CN119139411A

CN119139411A - Traditional Chinese medicine composition for promoting healing of diabetic foot chronic wound and preparation method thereof

Info

Publication number: CN119139411A
Application number: CN202411367233.5A
Authority: CN
Inventors: 林坚; 李雪卉
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2024-09-29
Filing date: 2024-09-29
Publication date: 2024-12-17

Abstract

The invention provides a traditional Chinese medicine composition for promoting healing of diabetic foot chronic wounds and a preparation method thereof, wherein the traditional Chinese medicine composition comprises the following raw materials, by weight, 15-60g of honeysuckle, 15-60g of cortex phellodendri, 15-60g of radix scrophulariae, 15-60g of rhizoma atractylodis, 10-40g of angelica sinensis, 10-40g of radix rehmanniae, 10-40g of pinellia ternate and 5-20g of liquorice. The invention adds flavor to the four wonderful branchia angan decoction based on the traditional Chinese medicine theory and the transcriptome sequencing result of clinical samples, generates an improved compound traditional Chinese medicine composition prescription of a new prescription, can effectively improve the wound healing rate of a diabetic foot animal model, reduces the serum inflammatory factor level, targets more targeted paths related to nervous system, hypoxia and blood circulation regulation compared with the original prescription, and has more advantages in the aspects of regulating inflammation, improving peripheral neuropathy, promoting wound healing and the like.

Description

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

Technical Field

The invention relates to the technical field of traditional Chinese medicine and traditional Chinese medicine compositions, in particular to a traditional Chinese medicine composition for promoting healing of chronic wounds of diabetic feet and a preparation method thereof.

Background

Diabetic foot ulcers (Diabetic foot ulcer, DFU) are one of the most common and serious complications of diabetes and one of the main causes of disability and mortality for patients. Studies have shown that about 1860 tens of thousands of people worldwide suffer from diabetic foot ulcers each year, of which about 20% result in amputation. The death rate of the DFU patient in 5 years is about 30%, and the death rate of the amputee in 5 years is above 70%, so that the life quality of the patient is seriously influenced, and great burden is brought to social medical treatment.

The clinical treatment at present mainly comprises basic treatment such as blood sugar reduction, blood pressure reduction, blood lipid regulation, nutrition support and the like, surgical treatment including wound repair and vascular remodeling, but has low long-term smoothness rate and high vascular restenosis risk, and can influence drug permeation if a patient has vascular calcification lesions, and in addition, some commonly used drugs such as vascular active drugs, broad-spectrum antibiotics, tricyclic antidepressants, anxiolytics and the like can improve vascular lesions, anti-infection and peripheral neuropathy, but can also increase bleeding risk, induce drug-resistant strains and cause adverse reactions of cardiovascular systems, central nervous systems and endocrine systems.

Compared with western medicine treatment, the traditional Chinese medicine has the advantages of multiple treatment methods, good treatment effect, less adverse reaction, personalized treatment and the like in the aspect of treating DFU. Research shows that various traditional Chinese medicines and active ingredients can regulate inflammation, inhibit oxidative stress, regulate glucose metabolism and the like through Wnt, nrf2/ARE, MAPK, NF- κB and other signal paths, so that the healing of the wound surface of diabetes is promoted, and compared with the conventional treatment, the comprehensive treatment of the traditional Chinese medicines has the advantages that the healing of the wound surface of a DFU patient is quickened, and the level of inflammatory factors of wound surface tissues is obviously reduced.

The four wonderful brave and An decoction is prepared from flos Lonicerae (Lonicera japonica Thunb), radix scrophulariae (Scrophularia Ningpoensis Hemsl), radix Angelicae sinensis (ANGELICAE SINENSIS Radix) and Glycyrrhrizae Radix (Radix Glycyrrhizae Uralensis) by mixing, and has effects of clearing heat, detoxicating, promoting blood circulation for removing blood stasis, and relieving pain. At present, the traditional Chinese medicine composition is commonly used for treating cardiovascular diseases, regulating blood lipid metabolism, resisting inflammation, promoting skin wound healing and the like. Although the curative effect of the four wonderful brave An decoction is undoubtedly, the dosage of the original prescription is too large, and the modern scientific principles and theoretical support still lack when the traditional Chinese medicine compound is clinically added or modified according to the symptoms and physique of different patients at present, which hinders the knowledge of the traditional Chinese medicine from the modern biomedical angle.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a traditional Chinese medicine composition for promoting the healing of diabetic foot chronic wounds and a preparation method thereof, the four wonderful branchlet's an decoction is flavored based on the traditional Chinese medicine theory and the transcriptome sequencing technology, a new prescription of the improved compound traditional Chinese medicine composition formula is generated, compared with the original prescription, the traditional Chinese medicine composition formula can effectively promote the healing of diabetic foot chronic wounds and reduce the serum inflammatory factor level, and the medicinal material dosage is relatively reduced.

In order to achieve the above object, the present invention adopts the technical scheme that:

The invention provides a traditional Chinese medicine composition for promoting healing of diabetic foot chronic wounds, which is prepared from the following raw materials in parts by weight:

15-60g of honeysuckle, 15-60g of phellodendron bark, 15-60g of radix scrophulariae, 15-60g of rhizoma atractylodis, 10-40g of angelica sinensis, 10-40g of radix rehmanniae, 10-40g of pinellia ternate and 5-20g of liquorice.

Preferably, the traditional Chinese medicine composition comprises the following raw materials in parts by weight:

30g of honeysuckle, 30g of phellodendron bark, 30g of figwort, 30g of rhizoma atractylodis, 20g of angelica, 20g of dried rehmannia root, 20g of pinellia tuber and 10g of liquorice.

The invention also provides a traditional Chinese medicine oral preparation for promoting the healing of the chronic wounds of the diabetic feet, which is prepared from the traditional Chinese medicine composition and the bulk drug containing the active ingredients of the traditional Chinese medicine composition.

Further, the traditional Chinese medicine oral preparation is a traditional Chinese medicine decoction.

The invention also provides a preparation method of the traditional Chinese medicine oral preparation for promoting the healing of the diabetic foot chronic wound, which is prepared by using the traditional Chinese medicine composition and the bulk drug containing the active ingredients of the traditional Chinese medicine composition.

Further, the preparation method of the traditional Chinese medicine decoction comprises the following steps:

S1, adding pure water into the traditional Chinese medicine composition to form a mixed raw material, soaking, heating and extracting, and filtering to obtain an initial liquid medicine;

s2, performing normal pressure concentration operation on the initial liquid medicine to generate the traditional Chinese medicine decoction.

Further, the weight ratio of the traditional Chinese medicine composition in the mixed raw materials to the pure water is 1:6 to 1:10.

Further, the step S1 comprises soaking the mixed raw materials for more than 2 hours, heating and boiling, turning to low fire, continuously maintaining heating and extracting for 40min, and filtering to retain the initial medicinal liquid and the medicinal residues.

Further, the step S2 comprises adding 6-10 times of pure water into the residue obtained by the first decoction in the step S1, heating to boil, continuously decocting with small fire for 40min, filtering, mixing the decoctions, and concentrating.

The beneficial effects of the invention are as follows:

The syndrome (diabetic foot ulcer) is caused by internal heat-toxin, qi and blood stasis and yin and blood deficiency, especially the exuberance of heat-toxin, which is indicated for heat-clearing and detoxicating, blood-activating and blood-nourishing, and collaterals-dredging and pain-relieving. The invention discloses an improved compound traditional Chinese medicine composition prescription for producing a new prescription by adding flavor to four wonderful branchlet and An decoction based on traditional Chinese medicine theory and transcriptome sequencing, wherein honeysuckle and phellodendron bark are used as monarch drugs in the prescription, honeysuckle is sweet and cold and cool and has the effect of clearing away heat and dispersing, clearing heat and penetrating through the head, leading evil out, not only can release heat toxin of viscera, but also can clear depressed heat of channels, aromatic and penetrating without evil, and corktree bark is bitter and cold, enters kidney, bladder and large intestine channel, can clear damp heat, release fire toxin, and both damp heat toxin and evil can clear yin and dispel heat, and the corktree bark and honeysuckle are combined to one side to directly reduce evil, so that the heat toxin can be cleared, figwort root is used as ministerial drug, the figwort root has the effect of nourishing yin and dispersing, the effect of heat, the atractylis used for clearing away heat and dispersing evil heat, the stomach, the bitter atractylodes rhizome is used for assisting Huang Baisao, the spleen is not to remove heat and toxin, and has the effect of nourishing yin and dispersing stagnation, and the bitter heat and removing both damp and cold are compatible, and the two drugs are compatible. The Chinese angelica root is warm and moist, has the effects of promoting blood circulation to remove blood stasis, circulating blood vessels, supplementing and nourishing yin and blood to nourish four ends, is an adjuvant drug, is bitter and cold in taste and cool and moist in nutrient and blood system, is specially used for clearing heat and cooling blood and nourishing yin, is pungent and warm in taste and dry in dryness, is used as an adjuvant drug for regulating cold and heat, regulating stomach, eliminating stagnation and relieving stuffiness, can prevent the ascending and descending of stomach qi caused by stomach cold, has the effect of ascending and descending, has the effect of restoring spleen and stomach ascending and descending, and is used as an adjuvant drug for assisting the heat and toxin of honeysuckle, and is used for harmonizing the other drugs with the Chinese angelica root and the figwort root. The whole formula can be used for treating cold and heat, and has the effects of clearing heat and detoxicating, promoting blood circulation and relieving pain.

On one hand, the invention inherits the advantages of the traditional Chinese medicine dialectical prescription, plays the characteristics of accurate modern molecular diagnosis and treatment, has convenient clinical application and definite curative effect, has higher treatment efficiency compared with the original four wonderful branchia angan decoction and can obviously reduce the serum inflammatory factor level. On the other hand, the improved four wonderful brave and An decoction traditional Chinese medicine composition adopts common medicinal materials as raw materials, has low cost and easily-found medicinal sources, effectively reduces the economic burden of patients, can be suitable for preparing a plurality of different dosage forms, has simple preparation process, and is suitable for large-scale industrial production.

Drawings

FIG. 1 is a graph showing the distribution of KAS-seq signal in genomic DNA of foot wound tissue samples of diabetic foot ulcer patients (DFU) and normal control group (Ctrl).

FIG. 2 shows the analysis of differential genes in the DFU and Ctrl groups, wherein panel A shows the unsupervised clustering heat map (heatmap) analysis of differential genes in the Ctrl and DFU groups Top100, and panel B shows the volcanic map analysis of two groups of differential expressed genes (up is up-regulated in the DFU group and down is down-regulated in the DFU group).

FIG. 3 is a functional analysis of downregulated genes in Ctrl and DFU groups, wherein panel A is a GO enrichment analysis dot plot of the upregulated genes and panel B is a GO enrichment analysis dot plot of the downregulated genes.

Fig. 4 is a diagram of the traditional Chinese medicine-active ingredient-target point regulation network of the original four wonderful brave and An decoction.

FIG. 5 is a schematic diagram of the key gene screening for the improvement of the Simiaoyangan soup. The A diagram is a Venn diagram of an intersection of KAS-seq, a target spot of original four wonderful Yongan decoction traditional Chinese medicine and a gene set related to diabetic foot ulcer in GENECARDS database, and the B diagram is a functional analysis dot diagram of 534 genes existing in KAS-seq data and external data sets but not contained in the target spot of original traditional Chinese medicine.

Fig. 6 is a graph of PPI networks of 110 differential genes enriched in pathways closely related to diabetic foot.

FIG. 7 is a ROC graph of accuracy in distinguishing DFU and Ctrl groups from 8 key genes, and B graph is the names, loci, fold differences and P values of the 8 key genes.

Fig. 8 is a diagram of a network of target regulation of the traditional Chinese medicine-active ingredient-target of the improved formulation of the four wonderful brave An decoction.

Fig. 9 is a functional analysis diagram of the original and modified traditional Chinese medicine targets, a diagram A is a functional analysis diagram of the original traditional Chinese medicine targets of the Simiao Yongan decoction, and a diagram B is a functional analysis diagram of the modified traditional Chinese medicine targets of the Simiao Yongan decoction.

Fig. 10 is a schematic diagram showing the effect of the original and modified formulations on promoting DFU wound healing at high, medium and low doses, as demonstrated by animal experiments in test example 1.

Fig. 11 shows wound healing rates of each group of mice in test example 1 at days 0, 3, 7, and 12 of administration.

Fig. 12 is a schematic diagram of the treatment effect of the modified formulation in test example 2, wherein animal experiments prove that the modified formulation has a wound healing effect at different time points for each group of mice, B is the wound healing rate of each group at day 7, and C is the wound healing rate of each group of mice at day 12.

FIG. 13 is a schematic diagram showing serum inflammatory factor levels of four groups of experimental animals after drug intervention in test example 2, wherein A is TNF-alpha level, B is IL-6 level, and C is IL-1β level.

Fig. 14 is a general technical roadmap of the invention.

Detailed Description

In order to more clearly illustrate the present invention, the present invention will be described in further detail below with reference to examples and with reference to the accompanying drawings. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and that this invention is not limited to the details given herein.

Examples

In connection with fig. 14, the present invention mainly includes the following aspects:

1. Sample collection

The 6 diabetic foot ulcer patients were collected from the patients who received treatment from the third hospital of Beijing university during the period of 2021 month 1 to 2021 month 12 as the DFU group, and the control group was collected from the foot wound granulation tissue of the contemporaneous 3 healthy volunteers as the control group (Ctrl). Checking patient bed information, arranging a sample information sheet, taking fresh granulation tissue at a foot wound, placing the fresh granulation tissue in liquid nitrogen, transporting the fresh granulation tissue to a laboratory, checking the sample information after receiving the fresh granulation tissue, and storing the sample at-80 ℃.

The sample collection procedure was in compliance with patient informed consent and medical ethical approval requirements.

2. Extraction of wound tissue N ₃ markers and DNA extraction

25Mg of tissue sample was taken, 500. Mu.L of 1 XPBS was added, minced as much as possible at 4℃and the tissue cell suspension was aspirated, centrifuged at 1500g for 5min, the above was discarded and the tissue cells were collected. 1mL of DMEM medium and 5mM N ₃ -ketol (2.75 mM,1.82 mL) were added to the tissue cells and incubated for 10min in a 37℃incubator with 5% CO ₂. The cultured cell suspension was centrifuged at 1500rpm for 3min, the medium was removed, 1mLPBS was added for resuspension, and centrifugation at 1500rpm for 3min was performed to remove PBS. Genomic DNA (Genomic DNA, gDNA) was extracted using PureLinkPM Genomic DNA MINI KIT kit.

3. Construction of KAS-seq library

1. Mu.g of genomic DNA was suspended in 95. Mu.L of DNA elution buffer, 5. Mu.L of 20mM DBCO-PEG4-biotin (DMSO solution, sigma, 760749), 25mM K ₃BO₃, 37℃incubated for 1.5h, and gently shaken. Then 5. Mu.L of RNase A (Thermo, 12091039) was added to the reaction mixture and incubated at 37℃for 5min. The biotinylated DNA was then recovered using the DNA Clean & Concentrator-5 kit (Zymo, D4013). gDNA was suspended in 100. Mu.L of enzyme-free water, set with Biorupter Pico at 30-on/30s-off for 30 cycles, broken down to a size of 150-350bp, 5% of the DNA fragment was saved as input, and the remaining 95% was incubated with 10. Mu.L of pre-washed Dynabeads MyOne STREPTAVIDIN C (Thermo, 65001) for 15min at room temperature to enrich for biotin-labeled DNA. The beads were washed and the DNA eluted by heating in 15. Mu.L of enzyme-free water at 95℃for 10 min. Eluted DNA and its corresponding input were used for library construction using the Accel-NGS methyl sequence DNA library kit (Swift, 30024). Libraries were sequenced on a Illumina Novaseq6000 platform using a double-ended 150bp format with the goal of 3000 thousand reads per library.

4. Raw data processing

The constructed library was quality controlled using Fast QC (version 0.11.5). The resulting raw sequencing data was annotated by comparison of bowtie2 (version 2.2.9) with the human genome (version hg 19) and the unconditioned data was filtered using SAMtools (version 1.3.1). The possible KAS-seq enrichment regions for each sample were identified using MACS2 (version 2.1.1), and the KAS-seq enrichment regions were annotated with genes by the CHIP SEEKER PACKAGE (version 1.20.0) package, and the genes that best fit the marker region were used for subsequent functional analysis.

5. Differential analysis

The differential genes between Ctrl and DFU groups were analyzed by the DEseq package in RStudio software, the cutoff values were set to P <0.05 and |log2fold change| >0.5, and the differential gene volcanic map was plotted by the ggPlot package.

6. Differential gene functional analysis

The difference genes between the two groups were analyzed for functional enrichment via the Metscape website (https:// metacape. Org /) and the ClueGO (version 2.5.5) and CluePedia (version 1.5.5) insert in Cytoscape.

7. Statistical analysis

The two sets of data were subjected to differential analysis using RStudio software, sangerbox website (http:// www.sangerbox.com/logic. Html) and GRAPHPED PRISM software, p <0.05 proving statistically significant. Mean ± standard deviation for metering data in clinical sample informationAnd (3) representing.

8. Improved four wonderful brave An decoction construction based on target spot and network pharmacology

Based on clinical queues, key targets and traditional Chinese medicine theory are screened, and the four wonderful brave An decoction is flavored by combining a network pharmacological technology. Based on TCMSP and ETCM databases, traditional Chinese medicines acting on key sites of diabetic foot are retrieved and added.

The method comprises the steps of firstly searching traditional Chinese medicines in the Simiao Yongan soup through a TCMSP database (https:// old. Tcmsp-e.com/tcmsp. Php), downloading to obtain effective components (Ingredients) of each medicinal material and targets (targets) acted by the effective components, setting a threshold value to be more than 30% when the effective components are screened, setting the bioavailability (OB%) to be more than 0.18, and converting TARGET NAME obtained through screening into a gene name form through a UniProt database (https:// www.uniprot.org /) and a Perl language for subsequent analysis, and further supplementing the effective components and the targets of the traditional Chinese medicines through a ETCM database.

9. Improved four wonderful brave An decoction based on network pharmacology effectiveness verification

The improved four-effect branchlet An decoction composed of key targets is screened out based on KAS-seq sequencing, and a 'traditional Chinese medicine-active ingredient-action target' regulation network of the improved prescription of the four-effect branchlet An decoction is obtained by using Cytoscape3.7.2 software so as to verify the novel effectiveness.

1. Sequencing data analysis for diabetic foot patients

Foot wound tissue of DFU patients and Ctrl groups was examined using KAS-seq sequencing technology. The results show that the KAS-seq signal intensity of the Ctrl group is generally higher than that of DFU patients (FIG. 1), and that both groups of signals are mainly distributed in the transcription active region of the genome, such as Transcription Start Site (TSS) and Transcription Termination Site (TTS). After analysis of both Ctrl and DFU, 2545 differential genes were obtained in total, and these differential genes were found to be able to distinguish between the two sets of samples well by unsupervised cluster analysis (fig. 2A). Of these differential genes, 1049 genes were significantly up-regulated in DFU patients and 1496 genes were significantly down-regulated in DFU patients (fig. 2B). Functional enrichment of up-and down-regulated differential genes, up-regulated genes are 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.), respectively (FIG. 3A), whereas down-regulated genes are enriched in diseases related to Wnt, cell response to hypoxia (Cellular response to decreased oxygen levels), hematopoietic Cell differentiation (Hematopoietic progenitor Cell differentiation), and Cell growth differentiation (Cell growth, columnar/cuboidal EPITHELIAL CELL differentiation), etc., in addition to Axon development, nervous system development (Regulation of nervous system development), and synaptic signaling and regulation, respectively (FIG. 3B).

2. Pharmacological description of the original recipe of the Simiaoyangan decoction

The four wonderful brave and tranquilization decoction has the effects of clearing heat and detoxicating, promoting blood circulation and relieving pain. It is indicated for gangrene due to excessive heat toxin. Dark red, slight swelling and burning of the affected limbs, ulceration and rotten odor, severe pain, or fever, thirst, red tongue and rapid pulse. It is used for treating thromboangiitis obliterans, phlebitis, ulcer of lower limb, sciatica, deep venous embolism of lower limb, etc. Honeysuckle flower, flos Lonicerae, sweet and cold in nature, is good at clearing heat and detoxicating, so it is used as a main drug, radix Angelicae sinensis, promoting blood circulation and removing blood stasis, radix scrophulariae, purging pathogenic fire and detoxicating, and Glycyrrhrizae radix, and it is used as an auxiliary drug together with flos Lonicerae to strengthen the effect of clearing heat and detoxicating. The four medicines are combined, so that the traditional Chinese medicine can clear away heat and toxic materials, promote blood circulation and remove blood stasis, and is a good formula for treating gangrene.

The application carries out network pharmacological analysis on the original prescription of the four wonderful Yongan decoction, and the result shows that the four medicinal materials of honeysuckle, figwort root, chinese angelica and liquorice contain 112 effective components in total and target 118 gene targets. The regulation network construction of the traditional Chinese medicine-active ingredient-action target point is carried out on the traditional Chinese medicine (figure 4).

3. Network pharmacology research and pharmacology description of traditional Chinese medicine composition of improved prescription of four wonderful branchlet' an decoction

The differentially expressed genes obtained by KAS-seq sequencing, the genes related to the disease in GENECARDS database and the target of the four wonderful branchia angan decoction were crossed, and for improvement on the basis of the original, the 534 genes contained in the KAS-seq data and the external dataset, but not contained in the original target of the traditional Chinese medicine, were selected for subsequent screening (FIG. 5A). Functional enrichment analysis (figure 5B) is carried out on the part of the genes, and the results show that the genes are enriched on signal paths closely related to diseases such as RAP1, MAPK, PI3K-Akt, inflammation, platelet activation, insulin secretion, lipid decomposition and the like, and the part of the genes are proved to contain potential targets for improving the four wonderful branchia angan soup.

The 534 genes were used for reverse search in TCMSP and ETCM databases, and a total of 110 genes could be targeted to the traditional Chinese medicine in the database (FIG. 6). Further screening by machine learning resulted in 8 markers that were able to distinguish DFU from Ctrl with accuracy of AUC value of 0.987 (fig. 7A), where CREB1, GRID2, pike, DRD2, ADORA1, TNFSF11, EDNRB expression was significantly down-regulated in diabetic foot patients and HTR1B expression was significantly up-regulated in diabetic foot patients (fig. 7B).

Based on the network pharmacological research result of TCMSP database, adding cortex Phellodendri, rhizoma Atractylodis, rehmanniae radix and rhizoma Pinelliae targeted to key target points into the original decoction, preparing improved decoction (hereinafter referred to as improved prescription), and performing drug effect verification.

The recipe compatibility of the improved recipe is analyzed as follows, the recipe is caused by the internal heat-toxin accumulation, qi and blood stasis and yin and blood deficiency, and among the three, the recipe is mainly used for clearing heat and detoxicating, activating blood and nourishing blood, and dredging collaterals and relieving pain. In the formula, honeysuckle and phellodendron are used as principal drugs, and the honeysuckle is sweet and cold, cool and has the effect of dispersing nature, clearing the middle-jiao, clearing heat, reaching, guiding evil out, not only can release the heat toxin of viscera, but also can clear the stagnated heat of meridians, and is fragrant, reaching without accumulating evil. Cortex Phellodendri is bitter and cold, and enters kidney, bladder and large intestine meridians, it can clear damp-heat, dispel fire toxin, clear damp-heat toxin and enter yin to dispel deficiency heat. The two medicines of phellodendron bark and honeysuckle are combined together, and the two medicines are dispersed for reducing pathogenic factors directly so as to clear heat toxin. Radix scrophulariae is used as a ministerial drug, and has the effects of assisting the honeysuckle to clear heat and detoxify, and combining the two drugs, namely clearing qi and eliminating pathogenic heat and resolving blood and heat toxin, and the radix scrophulariae has the effects of nourishing yin and resolving masses. The ministerial drugs are rhizoma atractylodis with bitter and warm properties to assist Huang Baisao in wetting and activating spleen, and are healthy and free from bitter taste of the intestines and stomach, and bitter and warm without fear of fire. The two medicines are compatible, the yin and yang are mutually compatible, the cold and heat are coordinated, the principal and subordinate symptoms are combined, so that heat and dampness are removed. The Chinese angelica root, radix Angelicae sinensis is used as an adjuvant drug for warming and moistening, promoting blood circulation and removing blood stasis, promoting blood circulation and nourishing yin and blood to moisten the four powders. The radix rehmanniae is bitter and cold, enters nutrient and blood system, is sweet and cool and moist, and is specially used for clearing heat and cooling blood and nourishing yin. Ban Xia is pungent, warm and dry in nature, and acts as an adjuvant to regulate its cold and heat, reduce adverse flow of qi and harmonize stomach, dispel stasis and relieve distention and fullness, and can prevent adverse flow of stomach qi due to stomach cold, and has the actions of ascending and descending to restore the normal ascending and descending of spleen and stomach. Glycyrrhrizae radix is used as adjuvant, and has effects of clearing heat and detoxicating, and harmonizing with radix Angelicae sinensis, radix scrophulariae, nourishing yin and promoting fluid production. The whole formula can be used for treating cold and heat, and has the effects of clearing heat and detoxicating, promoting blood circulation and relieving pain.

The applicant carries out network pharmacological analysis on the improved prescription, and the result shows that four medicinal materials of phellodendron, rhizoma atractylodis, rehmannia and pinellia contain 111 active ingredients in total, and newly targets 456 gene targets on the basis of the original prescription, and carries out regulation and control network construction of the traditional Chinese medicine-active ingredient-action targets (figure 8).

Functional analysis of traditional Chinese medicine targets of the original and improved prescriptions of the four wonderful branchings and the improved prescriptions shows that the targets of the original prescriptions are mainly enriched in various lipid metabolism related paths (figure 9A) which are closely related to occurrence and development of diabetic foot ulcers, such as fatty acid metabolism, lipid catabolism, unsaturated fatty acid metabolism and the like, while the targets of the improved prescriptions are enriched in more paths related to nervous systems, such as synaptic chemical transmission, post-synaptic signal transduction, neurotransmitter level regulation and the like, compared with the original prescriptions, the targets of the improved prescriptions are enriched in more paths related to nervous systems, such as synaptic chemical transmission, post-synaptic signal transduction, neurotransmitter level regulation and the like, and in addition, some paths related to hypoxia and blood circulation regulation are enriched (figure 9B), which suggests that the targets can play roles in regulating inflammation, improving peripheral neuropathy, promoting wound healing and the like through the related biological processes.

The following uses the Chinese medicinal composition of the invention to promote the wound healing of diabetic foot, and the drug effect of the Chinese medicinal composition is verified by random control experiments on animal models.

Test example 1

1. Grouping, modeling and administration of animal models

Study A DFU wound model was established using 6 week old male SPF C57BKS-DB (-/-) mice (DB/DB). All animals were fed a standard diet under standard 12 hour light/12 hour dark cycle conditions. Db/db mice with random blood glucose >16.7mmol/L were satisfactory after 1 week of adaptive feeding. After skin preparation, a circular mark with the diameter of 1cm is made on the middle of the back below the scapula of each group of mice, the skin is cut along the mark, the skin is deeply covered to fascia, and the mice are wrapped by a medical bandage after conventional disinfection, so that the mice are prevented from biting the wound.

21 Db/db mice were randomly divided into 7 groups, and the control group was perfused with normal saline, the four high group was perfused with primary side (24 g/kg,0.1mL/10 g), the four middle group was perfused with primary side medium dose (12 g/kg,0.1mL/10 g), the four low group was perfused with primary side low dose (6 g/kg,0.1mL/10 g), the four+ high group was perfused with modified side high dose (49.4 g/kg,0.1mL/10 g), the four+ middle group was perfused with modified side medium dose (24.7 g/kg,0.1mL/10 g), and the four+ low group was perfused with modified side low dose (12.3 g/kg,0.1mL/10 g). The wound surface area was measured on days 0, 3, 7, 12 after the intervention, and the wound healing rate was calculated (calculation formula: initial area-measured area at different time points)/initial area x 100%).

2. Preparation of Chinese medicinal decoction

The feed comprises the following raw materials in parts by weight:

Four high group including 60g of honeysuckle, 60g of figwort, 40g of Chinese angelica and 20g of liquorice;

the fourth group comprises 30g of honeysuckle, 30g of figwort, 20g of angelica and 10g of liquorice;

The fourth group comprises 15g of honeysuckle, 15g of radix scrophulariae, 10g of angelica and 5g of liquorice;

Four+high group, including honeysuckle 60g, phellodendron bark 60g, figwort 60g, atractylodes 60g, angelica 40g, dried rhizome of rehmannia 40g, pinellia 40g and licorice 20g;

Four + middle group, honeysuckle 30g, corktree bark 30g, figwort 30g, atractylodes rhizome 30g, chinese angelica 20g, dried rehmannia root 20g, pinellia tuber 20g and licorice root 10g;

four+low group, flos Lonicerae 15g, cortex Phellodendri 15g, radix scrophulariae 15g, rhizoma Atractylodis 15g, radix Angelicae sinensis 10g, radix rehmanniae 10g, rhizoma Pinelliae 10g, glycyrrhrizae radix 5g.

Adding 6-10 times of pure water into the decoction for soaking for 2 hr, decocting with strong fire for 40min, filtering to obtain filtrate, adding 6-10 times of pure water into the residue, decocting with weak fire for 40min, and filtering to obtain filtrate. Mixing the two filtrates, concentrating at high temperature to desired concentration volume, and storing at 4deg.C for short term before use.

3. Statistical method

The data of each group are compared by adopting single-factor analysis of variance, and the related data of animal experiments are visualized by GRAPHPAD PRISM software.

4. Experimental results

The wound healing rate at the different drug intervention time points was calculated for each group of mice by (initial area-area measured at the different time points)/initial area x 100% (fig. 10). From the results, the four high, four + medium and four + high groups had significantly higher wound healing rates than the control group at day 3, the four high and four + medium groups had significantly higher wound healing rates than the control group (table 1), the four medium, four high, four + medium and four + high groups had significantly higher wound healing rates than the control group at day 7, the four high group had significantly higher wound healing rates than the four low group (table 2), and the four high, four + low, four + medium and four + high groups had significantly higher wound healing rates than the control group at day 12, the four + medium group had significantly higher wound healing rates than the four low group (table 3).

Table 1. Cases where there was a significant difference in wound healing rates for groups of mice on day 3 (p < 0.05; p < 0.01)

Table 2. Cases where there was a significant difference in wound healing rates for groups of mice at day 7 (p < 0.05; p < 0.01)

Table 3 cases where there was a significant difference in wound healing rates for groups of mice at day 12 (p < 0.05; p < 0.01)

The result shows that the high, medium and low dose groups of the improved prescription can obviously promote the wound healing of the DFU animal model, and the curative effect of the improved prescription at the medium dose is optimal after comprehensive analysis.

Test example 2

1. Grouping, modeling and administration of animal models

Study A DFU wound model was established using 6-week-old male SPF C57BKS-DB (-/-) mice (DB/DB), and 6-week-old male SPF C57BKS-DB (+/+) mice (DB/m) as healthy groups. All animals were fed a standard diet under standard 12 hour light/12 hour dark cycle conditions. Db/db mice with random blood glucose >16.7mmol/L were satisfactory after 1 week of adaptive feeding. After skin preparation, a circular mark with the diameter of 1cm is made on the middle of the back below the scapula of each group of mice, the skin is cut along the mark, the skin is deeply covered to fascia, and the mice are wrapped by a medical bandage after conventional disinfection, so that the mice are prevented from biting the wound.

15 Db/db mice were randomly divided into 3 groups, ctrl group lavage saline, SMYAT group lavage primary (11.7 g/kg,0.1mL/10 g), SMYAT + group lavage modified (24.7 g/kg,0.1mL/10 g), db/m mice as Healthy groups (n=5) lavage saline. The wound surface areas were measured at the 0, 3, 7, 12 th after intervention, and the wound healing rate was calculated (calculation formula: initial area-measured area at different time points)/initial area 100%).

2. The preparation of the traditional Chinese medicine decoction is the same as in example 1.

3. Statistical method the same as in example 1.

4. Experimental results

The wound healing rate at the different drug intervention time points was calculated for each group of mice by (initial area-area measured at the different time points)/initial area x 100% (fig. 12A). From the results, the wound healing rate of the four wonderful branchlet An decoction modified party group at day 7 is obviously higher than that of the Ctrl group (P < 0.01) (fig. 12B), and the wound healing rate of the four wonderful branchlet An decoction modified party group at day 12 is obviously higher than that of the four wonderful branchlet An decoction original party group (P < 0.05) (fig. 12C), which shows that the modified party can obviously promote the wound healing of the DFU animal model compared with the original party.

Serum inflammatory factor levels were measured in 4 groups of mice by ELISA. Firstly, drawing a standard curve according to the concentration and absorbance values of a standard substance hole, wherein the curve equation and r2 values of TNF-alpha are y= 2.53215/[1+ (x/867.50466)/(1.36523 ] +0.02319 and 0.99982 respectively, the curve equation and r2 values of IL-6 are y= 9.95329/[1+ (x/1361.63947)/(1.09666 ] +0.03334 and 0.99992 respectively, and the curve equation and r2 values of IL-1 beta are y= 2.04456/[1+ (x/736.55342)/(1.51532 ] +0.04861 and 0.99952 respectively). The absorbance values for each well were taken into the curve equation and multiplied by a factor to calculate the inflammatory factor level, which showed that the levels of TNF- α (fig. 13A) and IL-6 (fig. 13B) were significantly lower in the modified party group than in the original party group (P < 0.05), and that IL-1β (fig. 13C) were slightly lower in the modified party group than in the original party group, but the differences were not statistically significant (p=0.0664).

The results show that the improved Simiaoyangan decoction has remarkable promotion effect on wound healing of diabetic foot model mice compared with the original prescription, can reduce the levels of serum IL-1 beta, IL-6 and TNF-alpha, and the functional analysis shows that the traditional Chinese medicine targets of the improved prescription are more enriched to the pathways related to nerve and synapse related signal pathways, blood circulation, vascular morphology and vascular function maintenance, and response to oxygen level reduction and response pathways to oxygen level, which are probably the main mechanisms of the improved prescription for treating diabetic foot ulcers, and the improved prescription may have more advantages in improving peripheral neuropathy compared with the original prescription.

It should be understood that the foregoing examples of the present invention are merely illustrative of the present invention and not limiting of the embodiments of the present invention, and that various other changes and modifications can be made by those skilled in the art based on the above description, and it is not intended to be exhaustive of all the embodiments of the present invention, and all obvious changes and modifications that come within the scope of the invention are defined by the following claims.

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 proportions by weight:

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.

4. 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 oral Chinese medicine 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 is characterized in that the step S1 comprises soaking the mixed raw materials for more than 2 hours, heating and boiling, then turning to low heat and continuing to heat and extract for 40 minutes, and retaining the initial medicinal solution and medicinal residues after filtering.

10. The preparation method according to claim 9 is characterized in that the step S2 comprises: adding 6-10 times the volume of pure water to the dregs after 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 over high heat to concentrate the decoction.