Application of cyclocarya paliurus extraction product in preparation of medicine for diabetic foot ulcer

Abstract

The invention discloses application of a cyclocarya paliurus extraction product in preparation of a medicine for diabetic foot ulcer. Rat experiments show that compared with a model group, the prepared cyclocarya paliurus extract product can improve and promote healing of diabetic wounds, and the treatment effect on infected wounds is superior to that of erythromycin ointment. The cyclocarya paliurus extract product can effectively reduce the levels of IL-6 and TNF-alpha in wound tissues, significantly improve the SOD activity and improve the MDA level, and promote the secretion of VEGF and EGF at the same time. The experimental result shows that the cyclocarya paliurus extract product disclosed by the invention can promote the healing of the diabetic foot ulcer, is a potential clinical treatment medicine for the diabetic foot ulcer, and has a wide clinical prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of application of traditional Chinese medicines, and in particular to application of a Cyclocarya paliurus extract product in the preparation of a medicine for diabetic foot ulcers. Background Art

[0002] Diabetic foot ulcer is a common complication of diabetes with high disabling rate, which has always been a difficult problem in clinical treatment. The mortality rate of patients with diabetic foot ulcer after amputation increases significantly with the course of the disease. The high disability and mortality rates of diabetic foot ulcer have a serious negative impact on patients and society, and have brought a heavy burden to patients and global public health. Therefore, therapies that try to avoid amputation and promote wound healing are still of great significance.

[0003] The typical characteristics of diabetic foot ulcer tissue damage are peripheral nerve and lower limb vascular lesions caused by long-term high sugar levels, as well as delayed wound healing caused by a combination of factors such as chronic inflammation, ischemia and hypoxia, oxidative stress, and repeated infections. Among them, the normal physiological processes of wound healing, such as coagulation, inflammation, granulation tissue formation, and extracellular matrix remodeling, will be disturbed or stagnant in diabetic foot ulcers, ultimately manifesting as a local chronic inflammatory state and delayed wound healing.

[0004] In order to cope with the multiple challenges brought by diabetic foot ulcers, new strategies combining multiple functions and pathways, such as reducing inflammatory response, improving oxidative stress and wound microenvironment, have been proposed to combat diabetic foot ulcers. Finding more effective and safer biomolecules has always been an important area of ​​research. Natural medicinal plants contain many active ingredients that have the potential to overcome the drug resistance of pathogenic microorganisms, can effectively treat diabetic foot ulcers in multiple ways, and may promote wound healing by establishing multiple favorable pathways such as anti-inflammatory and antibacterial.

[0005] Cyclocarya paliurus (Batal.) Iljinsk belongs to the genus Cyclocarya of the Juglandaceae family, also known as green money plum, mountain hemp willow, mountain tree, money tree, etc. At present, many basic and clinical studies have shown that Cyclocarya paliurus decoction or tea substitute oral administration has obvious advantages in the treatment of diabetes. However, there are no reports on the topical use of Cyclocarya paliurus and its extracts in the treatment of diabetic foot ulcers.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide the use of a Cyclocarya paliurus extract product in preparing a medicine for treating diabetic foot ulcers.

[0008] The present invention is achieved in that:

[0009] In a first aspect, the present invention provides a use of a Cyclocarya paliurus extract product in the preparation of a medicament for diabetic foot ulcers.

[0010] In an optional embodiment, the drug is an external medicine;

[0011] Preferably, the medicament comprises one of an ointment, a cream, a gel, a spray, a patch, a powder, a foam and a solution.

[0012] In an optional embodiment, the Cyclocarya paliurus extract product includes at least one of a Cyclocarya paliurus extract and a Cyclocarya paliurus squeezed juice.

[0013] In an optional embodiment, the Cyclocarya paliurus juice is filtered and sterilized before preparing the medicine;

[0014] Preferably, the Cyclocarya paliurus juice is prepared from fresh, green, smallest, fully expanded leaves picked from March to June.

[0015] In an optional embodiment, the Cyclocarya paliurus extract includes Cyclocarya paliurus water extract and Cyclocarya paliurus alcohol extract.

[0016] In an optional embodiment, the preparation method of the Cyclocarya paliurus extract includes: mixing crushed Cyclocarya paliurus leaves and an extractant, obtaining an extract after reflux extraction, concentrating and drying the extract under reduced pressure to obtain a powder extract, diluting and filtering the powder extract to obtain the Cyclocarya paliurus extract.

[0017] In an optional embodiment, the crushed Cyclocarya paliurus leaves have a particle size of 10 to 50 meshes;

[0018] Preferably, the volume mass ratio of the extractant to the Cyclocarya paliurus leaves in the reflux extraction step is 15 to 25 mL / g.

[0019] In an optional embodiment, the reflux extraction is performed once or twice or more. When the reflux extraction is performed twice or more, solid-liquid separation is performed after each reflux extraction to obtain a filtrate, and the filtrates obtained from each reflux extraction are combined to form the extract.

[0020] In an optional embodiment, the reflux extraction temperature is 110-130° C., and the time for a single reflux extraction is 0.5-2 h.

[0021] In an optional embodiment, the concentration of the powder extract in the diluted liquid is 50-200 mg / mL.

[0022] The present invention has the following beneficial effects:

[0023] The rat experiment showed that compared with the model group, the Cyclocarya paliurus extract product prepared by the present invention can improve and promote the healing of diabetic wounds, and some Cyclocarya paliurus extract products such as squeezed juice are better than erythromycin ointment in promoting the healing of diabetic wound infection. The Cyclocarya paliurus extract product can significantly reduce the levels of IL-6 and TNF-α in wound tissue, significantly improve SOD activity and MDA levels, and promote VEGF and EGF secretion. The above experimental results show that the Cyclocarya paliurus extract product disclosed in the present invention can promote the healing of diabetic foot ulcers, is a potential active ingredient in clinical treatment drugs for diabetic foot ulcers, and has broad clinical prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 The results of the antibacterial experiments of Cyclocarya paliurus juice (CPJ), Cyclocarya paliurus water extract (CPA), and Cyclocarya paliurus ethanol extract (CPE) in Experimental Example 1 are compared;

[0026] Figure 2 The changes of wound surface in different treatment groups in Experimental Example 2;

[0027] Figure 3 The expression of IL-6 in wound tissue of rats in each group in Experimental Example 2;

[0028] Figure 4 The expression of TNF-α in wound tissue of rats in each group in Experimental Example 2;

[0029] Figure 5 The SOD activity of wound tissues of rats in each group in Experimental Example 2;

[0030] Figure 6 The MDA levels of wound tissues of rats in each group in Experimental Example 2;

[0031] Figure 7 The blood sugar changes of rats in different treatment groups in Experimental Example 3;

[0032] Figure 8 The changes of infected wounds and wound healing rates in different treatment groups in Experimental Example 3;

[0033] Fig. 9The expression of IL-6 and TNF-α in the wound tissues of rats in each group in Experimental Example 3; (a) the expression of IL-6 in the wound tissues of rats in each group; (b) the expression of TNF-α in the wound tissues of rats in each group;

[0034] Fig.10 The expression of SOD activity and MDA level in the wound tissue of each group of rats in Experimental Example 3; (a) SOD activity in the wound tissue of each group of rats; (b) MDA level in the wound tissue of each group of rats;

[0035] Fig.11 The expression of VEGF and EGF levels in the infected wound tissues of rats in each group in Experimental Example 3; (a) VEGF activity in the wound tissues of rats in each group; (b) EGF levels in the wound tissues of rats in each group. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0037] An embodiment of the present invention provides an application of a Cyclocarya paliurus extract product in the preparation of a medicine for treating diabetic foot ulcers with or without infection.

[0038] The present invention discloses the use of a Cyclocarya paliurus extract product in the preparation of a drug for treating diabetic foot ulcers, and the drug is prepared as an active ingredient together with other pharmaceutically acceptable excipients to obtain a drug for treating diabetic foot ulcers. Compared with antibiotics, growth factors and other drugs used in the prior art, the drug has fewer adverse reactions, stable efficacy, low price, easy to obtain, simple and rapid preparation process, easy to control the quality of the finished product, convenient for large-scale industrial production, and can effectively exert anti-inflammatory and anti-oxidative stress effects, promote the regeneration of granulation tissue, and promote wound healing.

[0039] The rat experiment showed that compared with the model group, the Cyclocarya paliurus extract product prepared by the present invention can improve and promote the healing of diabetic wounds, and some Cyclocarya paliurus extract products such as squeezed juice are better than erythromycin ointment in promoting the healing of diabetic wound infection. The Cyclocarya paliurus extract product can significantly reduce the levels of IL-6 and TNF-α in wound tissue, significantly improve SOD activity and MDA levels, and promote VEGF and EGF secretion. The above experimental results show that the Cyclocarya paliurus extract product disclosed in the present invention can promote the healing of diabetic foot ulcers, is a potential active ingredient in clinical treatment drugs for diabetic foot ulcers, and has broad clinical prospects.

[0040] In an optional embodiment, the drug is an external medicine;

[0041] Preferably, the medicament comprises one of an ointment, a cream, a gel, a spray, a patch, a powder, a foam and a solution.

[0042] In an optional embodiment, the Cyclocarya paliurus extract product includes at least one of a Cyclocarya paliurus extract and a Cyclocarya paliurus squeezed juice.

[0043] In an optional embodiment, the Cyclocarya paliurus juice is filtered and sterilized before preparing the medicine;

[0044] Preferably, the Cyclocarya paliurus juice is prepared from fresh, green, smallest, fully expanded leaves picked from March to June.

[0045] Fresh, green, smallest, fully expanded young leaves of Cyclocarya paliurus picked from March to June are selected, washed and immediately squeezed with a juicer, or stored in a refrigerated vacuum state, and squeezed within 48 hours to obtain a coarse squeezed juice. The coarse squeezed juice is filtered through four layers of gauze and a disposable sterile syringe filter to obtain the Cyclocarya paliurus young leaf juice extract, which is then used to prepare a drug for diabetic foot ulcer infection.

[0046] The juice squeezing temperature may be room temperature, preferably 22±3° C., and the sterile syringe filter may be a disposable sterile syringe filter of 0.45 μm.

[0047] In an optional embodiment, the Cyclocarya paliurus extract includes Cyclocarya paliurus water extract and Cyclocarya paliurus alcohol extract.

[0048] In an optional embodiment, the preparation method of the Cyclocarya paliurus extract includes: mixing crushed Cyclocarya paliurus leaves and an extractant, obtaining an extract after reflux extraction, concentrating and drying the extract under reduced pressure to obtain a powder extract, diluting and filtering the powder extract to obtain the Cyclocarya paliurus extract.

[0049] In an optional embodiment, the crushed Cyclocarya paliurus leaves have a particle size of 10 to 50 meshes;

[0050] Preferably, the volume mass ratio of the extractant to the Cyclocarya paliurus leaves in the reflux extraction step is 15-25 mL / g, preferably 20.

[0051] In an optional embodiment, the reflux extraction is performed once or twice or more. When the reflux extraction is performed twice or more, solid-liquid separation is performed after each reflux extraction to obtain a filtrate. The filtrates obtained from each reflux extraction are combined to form the extract. Preferably, the reflux extraction is performed twice.

[0052] In an optional embodiment, the reflux extraction temperature is 110-130° C., and the single reflux extraction time is 0.5-2 h. Preferably, the temperature is 120° C., and the single reflux extraction time is 1 h.

[0053] In an optional embodiment, the concentration of the powder extract in the diluted liquid is 50-200 mg / mL, preferably 100 mg / mL.

[0054] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0055] Example 1

[0056] This embodiment provides a method for preparing a Cyclocarya paliurus aqueous extract, comprising:

[0057] Take the dried leaves of Cyclocarya paliurus, crush them and sieve them to get dry powder for later use;

[0058] Purified water and dry powder were mixed in a volume-to-mass ratio of 20, and refluxed at 120°C for 1 hour, extracted twice in total, the filtrates were combined, concentrated by rotary evaporation at 75°C to a volume concentration of 0.4 times, and then freeze-dried at -25°C until the weight no longer changed to obtain a dry powder extract with a calculated extraction rate of 13.8%.

[0059] Example 2

[0060] This embodiment provides a method for preparing juice squeezed from young leaves of Cyclocarya paliurus, comprising:

[0061] Fresh, green, smallest, fully expanded young leaves of Cyclocarya paliurus picked from March to June are picked, cleaned (dust and impurities are removed and selected), and a certain mass of Cyclocarya paliurus young leaves are squeezed to extract juice with a juicer, the coarse squeezed juice is filtered with 4 layers of gauze, and the supernatant is filtered through a 0.45 μm microporous filter membrane to obtain the Cyclocarya paliurus young leaf juice extract, and the Cyclocarya paliurus young leaf juice extract is further freeze-dried at -25°C until the weight no longer changes, to obtain a squeezed juice extract freeze-dried powder.

[0062] Example 3

[0063] This embodiment provides a preparation method of Cyclocarya paliurus ethanol extract, comprising:

[0064] Take the dried leaves of Cyclocarya paliurus, crush them and sieve them to get dry powder for later use;

[0065] Accurately weigh 15.0 g of powder and place it in a 500 mL round-bottom flask, reflux extract with 80% ethanol solution, the solid-liquid ratio is 1:20 (mass / volume), heat reflux at 120 ° C for 1 h, repeat the extraction twice, and concentrate the combined filtrate by 75 ° C rotary evaporator to 0.4 times the volume. Then, the extract is further freeze-dried at -25 ° C until the weight no longer changes to obtain a dry powder extract, and a Cyclocarya paliurus alcohol extract sample is obtained.

[0066] Experimental Example 1 In vitro antibacterial experiment

[0067] 1. Materials and reagents

[0068] 1.1 Drugs and reagents

[0069] Staphylococcus aureus (Qingdao Haibo Biotechnology Co., Ltd., ATCC25923), LB broth (Qingdao Haibo Biotechnology Co., Ltd., HB0128), LB agar medium (Qingdao Haibo Biotechnology Co., Ltd., HB0129), pipette tip (Lanjieke, 1000μL, 200μL, 20μL), EP tube (Lanjieke, 1.5mL), disposable plastic coating rod (Lanjieke, BS-PS-A), Gram staining solution (Qingdao Haibo Biotechnology Co., Ltd., HB8278), pipette (Dalong Xingchuang Experimental Instrument Co., Ltd.).

[0070] 1.2 Instruments

[0071] Biological safety cabinet (Thermo Fisher (Suzhou) Instrument Co., Ltd., 1374), biological microscope (Motic Industrial Group Co., Ltd., BA210), UPR series ultrapure water machine (Sichuan UPR Ultrapure Technology Co., Ltd., UPR-II-15TNZ), portable high-pressure steam sterilizer (Shanghai Shen'an Medical Equipment Factory, DSX-30L), constant temperature oscillator (Shanghai Yiheng Scientific Instrument Co., Ltd., THZ-98A), electric constant temperature incubator (Shanghai Yuejin Medical Equipment Co., Ltd., HH-B11·600-BS-Ⅱ) freeze dryer (Ningbo Xinzhi Biotechnology Co., Ltd.).

[0072] 1.3 Preparation of experimental solution

[0073] Preparation of experimental drug solutions: CPA, CPE and CPJ freeze-dried powders were dissolved in 10% DMSO solution, and solutions with a concentration of 100 mg / mL were prepared for use; erythromycin was prepared to a concentration of 1% for use, and all drug solutions were sterilized by filtration with a 0.45 μm sterile filter membrane.

[0074] 2. Experimental methods

[0075] 2.1 Bacterial recovery and subculture

[0076] Activation of bacterial strains: The entire operation was carried out in a sterilized biosafety cabinet. After 1-2 rings of Staphylococcus aureus (S. aureus) were dissolved and suspended according to the instructions, 1-2 rings of S. aureus were picked up with a bacterial inoculation stick, and regular lines were streaked on the LB solid culture medium prepared in advance. The culture dish was covered and placed in a 37°C bacterial incubator for 24 hours.

[0077] Preparation of bacterial suspension: Pick a single colony to prepare a bacterial suspension. After 24 hours of bacterial culture, take out the culture dish, disinfect it with 75% ethanol, and move it into the biosafety cabinet. Use a bacterial inoculation stick to pick a single colony, put it into the sterile LB liquid culture medium prepared in advance, plug the test tube with a stopper and place it on a bacterial shaker (150r / min, 37°C) for overnight culture, and then take 50μL of the expanded culture solution for subculture for 4 hours.

[0078] Gram staining verification: Take out the bacterial solution that has been shaken overnight on the shaker, observe the color and turbidity of the bacterial solution to preliminarily determine whether it is contaminated, and then perform routine microscopic examination on the bacteria. Centrifuge, discard the supernatant, collect the bacterial sediment, pick it and apply it to the center of the slide into a thin layer, and heat it slightly under an alcohol lamp to dry it quickly. Fix it by moving the slide back and forth quickly 3-5 times outside the flame of the alcohol lamp, and then add crystal violet staining solution to stain for 1 minute, and rinse off the staining solution with water. Add iodine solution, stain for 1 minute, and wash with water. Add decolorizing solution, shake for 10-30 seconds, until the remaining decolorizing solution does not appear purple, and immediately wash off the decolorizing solution with water to terminate the reaction. Add restaining solution, stain for 30-60 seconds, and wash with water. After drying, observe under an oil immersion lens (1000 times).

[0079] Adjust the concentration of bacterial solution: After confirming that the bacterial solution is free of contamination, centrifuge the bacterial solution and resuspend it in phosphate buffer saline (PBS). Test the OD value of 100 μL at 600 nm absorbance, dilute to OD value of 0.2, and then dilute 1000 times to obtain 10 6 CFU / mL bacterial solution.

[0080] 2.2 Inhibition zone test

[0081] Take 100 μL of bacterial suspension and spread it evenly on the plate; clamp the sterile Oxford cup and gently place it on the culture medium, so that the Oxford cup is completely attached to the culture medium, and then gently inject 100 μL of CPA, CPE and CPJ with a concentration of 100 mg / mL into the Oxford cup; diffuse the extract on the culture medium for 12 hours at 4°C, and then place the bacterial plate in a 37°C electric constant temperature incubator overnight. The diameter of the inhibition zone is measured using the cross method and recorded (the measurement result needs to subtract the diameter of the Oxford cup). The positive control group is 1% erythromycin, and the negative control group is sterile water. Three replicates are set for each treatment.

[0082] 3. Experimental results

[0083] The diameters of the inhibition zones of different Cyclocarya paliurus extracts are shown in Tables 1 and Figure 1It can be seen that the diameters of the inhibition zones of the three extracts against S. aureus increase with increasing concentrations, and there is statistical significance between the maximum and minimum concentrations of each extract, indicating that within the concentration range of 25-200 mg / mL, the antibacterial effect of the extract increases in a concentration-dependent manner.

[0084] Table 1 Antibacterial zone diameters of different Cyclocarya paliurus extracts

[0085]

[0086] Note: Different letters indicate statistically significant differences, P < 0.05.

[0087] Depend on Figure 1 From Table 1, we can see that:

[0088] The maximum inhibition zone diameter of CPA was 12.53±0.31mm at 200mg / mL, and the minimum inhibition zone diameter was 8.60±0.20mm at 25mg / mL. The antibacterial effect of CPA was concentration-dependent. In the concentration range of 25-200 mg / mL, the diameter of the inhibition zone showed an upward trend, and there were significant statistical differences among 200 mg / mL, 100 mg / mL, and 25 mg / mL. The diameter of the inhibition zone of CPE was the largest at 200 mg / mL, which was 13.93±0.90 mm. The diameter of the inhibition zone showed an upward trend at 25-100 mg / mL, which were 8.47±0.31 mm, 9.73±0.76 mm, and 10.80±0.53 mm, respectively, but the difference was not statistically significant. There was a significant difference in the diameter of the inhibition zone of 150 mg / mL at a concentration of 100 mg / mL. The inhibition zone of CPJ was the smallest at 25 mg / mL, which was 8.47±0.31 mm. There were significant differences in the concentration gradients of 25, 50, and 100 mg / mL. When the concentration was greater than 100 mg / mL, the increase in the diameter of the inhibition zone was not obvious.

[0089] At the same time, the antibacterial effects of Cyclocarya paliurus extracts extracted by different extraction methods at the same concentration were compared, and it was found that at concentrations of 25 and 50 mg / mL, there was no statistical difference in the antibacterial effects of each extract (P>0.05); at a concentration of 100 mg / mL, the antibacterial effect of CPJ extraction was significantly better than that of CPA and CPE (P<0.001); at 150 and 200 mg / mL, the diameter of the inhibition zone of CPA was significantly smaller than that of CPJ, while the antibacterial effect of CPE was between CPA and CPJ.

[0090] Experimental Example 2-Effect of the Cyclocarya paliurus aqueous extract prepared in Example 1 on non-infectious diabetic foot ulcers in rats

[0091] 1. Materials and reagents

[0092] 1.1 Animals

[0093] The rats in this embodiment are commercially available and purchased from Sichuan Chengdu Dashuo Experimental Animal Co., Ltd. They are all healthy male SD rats, SPF grade, 6 weeks old, and weigh (200±10) g. The experimental animal license number is SCXK (Sichuan) 2020-030. Feeding conditions: temperature is 22±3°C, humidity is 60±10%, and 12h light / 12h dark. Standard laboratory rat feed and purified water were given for adaptive feeding for one week. The study was approved by the Experimental Animal Ethics Committee of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, with the ethics approval number: 2023DL-032. All experimental research contents comply with the guidelines of the Chinese Ethics Committee on Animal Research.

[0094] 1.2 Drugs and reagents

[0095] Streptozotocin (STZ) (MP Biomedicals, 0210055701), sodium citrate buffer (Beijing Solebow Technology Co., Ltd., C1013), and Cyclocarya paliurus dry leaves were purchased from Nanjiang County People's Hospital, Bazhong, Sichuan.

[0096] 1.3 Instruments

[0097] Blood glucose meter, blood glucose test strips (Sinocare Biosensor Co., Ltd., GA-3, measuring range: 1.1-33.3 mmol / L, display HI when higher than 33.3 mmol / L), enzyme labeled detector (BioTek, Epoch, USA).

[0098] 1.4 Animal grouping and modeling

[0099] 40 healthy male SD rats were fed adaptively for one week, during which they were given ordinary feed and purified water. They were then randomly divided into a blank group of 8 and a modeling group of 32. The blank group maintained ordinary feed and purified water and freely consumed water. The modeling group rats were given a high-fat and high-sugar diet combined with STZ to induce diabetes: they were fed with a high-fat and high-sugar diet (67% rat basic material, 20% sugar, 10% lard, 2% cholesterol, 1% sodium cholate, provided by Chengdu Dashuo Biotechnology Co., Ltd.) and purified water for 14 days and then weighed. After fasting for 16 hours but not water, 1% STZ was injected intraperitoneally at a uniform speed of 50 mg / kg. The blood glucose of the rats was measured by blood sampling from the rat tail vein after 72h, 7 days, and 14 days, and rats with stable blood glucose ≥16.7mmol / L were selected as DM model rats. The rats in each group were anesthetized with 3% sodium pentobarbital, and a full-thickness skin defect was created with a disposable 10 mm skin biopsy sampler. The wound depth reached the muscle fascia layer. After gauze compression and hemostasis, a 10 mm (inner diameter) × 16 mm (outer diameter) × 2 mm (thickness) silicone gasket was fixed to the edge of the wound with 3M animal tissue glue. A simple foot ulcer rat wound model was constructed in the control group.

[0100] 2 Methods

[0101] 2.1 Intervention methods

[0102] After successful modeling, pure water was added to the sealed and refrigerated dry powder extract to dissolve it, and the extract was filtered with a sterile needle to prepare the Cyclocarya paliurus water extract with a mass fraction of 50 mg / mL, 100 mg / mL, and 200 mg / mL, which were used as the low-dose group (L-CPA), medium-dose group (M-CPA), and high-dose group (H-CPA), respectively. The ulcer wounds of rats in the control group and the model group were wiped once a day with an equal amount of normal saline. After wiping, the above-mentioned Cyclocarya paliurus water extract was dropped into the silicone gasket (about 0.2 mL), and the wounds were covered with six layers of medical gauze (4 cm × 4 cm) and fixed with a medical elastic bandage for 2 weeks.

[0103] 2.2 Observation indicators

[0104] 2.2.1 Wound healing rate

[0105] Dressings were changed every morning, the retention patch was removed, and after treatment, another layer of retention patch was covered on the wound site to exclude external contamination. No bacterial infection was induced, and the wound area on day 0 was taken as 100%. The wound area of ​​each group was quantified on days 7 and 14, and the size of the wound was calculated using Fiji Image J images.

[0106]

[0107] 2.2.2 Detection of IL-6 and TNF-α levels in granulation tissue of ulcer wound

[0108] ELISA was used to detect the levels of IL-6 and TNF-α in the granulation tissue of the ulcer wound. After the experimental rats were killed and the ulcer surface tissue was wrapped in tin foil and frozen in liquid nitrogen, an appropriate amount of the cut skin tissue was weighed, added to a 9-fold sterile PBS solution, ground and mixed thoroughly, and then the skin granulation tissue homogenate was centrifuged at 3000-4000 rpm at 4°C for 10 minutes, the supernatant was collected, and the test was completed in strict accordance with the instructions of the kit.

[0109] 2.2.3 Detection of SOD and MDA levels in granulation tissue of ulcer wound

[0110] Biochemical method was used to detect the SOD activity and MDA level of granulation tissue of ulcer wound. The frozen tissue was taken out and thawed on ice. According to the instructions of the kit, the superoxide dismutase (SOD) activity was calculated by colorimetric analysis of WST-1 product. Malondialdehyde (MDA) in the degradation product of lipid peroxidation can react with thiobarbituric acid under high temperature and acidic environment. The MDA content of skin granulation tissue of rats in each group was detected by thiobarbituric acid method.

[0111] 3 Results

[0112] (1) Wound healing changes and healing rate results are shown in Figure 2 , Table 2.

[0113] Table 2 Wound healing rate

[0114]

[0115] Note: Compared with the model group *P<0.05, **P<0.01

[0116] Depend on Figure 2 From Table 2, we can see that:

[0117] Compared with the control group, the wound tissue in the model group had significantly delayed healing; compared with the model group, the wound healing rates of the different doses of the Cyclocarya paliurus water extract groups of the present invention at 7 days were improved to varying degrees, and the medium-dose Cyclocarya paliurus water extract group was more significant (P<0.05); after 14 days of intervention, the wounds of the Cyclocarya paliurus water extract of the present invention were significantly healed compared with the model group, and the medium-dose Cyclocarya paliurus water extract of the present invention was compared with the low-dose group in promoting healing effect, indicating that the Cyclocarya paliurus water extract of the present invention can effectively promote the healing of diabetic ulcers, preferably at a concentration of 100 mg / mL.

[0118] (2) The experimental results of IL-6 and TNF-α levels in wound tissue are shown in Figure 3 , Figure 4 and Table 3.

[0119] Table 3 IL-6 and TNF-α levels in wound tissue

[0120]

[0121] Note: Compared with the model group *P<0.05, **P<0.01

[0122] Depend on Figure 3 , Figure 4 , Table 3 shows that:

[0123] Compared with the control group, the levels of TNF-α and IL-6 in the wound of rats in the model group were significantly increased (P<0.05), and the levels of TNF-α and IL-6 in the Cyclocarya paliurus water extract group of the present invention were significantly decreased (P<0.05); comparison of different dose groups showed that the healing effect of the medium dose group was better than that of the low dose group (P<0.05), indicating that the Cyclocarya paliurus water extract of the present invention can effectively reduce inflammatory factors on the wound surface and improve the inflammation of diabetic foot ulcers, and the medium dose group has the best effect.

[0124] (3) The experimental results of SOD activity and MDA expression level in wound tissue are shown in Figure 5 , Figure 6 , Table 4.

[0125] Table 4 SOD activity and MDA expression levels in wound tissue

[0126]

[0127] Note: Compared with the model group *P<0.05, **P<0.01

[0128] Depend on Figure 5 , Figure 6 From Table 4, we can see that:

[0129] Compared with the control group, the local SOD activity of the wound surface of the rats in the model group was significantly reduced, and the MDA level was significantly increased, indicating that the rats in the model group showed obvious oxidative stress. After 14 days of intervention, the water extract of the present invention significantly reduced the MDA level (P<0.05) and significantly increased the SOD activity, indicating that the extract of the present invention can effectively improve the oxidative stress state of diabetic foot ulcers and promote ulcer healing.

[0130] In summary, compared with the model group, the Cyclocarya paliurus water extract group of the present invention has significantly reduced levels of inflammatory factors IL-6 and TNF-α (P<0.05), significantly increased SOD activity (P<0.05), and significantly reduced MDA levels (P<0.05), which promotes the healing of diabetic wounds. This shows that the Cyclocarya paliurus water extract of the present invention has a positive therapeutic effect on diabetic foot ulcers, especially the inflammation and oxidative stress state of diabetic foot ulcers, and the effect of the medium-dose Cyclocarya paliurus water extract is better. The Cyclocarya paliurus water extract of the present invention can be further used in the treatment of human diabetic foot, and has broad clinical prospects.

[0131] Experimental Example 3 Testing the effect of the Cyclocarya paliurus extract product of the present invention on diabetic infectious ulcers in rats

[0132] 1. Materials and reagents

[0133] 1.1 Animals

[0134] The rats in this embodiment are commercially available and purchased from Sichuan Chengdu Dashuo Experimental Animal Co., Ltd. They are all healthy male SD rats, SPF grade, 6 weeks old, and weigh (200±10) g. The experimental animal license number is SCXK (Sichuan) 2020-030. Feeding conditions: temperature is 22±3°C, humidity is 60±10%, and 12h light / 12h dark. Standard laboratory rat feed and purified water were given for adaptive feeding for one week. The study was approved by the Experimental Animal Ethics Committee of the Affiliated Hospital of Chengdu University of Traditional Chinese Medicine, with the ethics approval number: 2023DL-032. All experimental research contents comply with the guidelines of the Chinese Ethics Committee on Animal Research.

[0135] 1.2 Drugs and reagents

[0136] Streptozotocin (STZ) (MP Biomedicals, 0210055701), sodium citrate buffer (Beijing Solebow Technology Co., Ltd., C1013), Staphylococcus aureus (Qingdao Haibo Biotechnology Co., Ltd., ATCC25923), fresh young leaves of Cyclocarya paliurus and dry leaves of Cyclocarya paliurus were purchased from Nanjiang County People's Hospital, Bazhong, Sichuan.

[0137] 1.3 Instruments

[0138] Blood glucose meter, blood glucose test strips (Sinocare Biosensor Co., Ltd., GA-3, measuring range: 1.1-33.3 mmol / L, display HI when higher than 33.3 mmol / L), enzyme labeled detector (BioTek, Epoch, USA).

[0139] 1.4 Animal grouping and modeling

[0140] 56 healthy male SD rats were fed adaptively for one week, during which they were given ordinary feed and purified water. They were then randomly divided into a blank group of 8 and a modeling group of 48 rats. The blank group maintained ordinary feed and purified water and freely consumed water. The modeling group rats were given a high-fat and high-sugar diet combined with STZ to induce diabetes: they were fed with a high-fat and high-sugar diet (67% rat basic feed, 20% sugar, 10% lard, 2% cholesterol, 1% sodium cholate, provided by Chengdu Dashuo Biotechnology Co., Ltd.) and purified water for 14 days and then weighed. After fasting for 16 hours but not water, 1% STZ was injected intraperitoneally at a uniform speed of 50 mg / kg. The blood glucose of the rats was measured by blood sampling from the rat tail vein after 72h, 7 days, and 14 days, and rats with stable blood glucose ≥16.7mmol / L were selected as DM model rats. The rats in each group were anesthetized with 3% sodium pentobarbital, and a full-thickness skin defect was created with a disposable 10 mm skin biopsy sampler. The wound depth reached the muscle fascia layer. After gauze was applied to stop bleeding, a 10 mm (inner diameter) × 16 mm (outer diameter) × 2 mm (thickness) silicone gasket was fixed to the edge of the wound with 3M animal tissue glue. 50 μL (2 × 106 CFU / mL) S. aureus was suspended in PBS and applied to the wound, and a retention patch was applied to prevent contamination by other bacteria to establish a wound infection model. A simple foot ulcer infection rat wound model was constructed in the control group.

[0141] 2 Methods

[0142] 2.1 Intervention methods

[0143] After successful modeling, erythromycin ointment was applied to the ulcer wound in the erythromycin ointment group (erythromycin ointment (national medicine standard H11021246, Beijing Shuangji Pharmaceutical Co., Ltd.), and recombinant human epidermal growth factor gel (national medicine standard S20020112, Guilin Huanovi Gene Pharmaceutical Co., Ltd.) was applied to the epidermal growth factor group. Pure water was added to the sealed and refrigerated dry powder extract and juice extract freeze-dried powder to dissolve, and sterile needle filtration was performed to prepare the dry powder extract or juice extract freeze-dried powder with a mass fraction of 100 mg / mL of Cyclocarya paliurus water extract, Cyclocarya paliurus alcohol extract, and Cyclocarya paliurus juice. The erythromycin ointment group, epidermal growth factor group, Cyclocarya paliurus water extract group, Cyclocarya paliurus alcohol extract group, and Cyclocarya paliurus juice group were recorded as EO, EGF, CPA, CPE, and CPJ groups, respectively.

[0144] The ulcer wounds of rats in the control group and the model group were wiped once a day with an equal amount of normal saline. After wiping, the above CPA, CPE and CPJ were dropped into the silicone gasket (about 0.2 mL), and the wounds were covered with six layers of medical gauze (4 cm × 4 cm) and fixed with medical elastic bandages for 2 weeks; or erythromycin ointment or recombinant human epidermal growth factor gel was applied after wiping, and fixed with medical elastic bandages for 2 weeks.

[0145] 2.2 Observation indicators

[0146] 2.2.1 Blood glucose levels in each group

[0147] After the experimental grouping and drug administration, blood sugar was measured once every week. The measurement method adopted the rat tail vein blood sampling method. First, the rat's tail was cleaned with water, and the moisture was absorbed with a cotton ball. The rat's tail was slowly massaged from the proximal end to the distal end of the tail to make the tip of the rat's tail fully congested. The tip of the tail was wiped with a sterilized cotton swab soaked in 75% ethanol to fully disinfect the blood sampling area. The tail was then wiped dry with a dry, clean, sterile cotton swab, and a disposable blood collection needle was used to pierce the congested area of ​​the tail tip (one needle for one mouse, and the blood collection needle was not reused). After the first drop of blood flowed out, it was wiped off with a clean, dry, sterilized cotton swab, and the second drop of blood was drawn with a blood glucose meter equipped with a clean blood glucose test strip. The blood glucose value displayed on the blood glucose meter was read in real time and accurately recorded.

[0148] 2.2.2 Wound healing rate

[0149] After 2 days of bacterial infection (marked as day 0), the dressing was changed every morning, the retention patch was removed, and after treatment, another layer of retention patch was covered on the wound site to exclude external contamination. The wound area on day 0 was set as 100%, and the wound area of ​​each group was quantified at the specified time, and the size of the wound was calculated using Fiji Image J images.

[0150]

[0151] 2.2.3 Detection of IL-6 and TNF-α levels in granulation tissue of ulcer wound

[0152] ELISA was used to detect the levels of IL-6 and TNF-α in the granulation tissue of the ulcer wound. After the experimental rats were killed and the ulcer surface tissue was wrapped in tin foil and frozen in liquid nitrogen, an appropriate amount of the cut skin tissue was weighed, added to a 9-fold sterile PBS solution, ground and mixed thoroughly, and then the skin granulation tissue homogenate was centrifuged at 3000-4000 rpm at 4°C for 10 minutes, the supernatant was collected, and the test was completed in strict accordance with the instructions of the kit.

[0153] 2.2.4 Detection of SOD and MDA levels in granulation tissue of ulcer wound

[0154] Biochemical method was used to detect the SOD activity and MDA level of granulation tissue of ulcer wound. The frozen tissue was taken out and thawed on ice. According to the instructions of the kit, the superoxide dismutase (SOD) activity was calculated by colorimetric analysis of WST-1 product. Malondialdehyde (MDA) in the degradation product of lipid peroxidation can react with thiobarbituric acid under high temperature and acidic environment. The MDA content of skin granulation tissue of rats in each group was detected by thiobarbituric acid method.

[0155] 2.2.5 Detection of VEGF and EGF levels in granulation tissue of ulcer wound

[0156] The expression of related healing factors (vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF)) in each group of rats was detected by ELISA. An appropriate amount of cut skin tissue was weighed and ground with 9-fold sterile PBS solution. Then the skin granulation tissue homogenate was centrifuged at 3000-4000rpm for 10 minutes at 4°C. The supernatant of the homogenate sample was collected and stored at 4°C. The VEGF and EGF contents in the skin granulation tissue of rats in each group were determined according to the instructions, and the final results were standardized according to the Bicinchoninic Acid Assay (BCA) method for the protein concentration detected.

[0157] 3 Results

[0158] (1) Changes in blood sugar in each group

[0159] The blood glucose levels of rats in different intervention groups were Figure 7 As shown, compared with the CON group, DM rats were in a stable hyperglycemic state during the experiment (P<0.01); during the 14-day treatment, there was no statistical difference in blood glucose between the rats in all intervention groups and the MOD group (P>0.05).

[0160] (2) Wound healing rate test results are shown in Figure 8 , Table 5.

[0161] Table 5 Comparison of wound healing rates in each group at different time points after treatment (%, )

[0162]

[0163] Note: Compared with the model group *P<0.05, **P<0.01

[0164] Depend on Figure 8 From Table 5, we can see that:

[0165] After wound modeling, the wounds of rats in the CON group were observed. After S. aureus infection, white pus was initially formed on the wounds of rats in the CON group at about 24 hours. At 48 hours, obvious purulent secretions could be seen, and redness and swelling appeared around the skin. The wounds of rats in the MOD group had less purulent secretions 1-2 days after bacterial induction, and the wound margins were slightly whiter than those in the CON group. The yellow-white secretions began to increase on the 3rd day. There was no statistical difference in the wound healing rates among the groups after the first three days of intervention (P>0.05). Compared with the CON group, the wound healing rate of rats in the MOD group was slow. On the 5th day of administration, the wound healing rate was significantly lower than that of the CON group, and yellow turbid pus could be seen on the wounds. The wound area was larger. Compared with the MOD group, the healing rates of the EO group, CPE group, and CPJ group were higher than those of the MOD group, and the difference was statistically significant (P<0.05 or P<0.01). The degree of redness and swelling of the wound margin was reduced compared with the previous one. After 7 days of administration, a small amount of yellow-white secretion was still visible in the EO and EGF groups, and light yellow and thin exudate was visible in the CPA group. New granulation tissue was visible in the CPA, CPE, CPJ intervention groups and the CON group, and the wounds contracted and healed significantly. The healing rates of rats after CPE and CPJ administration were significantly higher than those in the MOD group and the EO and EGF groups. In the comparison of different Cyclocarya paliurus extracts, CPJ had a higher ability to promote wound healing than CPA, and the difference was statistically significant (P < 0.05). There was no statistically significant difference between the Cyclocarya paliurus extract groups. On the 11th day of administration, the wound surface of the MOD group was covered with black scabs, and the healing rate was significantly lower than that of the CON group. The wounds of each intervention group of Cyclocarya paliurus were dry, with hair growth around them. The healing rate was significantly higher than that of the MOD group (P < 0.05), and there was no significant difference compared with the CON group. Compared with the different intervention methods, the healing rate of the EO group was significantly lower than that of the Cyclocarya paliurus groups at 11 days, and the healing rate of the EGF group was lower than that of the CPJ group, and the difference was statistically significant. There was no statistical difference in the healing rate among the Cyclocarya paliurus groups. On the 14th day of administration, inflammation was still visible at the junction of the wound and normal tissue in the MOD group. Compared with the MOD group, the wounds of the EGF group and the Cyclocarya paliurus groups were significantly healed (P < 0.05). The wounds of rats in the CPA, CPE, and CPJ groups healed significantly (P < 0.01), the scabs were absorbed and fell off, a small amount of unhealed was visible at the center of the circle, and there was almost no inflammatory reaction such as redness and swelling. The hair growth on the back was basically fully restored. Compared with the different intervention groups, the efficacy of the EO group was significantly inferior to that of the other groups, and the efficacy of CPJ was better than that of the EGF group and the CPA group.

[0166] (3) The experimental results of IL-6 and TNF-α levels in wound tissue are shown in Fig. 9 , Table 6.

[0167] Table 6 Expression of TNF-α and IL-6 inflammatory factors in the wound surface of rats in each group at different time points

[0168]

[0169] Note: Compared with the model group *P<0.05, **P<0.01

[0170] Depend on Fig. 9 From Table 6, we can see that:

[0171] Elisa method was used to detect the expression changes of IL-6 and TNF-α in the wound of rats. Compared with the CON group, the levels of TNF-α and IL-6 in the wound of rats in the MOD group were significantly increased (P<0.01); after 7 days of intervention, the levels of TNF-α and IL-6 in all intervention groups were significantly decreased compared with the MOD group. Compared with the intervention groups, CPJ could significantly reduce the level of TNF-α in the wound of rats infected with DM more than CPA, and the difference was statistically significant (P<0.05); in the IL-6 index, there was no statistical difference among the intervention groups; after 14 days, compared with the MOD group, except for the EO group, which did not significantly reduce the level of TNF-α, the levels of TNF-α and IL-6 in the other groups were significantly reduced. Compared with EO, CPJ can reduce the level of TNF-α in the late stage of wound healing, and it has a better effect on reducing the level of IL-6 than CPA and EGF.

[0172] (4) The experimental results of SOD activity and MDA expression level in wound tissue are shown in Fig.10 , Table 7.

[0173] Table 7 SOD activity and MDA expression levels in wound tissue

[0174]

[0175]

[0176] Note: Compared with the model group *P<0.05, **P<0.01

[0177] Depend on Fig.10 , Table 7 shows that:

[0178] In the wound tissue, compared with the CON group, the SOD activity of the MOD group was significantly reduced and the MDA level was significantly increased at 7d and 14d of wound healing, indicating that the MOD group rats showed obvious oxidative stress. The MDA level of each intervention group was significantly reduced at 7d, and the SOD activity was increased to varying degrees compared with the MOD group, among which the CPE and CPJ groups were more significant (P<0.05 or P<0.01). At 14d, the SOD and MDA levels of each intervention group were improved to varying degrees compared with the MOD group, among which the CPA and CPJ groups were more significant.

[0179] (5) Experimental results of VEGF and EGF expression levels in wound tissue are shown in Fig.11 , Table 8.

[0180] Table 8 Expression of VEGF and EGF in wounds of rats in each group at different time points

[0181]

[0182] Note: Compared with the model group *P<0.05, **P<0.01

[0183] Depend on Fig.11 , Table 8 shows that:

[0184] After 7 days of intervention, although the expression of VEGF in each intervention group and CON group increased to varying degrees, there was no statistically significant difference between the groups (P>0.05). In terms of EGF expression, compared with the CON group, the expression of EGF in the MOD group was in a low-level inhibition state at 7 days, and the EGF levels in the other intervention groups were significantly lower than that in the CON group at 7 days (P<0.05 or P<0.01). Compared with the MOD group, the EGF levels in each intervention group increased to varying degrees, and the CPJ group was more significant. After 14 days, VEGF and EGF in the MOD group were still in a state of secretion inhibition, and the VEGF level in the CON group increased compared with the previous level, while the EGF level decreased compared with the previous level. Compared with the MOD group, the VEGF and EGF levels in the CON and CPJ groups increased significantly at 14 days, and the EGF level in the CPE group was also significantly increased at 14 days.

[0185] In summary, different extracts of Cyclocarya paliurus can significantly reduce the secretion of inflammatory factors TNF-α and IL-6, improve oxidative stress status (SOD, MDA), and increase the expression of healing factors VEGF and EGF to promote the healing of DM-infected wounds through multiple pathways. At the same time, they make up for the shortcomings of EO and EGF in the application of DM-infected wounds at different stages and achieve relatively better therapeutic effects. This is the first report that external application of Cyclocarya paliurus can accelerate the wound healing process of DM animals.

[0186] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of Cyclocarya paliurus extract in the preparation of medicines for diabetic foot ulcers.

2. The use of the Cyclocarya paliurus extract product according to claim 1 in the preparation of a medicament for diabetic foot ulcers, characterized in that: The drug is an external-use drug; Preferably, the medicament comprises one of an ointment, a cream, a gel, a spray, a patch, a powder, a foam and a solution.

3. The use of the Cyclocarya paliurus extract product according to claim 1 in the preparation of a medicament for diabetic foot ulcers, characterized in that: The Cyclocarya paliurus extract product comprises at least one of a Cyclocarya paliurus extract and a Cyclocarya paliurus squeezed juice.

4. Use of the Cyclocarya paliurus extract product according to claim 3 in the preparation of a medicament for diabetic foot ulcers, characterized in that: The Cyclocarya paliurus juice is filtered and sterilized before preparing the medicine; Preferably, the Cyclocarya paliurus juice is prepared from fresh, green, smallest, fully expanded leaves picked from March to June.

5. Use of the Cyclocarya paliurus extract product according to claim 3 in the preparation of a medicament for diabetic foot ulcers, characterized in that: The Cyclocarya paliurus extract comprises Cyclocarya paliurus water extract and Cyclocarya paliurus alcohol extract.

6. Use of the Cyclocarya paliurus extract product according to claim 5 in the preparation of a medicament for diabetic foot ulcers, characterized in that: The preparation method of the Cyclocarya paliurus extract comprises: mixing crushed Cyclocarya paliurus leaves and an extractant, obtaining an extract after reflux extraction, concentrating and drying the extract under reduced pressure to obtain a powder extract, and diluting and filtering the powder extract to obtain the Cyclocarya paliurus extract.

7. Use of the Cyclocarya paliurus extract product according to claim 6 in the preparation of a medicament for diabetic foot ulcers, characterized in that: The particle size of the crushed Cyclocarya paliurus leaves is 10 to 50 meshes; Preferably, the volume mass ratio of the extractant to the Cyclocarya paliurus leaves in the reflux extraction step is 15 to 25 mL / g.

8. Use of the Cyclocarya paliurus extract product according to claim 6 in the preparation of a medicament for diabetic foot ulcers, characterized in that: The number of reflux extractions is one or more than two times. When the number of reflux extractions is more than two times, solid-liquid separation is performed after each reflux extraction to obtain a filtrate, and the filtrates obtained from each reflux extraction are combined to form the extract.

9. Use of the Cyclocarya paliurus extract product according to claim 6 in the preparation of a medicament for diabetic foot ulcers, characterized in that: The reflux extraction temperature is 110-130°C, and the single reflux extraction time is 0.5-2h.

10. Use of the Cyclocarya paliurus extract product according to claim 6 in the preparation of a medicament for diabetic foot ulcers, characterized in that: The concentration of the powder extract in the diluted liquid is 50 to 200 mg / mL.

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