Traditional Chinese medicine composition for promoting healing of chronic wound of diabetic foot and preparation method of traditional Chinese medicine composition

Abstract

The invention provides a traditional Chinese medicine composition for promoting chronic wound healing of diabetic foot and a preparation method of the traditional Chinese medicine composition. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 15 to 60g of honeysuckle flower, 15 to 60g of cortex phellodendri, 15 to 60g of radix scrophulariae, 15 to 60g of rhizoma atractylodis, 10 to 40g of radix angelicae sinensis, 10 to 40g of radix rehmanniae, 10 to 40g of rhizoma pinelliae and 5 to 20g of liquorice root. Based on the traditional Chinese medicine theory and the transcriptomics sequencing result of a clinical sample, the Simiaoyongan decoction is added, the improved compound traditional Chinese medicine composition of a new prescription is generated, the wound healing rate of a diabetic foot animal model can be effectively increased, the serum inflammatory factor level is reduced, and the curative effect is good. Compared with the original prescription, the traditional Chinese medicine target of the improved prescription is more targeted to pathways related to nervous system, hypoxia and blood circulation regulation, and the improved prescription has more advantages in the aspects of regulating inflammation, improving peripheral neuropathy, promoting wound healing and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine and Chinese medicine compositions, and in particular to a Chinese medicine composition for promoting the healing of chronic wounds of diabetic foot and a preparation method thereof. Background Art

[0002] Diabetic foot ulcer (DFU) is one of the most common and serious complications of diabetes, and one of the main causes of disability and death for patients. Studies have shown that approximately 18.6 million people suffer from diabetic foot ulcers each year worldwide, of which approximately 20% will result in amputation. The 5-year mortality rate of DFU patients is around 30%, while the 5-year mortality rate of amputation patients is over 70%, which seriously affects the quality of life of patients and also places a great burden on social medical care.

[0003] At present, clinical treatment mainly focuses on basic treatment, such as lowering blood sugar, lowering blood pressure, regulating blood lipids, nutritional support, etc.; surgical treatment includes wound repair and vascular remodeling, but the long-term postoperative patency rate is low and the risk of vascular restenosis is high. If the patient has vascular calcification lesions, it may also affect drug penetration; in addition, some commonly used drugs, such as vasoactive drugs, broad-spectrum antibiotics, tricyclic antidepressants and antianxiety drugs, although they can improve vascular lesions, resist infections and peripheral neuropathy, they may also increase the risk of bleeding, induce drug-resistant strains, and cause adverse reactions in the cardiovascular system, central nervous system and endocrine system.

[0004] Compared with Western medicine, Chinese medicine has more advantages in treating DFU, with various treatment methods, good treatment effects, few adverse reactions, and individualized treatment. Studies have shown that a variety of Chinese medicines and active ingredients can regulate inflammation, inhibit oxidative stress, and regulate glucose metabolism through signaling pathways such as Wnt, Nrf2 / ARE, MAPK, and NF-κB, thereby promoting diabetic wound healing; after comprehensive Chinese medicine treatment, compared with conventional treatment, the wound healing of DFU patients was accelerated, and the level of inflammatory factors in wound tissue was significantly reduced.

[0005] Simiao Yong'an Decoction comes from the New Edition of Proven Prescriptions. It is composed of Lonicera japonica Thunb, Scrophularia Ningpoensis Hemsl, Angelicae Sinensis Radix, and Licorice (Radix Glycyrrhizae Uralensis). It has the effects of clearing away heat and detoxifying, promoting blood circulation and removing blood stasis, and relieving pain. It is currently commonly used to treat cardiovascular diseases, regulate blood lipid metabolism, fight inflammation, and promote skin wound healing. Although the therapeutic effect of Simiao Yong'an Decoction is unquestionable, the dosage of the original prescription is too large, and the current clinical practice of adding or modifying the Chinese medicine compound according to the symptoms and constitution of different patients still lacks modern scientific principles and theoretical support, which hinders the understanding of Chinese medicine from the perspective of modern biomedicine. Summary of the invention

[0006] In order to address the deficiencies of the prior art, the present invention proposes a Chinese medicine composition for promoting the healing of chronic wounds of diabetic foot and a preparation method thereof. Based on the theory of traditional Chinese medicine and transcriptomics sequencing technology, Simiao Yong'an Decoction is flavored to produce a new prescription of an improved compound Chinese medicine composition, which can effectively promote the healing of chronic wounds of diabetic foot and reduce the level of serum inflammatory factors compared with the original prescription, and the dosage of medicinal materials is also relatively reduced.

[0007] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0008] The present invention provides a traditional Chinese medicine composition for promoting the healing of chronic wounds of diabetic foot, which is composed of the following raw materials in proportions by weight:

[0009] Honeysuckle 15-60g, Phellodendron chinense 15-60g, Scrophularia 15-60g, Atractylodes 15-60g, Angelica 10-40g, Rehmannia glutinosa 10-40g, Pinellia 10-40g, Licorice 5-20g.

[0010] Preferably, the Chinese medicine composition is composed of the following raw materials in the following weight proportions:

[0011] Honeysuckle 30g, Phellodendron 30g, Scrophularia 30g, Atractylodes 30g, Angelica 20g, Rehmannia glutinosa 20g, Pinellia 20g, Licorice 10g.

[0012] The present invention also provides a Chinese medicine oral preparation for promoting the healing of chronic wounds of diabetic foot, which is prepared using the Chinese medicine composition as described above and a raw material drug containing the active ingredients of the Chinese medicine composition.

[0013] Furthermore, the oral Chinese medicine preparation is a Chinese medicine decoction.

[0014] The present invention further provides a method for preparing a Chinese medicine oral preparation for promoting the healing of chronic wounds of diabetic foot, which is prepared using the Chinese medicine composition as described above and a raw material drug containing the active ingredients of the Chinese medicine composition.

[0015] Furthermore, the oral Chinese medicine preparation is a Chinese medicine decoction.

[0016] Furthermore, the preparation method of the Chinese medicine decoction comprises:

[0017] S1, adding pure water to the Chinese medicine composition to form a mixed raw material, soaking it, heating it for extraction, and filtering it to obtain an initial medicinal solution;

[0018] S2, performing a normal pressure concentration operation on the initial medicinal solution to generate a Chinese medicinal decoction.

[0019] Furthermore, the weight ratio of the Chinese medicine composition to pure water in the mixed raw material is 1:6 to 1:10.

[0020] Furthermore, the step S1 includes soaking the mixed raw materials for more than 2 hours, heating to boil and then reducing the heat to continue heating and extracting for 40 minutes, and retaining the initial medicinal solution and medicinal residues after filtering.

[0021] Furthermore, the step S2 comprises: adding 6-10 times the volume of pure water to the dregs after decoction in step S1, heating to boil and then turning to low heat to continue decocting for 40 minutes, filtering, and combining the decoctions obtained twice and then decocting over high heat to concentrate the decoction.

[0022] The beneficial effects of the present invention are:

[0023] The syndrome of this prescription (diabetic foot ulcer) is caused by internal heat and toxicity, stagnation of qi and blood, and deficiency of yin and blood, and among the three, the heat and toxicity are the main ones, and the treatment is suitable for clearing away heat and detoxification, promoting blood circulation and nourishing blood, and dredge meridians to relieve pain. The present invention adds flavors to Simiao Yong'an Decoction based on traditional Chinese medicine theory and transcriptomics sequencing to produce an improved compound Chinese medicine composition prescription with a new prescription, wherein: honeysuckle and phellodendron are reused as the main medicines in the prescription. Honeysuckle is sweet, cold, and refreshing, and has a dispersing nature. It has the power of dispersing in the clearing, clearing heat and penetrating, and leading the evil out. It can not only relieve the heat and toxicity of the internal organs, but also clear the stagnant heat of the meridians. It is aromatic and penetrating without containing evil; phellodendron is bitter and cold, enters the kidney, bladder, and large intestine meridians, and can not only clear damp heat, relieve fire toxins, but also damp heat toxins. It can clear pathogenic heat and enter the yin to reduce deficiency heat; Phellodendron chinense and honeysuckle are used together, one dispersing and the other descending, directly reaching the pathogenic factors and clearing away the heat and toxicity; Scrophularia ningpoensis is the assistant drug, which helps honeysuckle clear away heat and detoxify. The two drugs are used together, which can not only clear the pathogenic heat in the qi, but also resolve the heat and toxicity in the blood, and Scrophularia ningpoensis also has the effect of nourishing yin and dispersing stagnation; the assistant drug, Atractylodes lancea, which is bitter and warm, helps Phellodendron chinense to dry dampness and move the spleen, which strengthens the spleen without the bitterness of suppressing the stomach and intestines, and is bitter and warm without the risk of stirring up fire; the two drugs are compatible, yin and yang complement each other, cold and heat are coordinated, and both the symptoms and the root causes are taken into account, so that heat and dampness are eliminated. Angelica sinensis is warm and moist, promotes blood circulation and removes blood stasis, circulates blood vessels, nourishes yin and blood to moisten the extremities, and is an adjuvant; Rehmannia root is bitter and cold, enters the blood and blood, is sweet, cool and moist, and is specialized in clearing heat, cooling blood and nourishing yin; Pinellia ternata is pungent, warm and dry, and is used as an adjuvant to adjust the cold and heat, reduce adverse effects and harmonize the stomach, disperse knots and remove lumps, and can prevent the upward reversal of stomach qi caused by stomach cold, with both ascending and descending, to restore the normal ascending and descending of the spleen and stomach; Licorice is an adjuvant, firstly to help honeysuckle clear heat and detoxify, secondly to nourish yin and produce body fluid with Angelica sinensis and Scrophularia ningpoensis, and thirdly to harmonize the various medicines. The whole prescription uses both cold and hot to harmonize the yin and yang, and bitter and pungent to adjust the ascending and descending. Although the taste of the medicine is large, the amount is strong and specialized, and it plays the role of clearing heat and detoxifying, promoting blood circulation and relieving pain.

[0024] On the one hand, the present invention inherits the advantages of traditional Chinese medicine syndrome differentiation and prescription, and gives play to the characteristics of modern molecular diagnosis and precise treatment. It is convenient for clinical application and has a definite curative effect. Compared with the original Simiao Yong'an Decoction, it has a higher therapeutic efficiency and can significantly reduce the level of serum inflammatory factors. On the other hand, the improved Simiao Yong'an Decoction Chinese medicine composition of the present invention uses common medicinal materials as raw materials, which is low in cost and easy to find the source of medicine, effectively reducing the economic burden of patients, and can be applied to the preparation of a variety of different dosage forms. The preparation process is simple and suitable for large-scale industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The KAS-seq signal distribution characteristics in genomic DNA of foot wound tissue samples from patients with diabetic foot ulcer (DFU) and normal control group (Ctrl).

[0026] Figure 2Figure 2 is the differential gene analysis between the DFU and Ctrl groups, where Figure A is the unsupervised clustering heatmap analysis of the Top 100 differentially expressed genes between the Ctrl and DFU groups, and Figure B is the volcano plot analysis of the differentially expressed genes between the two groups (up means up-regulated in the DFU group, and down means down-regulated in the DFU group).

[0027] Figure 3 Figure 2 is the functional analysis of up-regulated genes in Ctrl and DFU groups, where Figure A is the GO enrichment analysis dot plot of up-regulated genes, and Figure B is the GO enrichment analysis dot plot of down-regulated genes.

[0028] Figure 4 This is the traditional Chinese medicine-active ingredient-target regulation network diagram of the original Simiao Yong'an Decoction.

[0029] Figure 5 Schematic diagram of the screening of key genes for the improvement of Simiao Yong'an Decoction. Figure A is a Venn diagram of the intersection of KAS-seq, the original Simiao Yong'an Decoction TCM targets, and the gene set related to diabetic foot ulcers in the Genecards database. Figure B is a functional analysis point diagram of 534 genes that exist in KAS-seq data and external datasets but are not included in the original TCM targets.

[0030] Figure 6 This is the PPI network diagram of 110 differentially expressed genes enriched in pathways closely related to diabetic foot.

[0031] Figure 7 These are the key genes screened from 110 genes through machine learning. Figure A is the ROC diagram of the accuracy of the eight key genes in distinguishing the DFU and Ctrl groups. Figure B is the name, site, difference fold and P value of the eight key genes.

[0032] Figure 8 This is the traditional Chinese medicine-active ingredient-target regulatory network diagram of the improved formula of Simiao Yong'an Decoction.

[0033] Fig. 9 This is the functional analysis of the Chinese medicine targets in the original and improved formulas. Figure A is the functional analysis dot diagram of the Chinese medicine targets in the original formula of Simiao Yong'an Decoction, and Figure B is the functional analysis dot diagram of the Chinese medicine targets in the improved formula of Simiao Yong'an Decoction.

[0034] Fig.10 This is a schematic diagram of the animal experiment used to verify the effects of the original and improved formulas in promoting DFU wound healing at high, medium and low doses in Test Example 1.

[0035] Fig.11 The wound healing rates of mice in each group in Test Example 1 at the 0th, 3rd, 7th and 12th days of administration.

[0036] Fig.12This is a schematic diagram of verifying the therapeutic effect of the improved prescription through animal experiments in Test Example 2. Figure A shows the wound healing status of each group of mice at different time points; Figure B shows the wound healing rate of each group on the 7th day; and Figure C shows the wound healing rate of each group of mice on the 12th day.

[0037] Fig.13 This is a schematic diagram of the levels of serum inflammatory factors in the four groups of experimental animals after drug intervention in Test Example 2, Figure A is the TNF-α level, Figure B is the IL-6 level, and Figure C is the IL-1β level.

[0038] Fig.14 It is the overall technical roadmap of the present invention. DETAILED DESCRIPTION

[0039] In order to explain the present invention more clearly, the present invention is further described in detail below in conjunction with embodiments and with reference to the accompanying drawings. It should be understood by those skilled in the art that the content described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0040] Example

[0041] Combination Fig.14 The present invention mainly includes the following aspects:

[0042] 1. Sample Collection

[0043] Granulation tissue samples of foot wounds of 6 patients with diabetic foot ulcers were collected from patients admitted to Peking University Third Hospital from January to December 2021 as the DFU group; the control group was collected from granulation tissue of foot wounds of 3 healthy volunteers during the same period as the control group (Ctrl). Check the patient bed information, organize the sample information sheet, take fresh granulation tissue from the foot wound, put it in liquid nitrogen, transport it to the laboratory, check the sample information after receiving it, and store the sample at -80℃.

[0044] The sample collection procedures were in compliance with the patient informed consent and medical ethics approval requirements.

[0045] 2. Extraction of wound tissue N3 markers and DNA extraction

[0046] Take 25 mg of tissue sample and add 500 μL of 1×PBS. Cut it as finely as possible at 4°C, aspirate the tissue cell suspension, centrifuge at 1500g for 5 minutes, discard the supernatant, and collect the tissue cells. Add 1 mL of DMEM culture medium and 5 mM N3-ketol (2.75 mM, 1.82 mL) to the tissue cells and incubate in a 37°C incubator with 5% CO2 for 10 minutes. Centrifuge the cultured cell suspension at 1500 rpm for 3 minutes, remove the culture medium, add 1 mL of PBS to resuspend, centrifuge at 1500 rpm for 3 minutes, and remove PBS. Use the PureLink PM Genomic DNA Mini Kit to extract genomic DNA (gDNA).

[0047] 3. KAS-seq library construction

[0048] Suspend 1 μg of genomic DNA in 95 μL of DNA elution buffer, add 5 μL of 20 mM DBCO-PEG4-biotin (DMSO solution, Sigma, 760749), 25 mM K3BO3, incubate at 37°C for 1.5 h, and shake gently. Then add 5 μL of RNase A (Thermo, 12091039) to the reaction mixture and incubate at 37°C for 5 min. Then use DNA Clean & Concentrator-5 kit (Zymo, D4013) to recover biotinylated DNA. The gDNA was suspended in 100 μL of enzyme-free water and fragmented into 150-350 bp in size using a Bioruptor Pico at 30-on / 30s-off for 30 cycles; 5% of the DNA fragments were saved as input, and the remaining 95% were incubated with 10 μL of pre-washed Dynabeads MyOne Streptavidin C1 (Thermo, 65001) at room temperature for 15 min to enrich the biotin-labeled DNA. The beads were washed and heated at 95°C for 10 min in 15 μL of enzyme-free water to elute the DNA. The eluted DNA and its corresponding input were used for library construction using the Accel-NGS Methyl Sequence DNA Library Kit (Swift, 30024). The library was sequenced on the Illumina Novaseq6000 platform using a double-end 150 bp mode, with the goal of obtaining 30 million reads per library.

[0049] 4. Raw Data Processing

[0050] The constructed library was quality controlled using Fast QC (version 0.11.5). The raw sequencing data were aligned and annotated with the human genome (version hg19) using bowtie2 (version 2.2.9), and the data that did not meet the conditions were filtered using SAMtools (version 1.3.1). MACS2 (version 2.1.1) was used to identify the possible KAS-seq enriched regions of each sample, and the CHIP seeker Package (version 1.20.0) was used to annotate the KAS-seq enriched regions, and the genes that best matched the marked regions were used for subsequent functional analysis.

[0051] V. Difference Analysis

[0052] The differentially expressed genes between the Ctrl group and the DFU group were analyzed using the DEseq2 package in the RStudio software, with the cutoff values ​​set to P<0.05 and |log2 Fold Change|>0.5, and the volcano plot of the differentially expressed genes was drawn using the ggPlot2 package.

[0053] VI. Functional analysis of differentially expressed genes

[0054] The differentially expressed genes between the two groups were analyzed for functional enrichment using the Metscape website (https: / / metascape.org / ) and the ClueGO (version 2.5.5) and CluePedia (version 1.5.5) plug-ins in Cytoscape.

[0055] 7. Statistical Analysis

[0056] The difference analysis between the two groups of data was performed using RStudio software, Sangerbox website (http: / / www.sangerbox.com / login.html) and GraphPed Prism 8 software, and p < 0.05 proved to be statistically significant. The measurement data in the clinical sample information were expressed as mean ± standard deviation express.

[0057] 8. Construction of improved Simiaoyong'an decoction based on target and network pharmacology

[0058] Based on the clinical cohort, key targets and TCM theories were screened, and Simiao Yong'an Decoction was flavored with network pharmacology technology. Based on the TCMSP and ETCM databases, Chinese medicines that act on key sites of diabetic foot were retrieved and added.

[0059] Firstly, the Chinese medicine in Simiao Yong'an Decoction was searched through the TCMSP database (https: / / old.tcmsp-e.com / tcmsp.php), and the active ingredients (Ingredients) and the targets (Target) of each medicinal material were downloaded; when screening the active ingredients, the threshold was set as bioavailability (OB%) greater than 30% and drug-likeness (DL) greater than 0.18; then the screened target name was converted into gene name form through the UniProt database (https: / / www.uniprot.org / ) and Perl language for subsequent analysis; in addition, the active ingredients and targets of Chinese medicine were further supplemented through the ETCM database.

[0060] IX. Effectiveness verification of improved Simiaoyong'an decoction based on network pharmacology

[0061] The improved Simiao Yong'an Decoction was screened out based on KAS-seq sequencing, and the "traditional Chinese medicine-active ingredients-target" regulatory network of the improved Simiao Yong'an Decoction was obtained using Cytoscape3.7.2 software to verify the effectiveness of the new prescription.

[0062] 1. Analysis of sequencing data of diabetic foot patients

[0063] KAS-seq sequencing technology was used to detect foot wound tissues of DFU patients and Ctrl group. The results showed that the KAS-seq signal intensity of Ctrl group was generally higher than that of DFU patients ( Figure 1 ), and the two groups of signals are mainly distributed in the transcriptionally active regions of the genome, such as the transcription start site (TSS) and the transcription termination site (TTS). After analyzing the Ctrl and DFU groups, a total of 2545 differentially expressed genes were obtained. Unsupervised cluster analysis showed that these differentially expressed genes can well distinguish the two groups of samples ( Figure 2 A). Among these differentially expressed genes, 1049 genes were significantly upregulated in DFU patients, and 1496 genes were significantly downregulated in DFU patients ( Figure 2B). Functional enrichment was performed on the up-regulated and down-regulated differentially expressed genes, respectively. The up-regulated genes were enriched in axon development, glycoprotein biosynthesis and metabolism-related processes (Glycoprotein biosynthetic process, glycosylation, Glycoprotein metabolic process, etc.), and immune cell differentiation and apoptosis-related pathways (Regulation of leukocyte apoptotic process, leukocyte apoptotic process, CD8-positive, α-βT cell differentiation, etc.) ( Figure 3 A), while the down-regulated genes were enriched in the nervous system-related pathways such as axon development, regulation of nervous system development, and synaptic signal transmission and regulation, as well as the disease-related pathways such as Wnt, cellular response to decreased oxygen levels, hematopoietic progenitor cell differentiation, and cell growth and differentiation (Cell growth, Columnar / cuboidal epithelial cell differentiation). Figure 3 B).

[0064] 2. Pharmacological description of the original Chinese medicine composition of Simiao Yong'an Decoction

[0065] The original formula of Simiao Yong'an Decoction has the effects of clearing away heat and detoxifying, promoting blood circulation and relieving pain. It is mainly used to treat gangrene with intense heat and toxicity. The affected limb is dark red, slightly swollen and burning, ulcerated and putrid, with severe pain, or fever, thirst, red tongue and rapid pulse. It is commonly used in the clinical treatment of thromboangiitis obliterans, phlebitis, lower limb ulcers, sciatica, deep vein thrombosis of the lower limbs, etc. Honeysuckle is sweet and cold and enters the heart. It is good at clearing heat and detoxifying, so it is used as the main medicine. Angelica promotes blood circulation and removes blood stasis, Scrophularia purges fire and detoxifies, and Licorice clears and removes all poisons. Honeysuckle is used to strengthen the power of clearing heat and detoxifying, and the dosage is not light, which is a good prescription for treating gangrene. The combination of the four medicines can not only clear away heat and detoxify, but also promote blood circulation and remove blood stasis.

[0066] This application conducted a network pharmacology analysis on the original formula of Simiao Yong'an Decoction. The results showed that the four medicinal materials of honeysuckle, Scrophularia, Angelica and Licorice contained a total of 112 active ingredients and targeted 118 gene targets. The regulatory network of traditional Chinese medicine-active ingredients-action targets was constructed ( Figure 4 ).

[0067] 3. Network pharmacology study and pharmacological description of the traditional Chinese medicine composition of the improved formula of Simiao Yong'an Decoction

[0068] The differentially expressed genes obtained by KAS-seq sequencing, the disease-related genes in the Genecards database, and the traditional Chinese medicine targets of Simiao Yong'an Decoction were intersected. In order to improve the original prescription, 534 genes included in the KAS-seq data and external datasets but not included in the traditional Chinese medicine targets of the original prescription were selected for subsequent screening ( Figure 5 A). Functional enrichment analysis was performed on these genes ( Figure 5 B), the results showed that it was enriched in signal pathways closely related to diseases, such as RAP1, MAPK, PI3K-Akt, inflammation, platelet activation, insulin secretion and lipid decomposition, proving that this part of genes contains potential targets for the improved Simiao Yong'an Decoction.

[0069] These 534 genes were used to reverse search the TCMSP and ETCM databases, and a total of 110 genes in the databases could be targeted to traditional Chinese medicine ( Figure 6 ). Further machine learning screening revealed 8 markers that could distinguish DFU from Ctrl with an AUC value of 0.987 ( Figure 7 A), among which the expressions of CREB1, GRID2, PIKERE, DRD2, ADORA1, TNFSF11, and EDNRB were significantly downregulated in patients with diabetic foot, and the expression of HTR1B was significantly upregulated in patients with diabetic foot ( Figure 7 B).

[0070] On this basis, based on the results of network pharmacology research in the TCMSP database, the traditional Chinese medicines Phellodendron chinense, Atractylodes macrocephala, Rehmannia glutinosa and Pinellia ternata, which target key targets, were added to the original formula of Simiao Yong'an Decoction to prepare an improved Simiao Yong'an Decoction Chinese medicine composition (hereinafter referred to as the improved formula), and the efficacy was verified.

[0071] The formula compatibility of the modified prescription is analyzed as follows: The syndrome of this prescription (diabetic foot ulcer) is caused by internal heat and toxicity, stagnation of qi and blood, and deficiency of yin and blood. Among the three, the heat and toxicity are the main ones. The treatment should be to clear away heat and detoxify, promote blood circulation and nourish blood, and relieve pain. Honeysuckle and Phellodendron are used as the main drugs in the prescription. Honeysuckle is sweet, cold, and refreshing, and has a dispersing nature. It has the power of dispersing and penetrating in the clearing, clearing heat and penetrating, and leading the evil out. It can not only relieve the heat and toxicity of the internal organs, but also clear the stagnant heat in the meridians. It is fragrant and penetrating without containing evil. Phellodendron is bitter and cold, and enters the kidney, bladder, and large intestine meridians. It can not only clear damp heat, relieve fire toxicity, and clear damp heat and toxic evil, but also enter the yin to relieve deficiency heat. Phellodendron and honeysuckle are used together, one dispersing and one descending, directly reaching the pathogenic evil, so that the heat and toxicity can be cleared. Scrophularia is a ministerial drug, which helps honeysuckle to clear away heat and detoxify. The two drugs are used together to clear away evil heat in the qi and detoxify the blood, and Scrophularia has the effect of nourishing yin and dispersing stagnation. The ministerial drug uses bitter and warm Atractylodes to help Phellodendron chinense to dry dampness and move the spleen, which is healthy and moving without the bitterness of suppressing the stomach and intestines, and bitter and warm without the risk of stirring fire. The two drugs are compatible, yin and yang are balanced, cold and heat are coordinated, and both the symptoms and the root are taken into account, so that heat is removed and dampness is eliminated. Angelica is warm and moist, promotes blood circulation and removes blood stasis, circulates blood vessels, nourishes yin and blood to moisten the four extremities, and is an adjuvant. Rehmannia root is bitter and cold, enters the blood and blood, is sweet, cool and moist, and specializes in clearing heat, cooling blood and nourishing yin. Pinellia is pungent, warm and dry. As an adjuvant, it adjusts its cold and heat, descends the adverse and harmonizes the stomach, disperses stagnation and removes lumps, and can prevent the stomach qi from going up due to stomach cold, with both ascending and descending, so as to restore the normal ascending and descending of the spleen and stomach. Licorice is used as an adjuvant, firstly to help honeysuckle clear away heat and detoxify, secondly to nourish yin and produce body fluid with angelica and Scrophularia, and thirdly to harmonize the other medicines. The whole prescription uses both cold and hot to harmonize yin and yang, and bitter and pungent to adjust the rise and fall. Although the taste of the medicine is large, the amount is strong and the effect is concentrated, which can clear away heat and detoxify, promote blood circulation and relieve pain.

[0072] The applicant conducted a network pharmacology analysis on the improved formula, and the results showed that the four medicinal materials of Phellodendron chinense, Atractylodes macrocephala, Rehmannia glutinosa and Pinellia ternata contained a total of 111 active ingredients, and 456 new gene targets were targeted on the basis of the original formula, and a regulatory network of traditional Chinese medicine-active ingredients-action targets was constructed ( Figure 8 ).

[0073] Functional analysis of the traditional Chinese medicine targets of the original and improved Simiao Yong'an Decoction formulas showed that the targets of the original formula were mainly enriched in fatty acid metabolism, lipid catabolism, unsaturated fatty acid metabolism and other lipid metabolism-related pathways that are closely related to the occurrence and development of diabetic foot ulcers ( Fig. 9 A); compared with the original formula, the targets of the modified formula were enriched in pathways related to lipid metabolism, and more pathways related to the nervous system, such as synaptic chemical transmission, postsynaptic signal transduction, and neurotransmitter level regulation; in addition, some pathways related to hypoxia and blood circulation regulation were also enriched ( Fig. 9 B), suggesting that it may play a role in regulating inflammation, improving peripheral neuropathy and promoting wound healing through the above-mentioned related biological processes.

[0074] The Chinese medicine composition of the present invention is used to promote wound healing of diabetic foot, and the efficacy of the Chinese medicine composition is verified by a randomized controlled experiment on an animal model.

[0075] Test Example 1

[0076] 1. Animal model grouping, modeling and drug administration

[0077] The study used 6-week-old male SPF C57BKS-DB(- / -) mice (db / db) to establish a DFU wound model. All animals were placed under standard 12-hour light / 12-hour dark cycle conditions and fed a standard diet. After one week of adaptive feeding, db / db mice with random blood glucose>16.7mmol / L met the requirements. After skin preparation, a circular mark with a diameter of 1 cm was made in the middle of the back below the shoulder blade of each group of mice. The skin was cut along the mark to the depth of the fascia, and after routine disinfection, it was bandaged with a medical bandage to prevent the mice from scratching and biting the wound.

[0078] Twenty-one db / db mice were randomly divided into seven groups: the control group was gavaged with normal saline, the four high group was gavaged with the original formula (24g / kg, 0.1mL / 10g), the four medium group was gavaged with the original formula (12g / kg, 0.1mL / 10g), the four low group was gavaged with the original formula (6g / kg, 0.1mL / 10g), the four + high group was gavaged with the modified formula (49.4g / kg, 0.1mL / 10g), the four + medium group was gavaged with the modified formula (24.7g / kg, 0.1mL / 10g), and the four + low group was gavaged with the modified formula (12.3g / kg, 0.1mL / 10g). The wound area was measured on the 0th, 3rd, 7th, and 12th day after intervention, and the wound healing rate was calculated (the calculation formula is: (initial area-measured area at different time points) / initial area*100%).

[0079] 2. Preparation of Chinese Herbal Decoction

[0080] Composed of the following raw materials by weight:

[0081] Four high group: honeysuckle 60g, Scrophularia 60g, Chinese angelica 40g, liquorice 20g;

[0082] Group IV: 30g honeysuckle, 30g Scrophularia, 20g Chinese angelica, 10g liquorice;

[0083] Four low group: honeysuckle 15g, Scrophularia 15g, Chinese angelica 10g, liquorice 5g;

[0084] Four + high group: honeysuckle 60g, phellodendron 60g, Scrophularia 60g, Atractylodes 60g, Angelica 40g, Rehmannia 40g, Pinellia 40g, Licorice 20g;

[0085] Four + medium group: honeysuckle 30g, phellodendron 30g, Scrophularia 30g, Atractylodes 30g, Angelica 20g, Rehmannia glutinosa 20g, Pinellia 20g, Licorice 10g;

[0086] Four + low group: honeysuckle 15g, phellodendron 15g, Scrophularia 15g, Atractylodes 15g, Angelica 10g, Rehmannia glutinosa 10g, Pinellia 10g, Licorice 5g.

[0087] The medium dose is calculated as 9.1 times the daily dose for adults, and then added to 6-10 times the volume of pure water for 2 hours before decoction. After boiling over high heat, turn to low heat and decoct for 40 minutes, and filter out the filtrate for later use; add 6-10 times the volume of pure water to the residue again, boil over high heat, turn to low heat and decoct for 40 minutes, and filter out the filtrate. Mix the two filtrates, concentrate at high temperature to the required concentration volume, and store at 4°C for a short period of time before use.

[0088] 3. Statistical Methods

[0089] The data of each group were compared by one-way analysis of variance, and the data related to the animal experiments were visualized by GraphPad Prism8 software.

[0090] IV. Experimental Results

[0091] The wound healing rate of each group of mice at different drug intervention time points was calculated by (initial area - measured area at different time points) / initial area*100% Fig.10 ). From the results, on the 3rd day, the wound healing rates of the four high groups, four + medium and four + high groups were significantly higher than those of the control group, and the wound healing rates of the four high groups and four + medium groups were significantly higher than those of the control group (Table 1); on the 7th day, the wound healing rates of the four medium groups, four high groups, four + medium and four + high groups were significantly higher than those of the control group, and the wound healing rate of the four high group was significantly higher than that of the four low groups (Table 2); on the 12th day, the wound healing rates of the four high groups, four + low, four + medium and four + high groups were significantly higher than those of the control group, and the wound healing rate of the four + medium group was significantly higher than that of the four low groups (Table 3).

[0092] Table 1: Significant differences in wound healing rates among mice groups on day 3 (*: p<0.05, **: p<0.01)

[0093]

[0094]

[0095] Table 2: There were significant differences in the wound healing rates of mice in each group on day 7 (*: p<0.05, **: p<0.01)

[0096]

[0097] Table 3: There were significant differences in the wound healing rates of mice in each group on day 12 (*: p<0.05, **: p<0.01)

[0098]

[0099] The results showed that the high, medium and low dose groups of the modified prescription could significantly promote wound healing in the DFU animal model, and after comprehensive analysis, the modified prescription had the best therapeutic effect at the medium dose.

[0100] Test Example 2

[0101] 1. Animal model grouping, modeling and drug administration

[0102] The study used 6-week-old male SPF C57BKS-DB(- / -) mice (db / db) to establish a DFU wound model, and 6-week-old male SPF C57BKS-DB(+ / +) mice (db / m) were used as the healthy group. All animals were placed under standard 12-hour light / 12-hour dark cycle conditions and fed a standard diet. After one week of adaptive feeding, db / db mice with random blood glucose>16.7mmol / L met the requirements. After skin preparation, a circular mark with a diameter of 1 cm was made in the middle of the back below the shoulder blade of each group of mice. The skin was cut along the mark to the depth of the fascia, and after routine disinfection, it was bandaged with a medical bandage to prevent the mice from scratching and biting the wound.

[0103] Fifteen db / db mice were randomly divided into three groups: the Ctrl group was gavaged with normal saline, the SMYAT group was gavaged with the original formula (11.7 g / kg, 0.1 mL / 10 g), the SMYAT+ group was gavaged with the modified formula (24.7 g / kg, 0.1 mL / 10 g), and the db / m mice were gavaged with normal saline as the Healthy group (n=5). The wound area was measured at 0, 3, 7, and 12 days after intervention, and the wound healing rate was calculated (the calculation formula was: (initial area - area measured at different time points) / initial area * 100%).

[0104] 2. Preparation of Chinese herbal medicine decoction: Same as Example 1.

[0105] 3. Statistical method: Same as Example 1.

[0106] IV. Experimental Results

[0107] The wound healing rate of each group of mice at different drug intervention time points was calculated by (initial area - measured area at different time points) / initial area*100% Fig.12 A). From the results, the wound healing rate of the modified Simiao Yong'an Decoction group was significantly higher than that of the Ctrl group on the 7th day (P<0.01) ( Fig.12 B); On the 12th day, the wound healing rate of the modified Simiao Yong'an Tang group was significantly higher than that of the original Simiao Yong'an Tang group (P<0.05) ( Fig.12 C), indicating that compared with the original recipe, the improved recipe can significantly promote wound healing in the DFU animal model.

[0108] The levels of serum inflammatory factors in the four groups of mice were detected by ELISA. First, the standard curve was drawn according to the concentration and absorbance value of the standard wells. The curve equation and r2 value of TNF-α were y=2.53215 / [1+(x / 867.50466)^-1.36523]+0.02319 and 0.99982, respectively; the curve equation and r2 value of IL-6 were y=9.95329 / [1+(x / 1361.63947)^-1.09666]+0.03334 and 0.99992, respectively; the curve equation and r2 value of IL-1β were y=2.04456 / [1+(x / 736.55342)^-1.51532]+0.04861 and 0.99952, respectively. The absorbance value of each well was put into the curve equation and multiplied by the coefficient to calculate the level of inflammatory factors. The results showed that TNF-α( Fig.13 A) and IL-6 ( Fig.13 B) was significantly lower than that in the original prescription group (P<0.05); the IL-1β( Fig.13 C) level was slightly lower than that of the original group, but the difference was not statistically significant (P = 0.0664).

[0109] The results showed that the improved Simiao Yong'an Decoction had a significant promoting effect on wound healing in diabetic foot model mice compared with the original prescription, and could reduce the levels of serum IL-1β, IL-6 and TNF-α; functional analysis showed that the Chinese medicine targets of the improved prescription were more enriched in nerve and synapse-related signaling pathways, blood circulation, vascular morphology and vascular function maintenance-related pathways, as well as responses to decreased oxygen levels and response pathways to oxygen levels. This may be the main mechanism of the improved prescription in the treatment of diabetic foot ulcers. Compared with the original prescription, the improved prescription may have more advantages in improving peripheral neuropathy.

[0110] Obviously, the above embodiments of the present invention are merely examples to more clearly illustrate the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A Chinese medicine composition for promoting the healing of chronic wounds of diabetic foot, comprising the following raw materials in the following weight proportions: Honeysuckle 15-60g, Phellodendron chinense 15-60g, Scrophularia 15-60g, Atractylodes 15-60g, Angelica 10-40g, Rehmannia glutinosa 10-40g, Pinellia 10-40g, Licorice 5-20g.

2. The Chinese medicine composition according to claim 1, characterized in that: The Chinese medicine composition is composed of the following raw materials in the following weight proportions: Honeysuckle 30g, Phellodendron 30g, Scrophularia 30g, Atractylodes 30g, Angelica 20g, Rehmannia glutinosa 20g, Pinellia 20g, Licorice 10g.

3. A Chinese medicine oral preparation for promoting the healing of chronic wounds of diabetic foot, prepared using the Chinese medicine composition according to claim 1 or 2 and a raw material drug containing the active ingredient of the Chinese medicine composition. The oral Chinese medicine preparation according to claim 3 , which is a Chinese medicine decoction.

5. A method for preparing a Chinese medicine oral preparation for promoting the healing of chronic wounds of diabetic foot, which is prepared using the Chinese medicine composition according to claim 1 or 2 and a raw material drug containing the active ingredient of the Chinese medicine composition.

6. The preparation method according to claim 5, characterized in that: The Chinese medicine oral preparation is a Chinese medicine decoction.

7. The preparation method according to claim 6, characterized in that: The preparation method of the Chinese medicine decoction comprises: S1, adding pure water to the Chinese medicine composition to form a mixed raw material, soaking it, heating it for extraction, and filtering it to obtain an initial medicinal solution; S2, performing a normal pressure concentration operation on the initial medicinal solution to generate a Chinese medicinal decoction.

8. The preparation method according to claim 7, characterized in that: The weight ratio of the Chinese medicine composition to pure water in the mixed raw material is 1:6 to 1:

10.

9. The preparation method according to claim 7, characterized in that: The step S1 comprises soaking the mixed raw materials for more than 2 hours, heating and boiling, then reducing the heat to continue heating and extracting for 40 minutes, and filtering and retaining the initial medicinal solution and medicinal residues.

10. The preparation method according to claim 9, characterized in that: The step S2 comprises: adding 6-10 times the volume of pure water to the dregs of the decoction in step S1, heating and boiling, then turning to low heat and continuing to decoct for 40 minutes, filtering, and combining the decoctions obtained from the two decoctions and then decocting with high heat to concentrate the decoction.

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