous resin by 6 cm-10 cm.

[0038] In another preferred embodiment, the standing time is 10 h-20 h.

[0039] In another preferred embodiment, the washing of the adsorption column comprises the following method: (2-0-1) Wash the filter core of the adsorption column with methanol, ethyl acetate in turn, and then wash it again with ethanol.

[0040] In another preferred embodiment, the step (2) further comprises the following post-treatment method: (2-2) After concentrating the filtrate containing the effective part of S. nudiflora obtained in step (2-1), the effective part is obtained.

[0041] In another preferred embodiment, in step (2-2), the concentration is by rotary evaporation.

[0042] In another preferred embodiment, the parameters of rotary evaporation are: pressure 180~80 hPa, temperature 50~55℃, and rotation speed 40r~80r.

[0043] In another preferred embodiment, the method for preparing the effective part of S. nudiflora comprises the following steps: (n1) soaking the original material of Callicarpa nudiflora in a first solvent for 10-15 hours, extracting 2-4 times, combining the extract, and then removing the solvent under reduced pressure after concentration, drying to obtain the extract of Callicarpa nudiflora; (n2) dissolving the extract of Callicarpa nudiflora in methanol, and then separating and column chromatographing the extract using macroporous resin with water, 15% ethanol and 30% ethanol as eluent in sequence to obtain the effective part of Callicarpa nudiflora.

[0044] In a third aspect of the present application, a pharmaceutical composition is provided, which comprises: (a) a therapeutically effective amount of active ingredient, which is verbascoside or the effective part of Callicarpa nudiflora according to the first aspect of the present application, wherein the effective part contains 50%-90% verbascoside; (b) a pharmaceutically acceptable carrier.

[0045] In another preferred embodiment, the pharmaceutically acceptable carrier is selected from the group consisting of emulsifiers, excipients, fillers, diluents, binders, humectants, lubricants, glidants, anti-adherents, antioxidants, anti-caking agents, buffers, chelating agents, preservatives, solubilizers, stabilizers, viscosity regulators, transdermal penetration enhancers, or combinations thereof.

[0046] In another preferred embodiment, the preparation of the pharmaceutical composition is a topical preparation.

[0047] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of cream, ointment, tincture, gel, patch, paste, aerosol, spray, powder, liniment, paint, coating agent, gel.

[0048] In another preferred embodiment, the dosage form of the pharmaceutical composition is a cream.

[0049] In another preferred embodiment, when the dosage form of the pharmaceutical composition is a cream, the content of the effective part is 5%-15%, preferably 7%-10%.

[0050] In another preferred embodiment, the cream is a W / O (water-in-oil) cream.

[0051] In another preferred embodiment, the oil phase contains mineral oil, higher fatty alcohol.

[0052] In another preferred embodiment, the mineral oil is selected from the group consisting of liquid paraffin, vaseline, or combinations thereof.

[0053] In another preferred embodiment, the higher fatty alcohol is selected from the group consisting of cetyl alcohol, stearyl alcohol, or combinations thereof.

[0054] In another preferred embodiment, the water phase contains water, emulsifier, suspending agent.

[0055] In another preferred embodiment, the emulsifier is selected from the group consisting of sodium lauryl sulfate, sodium laureth sulfate, acylsulfate, acylsulfate, Tween-20, Tween-80, preferably sodium lauryl sulfate.

[0056] In another preferred embodiment, the humectant is selected from the group consisting of glycerin, propylene glycol, butylene glycol, hexylene glycol butylene glycol, hexylene glycol, preferably glycerin.

[0057] In another preferred embodiment, the cream comprises the following ingredients: octadecanol, white petrolatum, liquid paraffin, active site, glycerin, water.

[0058] In another preferred embodiment, the method for preparing the cream comprises the following steps: (s1) providing oil phase material and water phase material; (s2) dissolving the oil phase material and the water phase material respectively at T1, and then mixing the oil phase with the water phase, stirring until emulsification; (s3) continuing stirring at T2 to obtain the cream.

[0059] In another preferred embodiment, the T1 is 60-100°C, preferably 70-85°C.

[0060] In another preferred embodiment, the mixing of the oil phase with the water phase is adding the water phase into the oil phase.

[0061] In another preferred embodiment, the water phase is added into the oil phase in a dropwise manner.

[0062] In another preferred embodiment, the emulsification time is 4-10 minutes.

[0063] In another preferred embodiment, the T2 is room temperature.

[0064] In another preferred embodiment, the cream is stored at 0-10°C, preferably 2-5°C.

[0065] In a fourth aspect of the present application, there is provided a use of the active site as described in the first aspect of the present application, or the pharmaceutical composition as described in the third aspect of the present application, for the preparation of a medicament for preventing and / or treating diabetic wound ulcer.

[0066] In another preferred embodiment, the diabetic wound ulcer is selected from the group consisting of diabetic foot, diabetic skin ulcer, or a combination thereof.

[0067] In another preferred embodiment, the diabetic wound ulcer has one or more characteristics selected from the group consisting of delayed wound healing, increased risk of infection, tissue necrosis.

[0068] In another preferred embodiment, the diabetic wound ulcer is a wound ulcer caused by microangiopathy, neuropathy, decreased immune function, blood circulation disorder.

[0069] In another preferred embodiment, the prevention and / or treatment of diabetic wound ulcer comprises a feature of accelerating the healing rate of diabetic wound ulcer.

[0070] In a fifth aspect of the present application, there is provided a use of verbascoside for the preparation of a medicament for the prevention and / or treatment of diabetic wound ulcer.

[0071] In another preferred embodiment, the verbascoside is derived from a plant selected from the group consisting of: Callicarpa nudiflora, Clerodendrum fortunatum, Rehmannia glutinosa, Verbascum thapsus, Cistanche deserticola, Rosmarinus officinalis, Perilla frutescens, Plantago asiatica.

[0072] In another preferred embodiment, the diabetic wound ulcer is selected from the group consisting of: diabetic foot, diabetic skin ulcer, or a combination thereof.

[0073] In another preferred embodiment, the medicament comprises a pharmaceutically acceptable carrier.

[0074] In another preferred embodiment, the carrier is selected from the group consisting of: emulsifier, excipient, filler, diluent, binder, humectant, lubricant, glidant, anti-adherent, antioxidant, anti-caking agent, buffer, chelating agent, preservative, co-solvent, stabilizer, viscosity modifier, transdermal penetration enhancer, or a combination thereof.

[0075] In another preferred embodiment, the medicament is formulated as a topical formulation.

[0076] In another preferred embodiment, the dosage form of the medicament is selected from the group consisting of: cream, ointment, tincture, gel, patch, paste, aerosol, spray, powder, liniment, paint, film, gel.

[0077] In another preferred embodiment, the dosage form of the medicament is a cream.

[0078] In another preferred embodiment, when the dosage form of the medicament is a cream, the verbascoside is present in an amount of 5 wt% to 15 wt%, preferably 7 wt% to 10 wt%.

[0079] It should be understood that, within the scope of the present application, each of the technical features described above and each of the technical features described in detail hereinafter (e.g., in the examples) can be combined with each other to form a new or preferred technical solution. Due to the limited space, they are not listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0080] Figure 1 The comparison of the component liquid phase of the effective site and the standard liquid phase of each component is shown.

[0081] Figure 2 The 1H NMR spectrum of verbascoside is shown, where A is the 1H NMR spectrum of the main peak in the effective fraction and B is the 1H NMR spectrum of the verbascoside standard.

[0082] Figure 3 The mass spectrum of the main peak in the effective region is displayed.

[0083] Figure 4 A representative diagram of wound healing in mice is shown.

[0084] Figure 5 A quantitative graph of wound healing rate in mice is shown. Detailed Implementation

[0085] Through extensive and in-depth research, including numerous screenings and tests, the inventors have provided an effective component of *Callicarpa nudiflora*, its preparation method, and its applications. The inventors have for the first time discovered an effective component of *Callicarpa nudiflora* and that verbascoside can prevent and / or treat diabetic wound ulcers, and based on this discovery, the present invention was completed.

[0086] The present invention discloses that the effective component powder and the cream preparations prepared in the form of "10% effective component", "5% effective component", "7% verbascoside", and "3.5% verbascoside" have therapeutic effects on diabetic foot ulcers and are expected to become symptomatic drugs for the treatment of diabetic foot ulcers.

[0087] the term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0088] 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”.

[0089] Verbascoside Verbascoside, with the molecular formula C 29 H 36 O 15 It is widely distributed in the plant kingdom. The following are some of the main source plants: Gynostemma pentaphyllum, Rehmannia glutinosa, Cistanche deserticola, and Mullein. It has also been found in many medicinal plants such as Ligustrum lucidum, Taraxacum mongolicum, Callicarpa japonica, and Forsythia suspensa.

[0090] Verbascoside can effectively scavenge free radicals (such as superoxide anions and hydroxyl radicals) and inhibit lipid peroxidation. Its antioxidant capacity is even stronger than some classic antioxidants. Due to its powerful antioxidant and anti-inflammatory properties, it has shown protective effects in various neurodegenerative disease models (such as Alzheimer's disease and Parkinson's disease), reducing β-amyloid (Aβ)-induced neurotoxicity and protecting neurons from damage. Pharmaceutical Compositions and Administration The present invention provides a pharmaceutical composition containing the compound of the present invention as an active ingredient.

[0091] The pharmaceutical compositions of the present invention comprise, within a safe and effective range, the compound of the present invention or a pharmacologically acceptable salt thereof, and a pharmacologically acceptable excipient or carrier. "Safe and effective range" refers to an amount of the compound sufficient to significantly improve the condition without causing serious side effects.

[0092] "Pharmaceutically acceptable carriers" refers to one or more compatible solid or liquid fillers or gelling substances that are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with and with the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween®), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0093] The main method of administration for the compounds or pharmaceutical compositions of the present invention is local administration.

[0094] Dosage forms of the compounds of this invention for topical administration include ointments, powders, and patches. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0095] When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to the mammal (such as a human) requiring treatment. The dosage administered is the pharmaceutically considered effective dose. For a person weighing 60 kg, the daily dose is typically 1–2000 mg, preferably 20–500 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of the skills of a skilled physician.

[0096] Preparation of effective component powder Weigh 150-170 g of macroporous resin and soak it in anhydrous ethanol. Take a 1 L adsorption column, wash the filter element with 0.5 L each of methanol and ethyl acetate, wash again with 1 L of anhydrous ethanol, and then pour the resin into the column. The resin should be filled to about 8 cm above the filter element with anhydrous ethanol. Cover with a rubber stopper and let stand for 12 h. Rinse with ultrapure water until the filtrate is free of ethanol. Take 2 g of crude extract powder of *Callicarpa nudiflora*, dissolve it in methanol (not exceeding 30 mL), and add it dropwise along the column wall using a dropper. Add quartz sand. Pass the column through 1.5 L of ultrapure water, 1 L of 15% ethanol, and 2 L of 30% ethanol.

[0097] Collect 2 L of 30% ethanol per column, collecting 50 mL portions in 23-40 50 mL centrifuge tubes. Concentrate under reduced pressure using a rotary evaporator (180-80 hPa, 50-55℃, 60 rpm) to obtain 239 mg of the effective fraction powder. Further separation and purification of the crude extract of *Callicarpa nudiflora* yields the effective fraction with anti-aging effects, with a more refined composition.

[0098] The main advantages of this invention include: (1) This invention first discovered that the effective parts of Callicarpa nudiflora and verbascoside have excellent biological activity, and as a topical preparation, they can effectively treat diabetic wound ulcers.

[0099] (2) The preparation method of the present invention is simple and low in cost. The entire process is carried out at a low temperature, which does not damage the active ingredients and the extraction efficiency of the active ingredients is high.

[0100] 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 or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0101] Example 1: Wound healing experiment in diabetic mice I. Extraction of Crude Extract from Callicarpa nudiflora Take 100g of dried leaves of *Callicarpa nudiflora* and add 5 L of 75% ethanol, ensuring all leaves are immersed in the ethanol. Soak for 12 hours each time, then filter using a Buchner funnel to remove impurities. Collect the filtrate. Repeat this process three times to obtain 15 L of filtrate. Concentrate the filtrate under reduced pressure using a rotary evaporator (250~80 hPa, 50~55℃, 60r) until it reaches a paste-like consistency. Dry the paste in a vacuum drying oven (55℃, Pa < 0.05hPa) for 24 hours to obtain 16 g of crude *Callicarpa nudiflora* extract solid powder, which should be stored at -20℃.

[0102] II. Macroporous resin enrichment Weigh 150-170 g of macroporous resin and soak it in anhydrous ethanol. Take a 1 L adsorption column, wash the filter element with 0.5 L each of methanol and ethyl acetate, wash again with 1 L of anhydrous ethanol, and then pour the resin into the column. The resin should be filled to about 8 cm above the filter element with anhydrous ethanol. Cover with a rubber stopper and let stand for 12 h. Rinse with ultrapure water until the filtrate is free of ethanol. Take 2 g of crude extract powder of *Callicarpa nudiflora*, dissolve it in methanol (not exceeding 30 mL), and add it dropwise along the column wall using a dropper. Add quartz sand. Pass the column through 1.5 L of ultrapure water, 1 L of 15% ethanol, and 2 L of 30% ethanol.

[0103] Collect 2 L of 30% ethanol from the column, collecting 23-40 50 mL centrifuge tubes, and rotary evaporate (180-80 hPa, 50-55℃, 60 rpm) to obtain 239 mg of the effective fraction powder. Store at -20℃. The liquid phase of the effective fraction and the comparison of the standard solutions of each component are as follows: Figure 1 As shown.

[0104] The main peak with the highest abundance and retention time of 8.78 min in the effective fraction was separated and analyzed by 1H NMR and mass spectrometry to confirm that the component was verbascoside ( Figure 2 The above are the 1H NMR spectra of verbascoside, where A is the 1H NMR spectrum of the main peak in the effective fraction and B is the 1H NMR spectrum of the verbascoside standard. Figure 3 (Mass spectrometry of the main peak in the effective fraction).

[0105] III. Preparation of W / O Cream The oil phase and aqueous phase materials were weighed separately into different beakers and placed in a water bath at a constant temperature of 80 °C until dissolved. The aqueous phase was added dropwise to the oil phase multiple times, and the mixture was stirred continuously at 80 °C in the beaker until emulsification was achieved for 5 minutes. Then, the mixture was stirred at room temperature until condensation and stored at 4 °C. Table 1 shows the composition of the oil phase and aqueous phase materials.

[0106] Table 1 Composition of oil phase and aqueous phase materials IV. Wound Healing Experiment in Diabetic Mice Mouse model of diabetes Mold making materials: Streptomycin (STZ), citrate buffer (pH=4.5), 300 μL syringe, 5% glucose solution.

[0107] Experimental steps: Day 0 of modeling: Remove the food from the mice, but retain the water (fasting for more than 12 hours).

[0108] Day 1 of modeling: Prepare a 5 mg / mL STZ solution. Quickly weigh 7.5 mg STZ into a 2 mL EP tube wrapped in aluminum foil. Measure 14 mL of sterile citrate buffer into a sterile 15 mL centrifuge tube. Place both tubes in a cold bath and transfer them to the SPF animal room. STZ solution is unstable and must be prepared fresh each time. Prepare STZ solutions for each group (2 mL EP tube with 1.5 mL citrate buffer). Inject 10 x μL of STZ solution per mouse (x g of fasting body weight), i.e., the modeling dose per mouse is 50 mg / kg STZ. Inject each tube of STZ solution within 30 minutes (protected from light). The non-modeling group is injected with 10 x μL of citrate buffer. After injection, maintain a fasting state. Feed is given 1 hour after injection, followed by 5% glucose solution 2 hours later, until the next fasting period, after which normal drinking water is provided.

[0109] Days 2-5 of modeling: Repeat the procedures of day 1, observe the mice's condition daily, and ensure they have access to plenty of water.

[0110] Days 6 to 14 after modeling: Mice ate and drank normally.

[0111] Days 15-19 of modeling: Fasting blood glucose was measured daily. Successful diabetes modeling was considered to have been achieved when fasting blood glucose was greater than 11.1 mmol / L for several consecutive days.

[0112] 2. Trauma modeling and drug administration in diabetic mice Diabetic mice were evenly divided into four groups according to their fasting blood glucose levels: a model group, a 10% effective fraction group, a 5% effective fraction group, a 7% verbascoside group, and a 3.5% verbascoside group, with six mice in each group. Six unmodeled control mice were also included as a blank group.

[0113] The back hair of mice was removed using an animal shaver. Hair removal cream was applied for 5-10 minutes, wiped off with a clean tissue, and then the mice were rinsed with saline solution and dried. Mice were anesthetized with 3.5% isoflurane and maintained anesthesia with 1.5% isoflurane. After anesthesia, the skin was wiped with an alcohol swab. Incisions were made on the dorsal side of both sides of the midline using a 6mm live puncture device. The wound edges were lifted with forceps, and the edges were trimmed with microsurgical scissors. The white fascia layer beneath the wound was grasped with forceps, and the scalpel was used to trim it, creating two full-thickness wounds penetrating the adipose tissue. From day 1 to day 12 post-surgery, 20 mg of cream was applied to each wound daily according to the group. Photos were taken on the day of surgery and every two days post-surgery. A fixed-size metal ring was placed over the wound, ensuring the wound was within the ring, before taking the photo to correct the actual proportions of each wound photograph. Figure 4 ).

[0114] 3. Results of experiments on diabetic trauma mice Quantitative statistical chart of mouse traumatic wounds The area of ​​each wound was quantified using ImageJ, and the ratio of wound area on days 2, 4, 6, 8, 10, and 12 to that on day 0 of the model was calculated. With the wound area on day 0 as 100%, all data were normalized, and quantitative analysis was performed, plotted, and tabulated. Results showed that in the STZ-induced diabetic mouse wound model, administration of two different doses of verbascoside and the effective site indicated that, starting on day 4, except for the model group, the skin could be observed to converge inward from the wound center, showing a positive trend in promoting wound healing. Simultaneously, the wound site in the model group was pale red compared to other groups on days 4 and 6, indicating inhibited wound angiogenesis and prolonged inflammatory response time. On day 8, the wound healing effect in the treatment group was significantly improved, and the healing rate was significantly higher than that in the model group. Figure 5 (Table 2) It can even have better wound healing effects than unmodeled blank mice.

[0115] These findings indicate that the active ingredient and effective fraction can effectively promote the healing of diabetic wounds and have promising application prospects in areas such as diabetic foot and wound ulcers.

[0116] Table 2 Relative area of ​​traumatic wounds discuss To address the problem of the lack of effective drugs for diabetic foot ulcers, the inventors discovered that after alcohol extraction to obtain a crude extract of Callicarpa nudiflora, followed by three elutions with macroporous resin, the effective fraction of Callicarpa nudiflora (containing 50% to 90% verbascoside) can be efficiently prepared.

[0117] The effective components and verbascoside described in this invention are mainly applied to diabetic wound ulcers through local topical administration (e.g., creams, sprays, or gels for external use on wounds). They have no therapeutic effect on systemic glucose metabolism disorders, but are mainly used to treat lesions caused by diabetic complications in the feet.

[0118] In an STZ-induced diabetic mouse wound model, the inventors unexpectedly discovered that the effective components of *Callicarpa nudiflora* and verbascoside prepared according to this invention can treat non-healing diabetic wounds. After applying the effective components and verbascoside to the wounds of diabetic mice, the degree of wound healing was significantly better than that of the model group. Furthermore, after applying high doses of the effective components of *Callicarpa nudiflora* and verbascoside, the degree of wound healing was better than that of the unmodeled control group on day 8, and comparable to that of the control group on day 10. This demonstrates that the effective components of *Callicarpa nudiflora* and verbascoside described in this invention have an excellent therapeutic effect on diabetic wound ulcers.

[0119] 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. An effective part of a naked-flowered Callicarpa, wherein, The effective fraction contains 50 wt% to 90 wt% verbascoside.

2. The effective part as described in claim 1, characterized in that, The effective parts of the naked-flowered Callicarpa are obtained by extracting from the raw materials of naked-flowered Callicarpa selected from the group consisting of: leaves, roots, stems, or combinations thereof; preferably, the leaves of naked-flowered Callicarpa.

3. The effective part as described in claim 1, characterized in that, The effective fraction is prepared by the following method, the method comprising: (1) Take the raw material of Callicarpa nudiflora, add alcohol solution and soak it to obtain crude extract of Callicarpa nudiflora; (2) The crude extract of Callicarpa nudiflora was separated by column chromatography using macroporous resin by sequentially using the first eluent, the second eluent, and the third eluent to obtain the effective fraction of Callicarpa nudiflora.

4. A method for preparing the effective part of Callicarpa nudiflora as described in claim 1, characterized in that, The method includes the following steps: (1) Take the raw material of Callicarpa nudiflora, add alcohol solution and soak it to obtain crude extract of Callicarpa nudiflora; (2) The crude extract of Callicarpa nudiflora was separated by column chromatography using macroporous resin by sequentially using the first eluent, the second eluent, and the third eluent to obtain the effective fraction of Callicarpa nudiflora.

5. The method as described in claim 4, characterized in that, The method has one or more features selected from the group consisting of: A. The alcohol solution is a C1-C5 alcohol solution selected from the group consisting of methanol solution, ethanol solution, n-propanol solution, isopropanol solution, n-butanol solution, isobutanol solution or tert-butanol solution, preferably an ethanol solution, more preferably a 90-20 vol% ethanol solution; B. The mass-to-volume ratio of the raw material of Callicarpa nudiflora to the alcohol solution is 1 g: 10-100 mL; C. The first eluent is water, and the mass-to-volume ratio of the crude extract of Callicarpa nudiflora to the first eluent is 1 g: 0.5-6 L, preferably 1 g: 0.5-3 L; D. The second eluent is selected from the group below: C1-C5 alcohol solution, preferably ethanol solution, more preferably 30-10 vol% ethanol solution, and the mass-volume ratio of the crude extract of Callicarpa nudiflora to the second eluent is 1 g: 0.2-6 L, preferably 1 g: 0.5-3 L; E. The third eluent is selected from the group consisting of C1-C5 alcohol solutions, preferably ethanol solutions, more preferably 60-30 vol% ethanol solutions, and the mass-volume ratio of the crude extract of Callicarpa nudiflora to the third eluent is 1 g:0.5-6 L, preferably 1 g:1-3 L.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (a) A therapeutically effective amount of an active ingredient, said active ingredient being verbascoside or the effective fraction of Callicarpa nudiflora as described in claim 1, wherein said effective fraction contains 50 wt% to 90 wt% verbascoside. (b) Pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage form of the pharmaceutical composition is selected from the group consisting of: creams, ointments, tinctures, gels, patches, pastes, aerosols, sprays, powders, liniments, coatings, films, and gels.

8. Use of an effective fraction as described in claim 1, or a pharmaceutical composition as described in claim 5, characterized in that, Used to prepare medicines for the prevention and / or treatment of diabetic wound ulcers.

9. The use of a verbascoside, characterized in that, Used to prepare medicines for the prevention and / or treatment of diabetic wound ulcers.

10. The use as described in any one of claims 8 or 9, characterized in that, The diabetic wound ulcer is selected from: diabetic foot, diabetic skin ulcer, or a combination thereof.

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