Fingerprint spectrum of compound tung leaf burn oil, detection method of effective components with symbolic characteristics and application

Abstract

The invention discloses a fingerprint spectrum of compound tung leaf burn oil, a detection method of effective components with symbolic characteristics and application of the fingerprint spectrum. The detection method of the fingerprint spectrum comprises the following steps: preparing a compound tung leaf burn oil sample test solution and a standard control solution, and constructing the fingerprint spectrum of the compound tung leaf burn oil by adopting HPLC (High Performance Liquid Chromatography); hPLC (High Performance Liquid Chromatography) chromatographic conditions for constructing the fingerprint spectrum of the compound tung leaf burn oil are as follows: an octadecyl pure water-resistant analytical column is used as a chromatographic column, a mobile phase is methanol (A)-0.08-0.12% ammonium acetate (B), the flow velocity is 0.5-0.7 mL / min, and the sample size is 5-15 mu L. The method is systematic and reliable in result, and a scientific screening method is provided for quality control of the compound tung leaf burn oil.

Description

Technical Field

[0001] The invention belongs to the technical field of quality detection of traditional Chinese medicine compound, and in particular relates to a detection method and application of a fingerprint spectrum and characteristic active ingredients of compound tung leaf burn oil. Background Art

[0002] Chronic Refractory Wounds (CRW), commonly known as skin ulcers, are internationally defined as wounds that cannot be completely repaired in anatomy and function through a normal, orderly and timely repair process. CRWs impose a great burden on individuals, the healthcare system and the entire society, but are often underestimated. The demand for treatment of various types of wounds in my country is about 100 million people each year, of which CRW patients reach 30 million, and it has become a major chronic disease that seriously affects people's health. The inability of a wound to heal normally depends on many factors, such as wound size, specific causes, individual differences, and physical health status. Usually, a wound that cannot shrink by 50% for more than 1 month is considered CRW.

[0003] Epidemiological surveys show that with the rapid increase in the aging population, CRW has changed from "traumatic type" to "disease type", among which diabetic ulcer (DU) is the most common. In my country, the incidence of foot ulcers in patients with type 2 diabetes over 50 years old is 8.1% within one year, the recurrence rate within one year after cure is as high as 31.6%, the annual mortality rate is 14.4%, and the amputation rate due to foot ulcers is 7.2%. Therefore, judging from the current development status of DU alone, the prevention and treatment of CRW is urgent.

[0004] Modern research has confirmed that persistent high-sugar status and inflammatory response are the key causes of prolonged DU. Vascular endothelial dysfunction, as a key event in the early stages of DU, has also received increasing attention. During the development of DU, long-term, excessive inflammatory responses can lead to endothelial cell dysfunction and apoptosis, which in turn triggers pathological changes in peripheral nerves and blood vessels. Coupled with long-term exposure to a high-sugar environment, the body faces multiple challenges such as abnormal hemodynamics, increased blood viscosity, and mitochondrial dysfunction. These factors will aggravate the degree of vascular endothelial damage, making DU wound healing more difficult. Therefore, repairing endothelial cell function undoubtedly provides a strategy for improving DU.

[0005] Compound tung leaf burn oil is a secret medicine developed by the Yao family of the Tujia ethnic group (National Medicine Standard No.: Z20063825). It has a simple prescription and consists of only two medicines: Paulownia fortunei (Seem.) Hemsl. and sesame oil. It is currently the only Tujia ethnic innovative medicine and belongs to the third category of new Chinese medicine. It has the functions of "clearing away heat and detoxifying, reducing swelling and relieving pain, removing rot and regenerating flesh". It generally does not leave scars for deep burns and scalds, and can cure small-area III degree burns. It is safe and has no toxic side effects. At the same time, the drug has also been tried clinically for CRW caused by various reasons such as diabetic foot, pressure sores, bedsores, and perianal abscesses after surgery, with significant efficacy. Studies have found that some active ingredients in compound tung leaf burn oil can promote wound healing by repairing vascular endothelial cell damage.

[0006] At present, the drug registration standard of compound tung leaf burn oil has been officially included in the 77th volume of the "National Drug Standard New Drug Standard". This standard uses high performance liquid chromatography to detect the content of ursolic acid and oleanolic acid, and uses this as a quality control indicator, with octadecylsilane bonded silica gel as the filler; acetonitrile-methanol-0.5% ammonium acetate solution (61:18:21) as the mobile phase; the detection wavelength is 210nm; the column temperature is 35℃. However, it is worth noting that compound tung leaf burn oil is carefully prepared from two medicinal materials, white paulownia leaves and sesame oil. Among them, ursolic acid and oleanolic acid are only present in white paulownia leaves, while the active ingredients contained in sesame oil are not included in the current detection standards. From the perspective of quality markers of traditional Chinese medicine, the iconic ingredients should follow the five core principles of characteristic, measurability, traceability, relevance to traditional Chinese medicine theory and effectiveness. Based on the above principles, it is not difficult to find that there is still room for improvement in the existing registration standards for compound tung leaf burn oil drugs.

[0007] As a traditional Chinese medicine, compound tung leaf burn oil has the characteristics of multiple components and multiple targets. The following prior arts respectively disclose the high performance liquid phase detection methods of the four components in compound tung leaf burn oil, but they cannot be designated as characteristic components.

[0008] The patent with the publication number CN115060840A discloses a quality control method for a Paulownia tungii leaf sesame oil preparation, which quantitatively analyzes sesamin and sesamolin. The chromatographic column is Waters XBridge C18 (4.6mm×150mm, 3.5μm); gradient elution is performed, during which the mobile phase A is 5% methanol, the mobile phase B is methanol, and the proportion of mobile phase B changes as follows: liquid phase conditions: 0-12min: 50-100% methanol, 12-25min: 100-100% methanol, 25-26min: 100-50% methanol, 26-36min: 50-50% methanol; column temperature 30°C; flow rate 1.0mL / min; injection volume 20μL; detection wavelength 300nm. This method can only detect sesamin and sesamolin, which are components of sesame oil, and cannot be used as a quality marker for compound Paulownia leaf burn oil. Summary of the invention

[0009] In view of the above technical problems, the purpose of the present invention is to provide a method for detecting the fingerprint of compound tung leaf burn oil, accurately detect the characteristic components of the compound tung leaf burn oil, and improve the detection standard of the compound tung leaf burn oil. The method determines its markers from multiple aspects such as characteristic, measurability, and effectiveness.

[0010] The invention also discloses a method for detecting the characteristic active ingredients of the compound tung leaf burn oil, and accurately detects the characteristic active ingredients in the compound tung leaf burn oil.

[0011] The invention also discloses the application of the characteristic active ingredients of the compound tung leaf burn oil in endothelial cell damage caused by inflammation and high sugar. A single characteristic active ingredient can play the role of the compound tung leaf burn oil and has broad application prospects.

[0012] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0013] A method for detecting the fingerprint spectrum of compound tung leaf burn oil comprises the following steps:

[0014] A sample solution of the compound tung leaf burn oil was prepared, and a fingerprint of the compound tung leaf burn oil was constructed by HPLC chromatography;

[0015] The HPLC chromatographic conditions for constructing the fingerprint of the compound tung leaf burn oil include: using an octadecyl pure water-resistant analytical column chromatographic column, the mobile phase is methanol (A)-0.08-0.12% ammonium acetate (B), the flow rate is 0.5-0.7mL / min, and the injection volume is: 5-15μL; preferably, the detection wavelength of the HPLC chromatogram in S2 is: 0-12min, λmax is 240-270nm; 12-40min, λmax=205-215nm.

[0016] In a preferred embodiment of the present invention, the HPLC chromatogram for constructing the fingerprint of the compound tung leaf burn oil adopts gradient elution, and the parameters of the gradient elution are: 0-5min, 80-85% (A); 5-20min, 85-90% (A); 20-35min, 85-90% (A); 35-36min, 90-85% (A); 36-40min, 85-80% (A).

[0017] The HPLC chromatographic conditions of the fingerprint spectrum of the present application can simultaneously measure the characteristic components in Paulownia alba leaves and sesame oil, and determine their markers from multiple aspects such as characteristic, measurability, and effectiveness.

[0018] In a preferred embodiment of the present invention, the chromatographic column used in constructing the fingerprint of the compound tung leaf burn oil is a Supersil AQ-C18 chromatographic column.

[0019] The concentration of the standard control solution is 0.5-1.0 mg / mL, and the standard control solution includes sesamin, sesamolin, linoleic acid, maslinic acid, corosolic acid, linolenic acid, oleanolic acid, and ursolic acid.

[0020] In a preferred embodiment of the present invention, before constructing the fingerprint of the compound tung leaf burn oil, the method further comprises:

[0021] S1. Use UPLC-Q-TOF-MS to detect and record the total ion flow chart of the test solution of the compound tung leaf burn oil sample, analyze the relative molecular mass of each component, and infer the chemical composition, and prepare the inferred chemical composition in S1 as a standard control solution;

[0022] The chromatographic conditions in S1 include: the chromatographic column is a C18 column, the mobile phase is 0.08-0.12% formic acid water (A)-acetonitrile (B), the flow rate is 0.3-0.5 mL / min, the column temperature is 20-30°C, and the injection volume is 1.0-4.0 μL; preferably, the mobile phase adopts gradient elution, and the gradient elution parameters are as follows: 0-10 min, 95% (A); 10-20 min, 85-65% (A); 20-30 min, 65-45% (A); 30-40 min, 45-15% (A); 40-50 min, 15-5% (A); further preferably, the chromatographic column in S1 is: Agilent ZORBAX Eclipse Plus C18.

[0023] In a preferred embodiment of the present invention, the mass spectrometry conditions in S1 include: detection in electrospray positive and negative ion modes, a primary mass spectrometry scanning range: m / z 100-1700, nitrogen as a solution drying gas, a temperature of 310-330°C, a flow rate of 6-7L / min, a sheath gas temperature of 340-360°C, a capillary voltage of 3.5-4.5kv, and a fragmentation voltage of 140-160V; preferably, an Agilent standard tuning solution ESI-L Low Concentration TuningMix is ​​used to calibrate the standard mass number before mass spectrometry injection analysis.

[0024] In a preferred embodiment of the present invention, the preparation of the compound tung leaf burn oil sample test solution includes the following preparation method: taking the compound tung leaf burn oil sample, extracting it with methanol, concentrating it, fixing the volume, and centrifuging it.

[0025] In a preferred embodiment of the present invention, when preparing the compound tung leaf burn oil sample test solution, the volume of methanol is 15 to 30 times the volume of the compound tung leaf burn oil sample, and the number of methanol extractions is 2 to 5 times.

[0026] In a preferred embodiment of the present invention, the mass of the compound tung leaf burn oil sample is 0.5-1.5 g, the volume of the fixed volume is 5-15 mL, and the centrifugal speed is 10000-14000 r / min.

[0027] In a preferred embodiment of the present invention, constructing the fingerprint of the compound tung leaf burn oil also includes predicting the characteristic values, contribution rates and initial factor loading matrix of the components in the compound tung leaf burn oil sample through OPLS-DA, predicting the VIP value, taking VIP>0.8 as a medium impact, and screening out characteristic components; preferably, the characteristic components are hawthorn acid, corosolic acid, oleanolic acid, ursolic acid, sesame, linolenic acid and linoleic acid.

[0028] The present invention also discloses a method for detecting the characteristic active ingredients of the compound tung leaf burn oil, including a method for detecting the fingerprint spectrum of the compound tung leaf burn oil, and further comprising the following steps:

[0029] The content of the characteristic active ingredients is calculated by fingerprint spectrum, and the characteristic active ingredients include sesamin, maslinic acid, corosolic acid, oleanolic acid and ursolic acid.

[0030] In a preferred embodiment of the present invention, the characteristic active ingredients are docked with target proteins by molecular docking technology, and the characteristic active ingredients are obtained by analyzing the complex and clustering the binding energy of the complex; preferably, the target protein is the core target protein in the IL-17 signaling pathway, and further preferably, the core target proteins in the IL-17 signaling pathway include IL-17A, NF-κBp65 and AP-1.

[0031] The endothelial cell injury model verified that these ingredients can not only effectively inhibit the IL-17-mediated NF-κB / AP-1 signaling pathway and downregulate the expression of inflammatory factors, but also promote cell migration and accelerate wound healing.

[0032] The present invention also discloses the application of the characteristic active ingredients of the compound tung leaf burn oil in endothelial cell damage caused by inflammation and high sugar, wherein the characteristic active ingredients include one or more of maslinic acid, corosolic acid, oleanolic acid, ursolic acid and sesame; preferably, the intervention concentration of the characteristic active ingredients is 1 to 100 μM, and the intervention time is 18 to 36 h; further preferably, the intervention concentration is 1 to 10 μM.

[0033] A human microvascular endothelial cell model of endothelial function injury was established, and the characteristic active ingredients were used for therapeutic intervention.

[0034] In a preferred embodiment of the present invention, the steps of constructing a human microvascular endothelial cell endothelial function injury model include: scratching the human microvascular endothelial cells, and then co-inducing the scratched human microvascular endothelial cells with 1 to 3 μg / mL lipopolysaccharide and 20 to 30 mM glucose, respectively, and the combined induction time is 18 to 36 hours.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] The invention provides a fingerprint spectrum of the compound tung leaf burn oil and a detection method and application of characteristic active ingredients, clarifies the differential characteristic ingredients between batches of the compound tung leaf burn oil, and the method is systematic and the result is reliable, thus providing a scientific screening method for the quality control of the compound tung leaf burn oil. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 .UPLC-Q-TOF-MS total ion chromatogram.

[0038] Figure 2 .Multivariate analysis of 10 batches of compound tung leaf burn oil extracts.

[0039] (A) HPLC fingerprint of 10 batches of compound tung leaf burn oil extracts. (B) HPLC graphs of mixed standard, sesame oil and compound tung leaf burn oil; 1, sesamin; 2, sesamelin; 3, peak-3; 4, linoleic acid; 5, hawthorn acid; 6, corosolic acid; 7, linolenic acid; 8, peak-8; 9, oleanolic acid; 10, ursolic acid; 11, peak-11; (C) PCA scores of 10 batches of compound tung leaf burn oil samples. (D) Cluster analysis of 10 batches of compound tung leaf burn oil samples; (E) VIP values ​​of 10 batches of compound tung leaf burn oil.

[0040] Figure 3 . Heat map and cluster analysis of docking binding energy between the seven components of compound tung leaf burn oil and the characteristic proteins of IL-17 signaling pathway.

[0041] Figure 4 .Molecular docking of the iconic candidate components of compound tung leaf burn oil with the core protein.

[0042] (A) Molecular docking of TYY candidate components with IL-17A (PDB ID: 8uss); (B) Molecular docking of TYY candidate components with NF-κBp65 (PDB ID: 6nv2); (C) Molecular docking of TYY candidate components with AP-1 (PDB ID: 1a02).

[0043] Figure 5 . Effects of high glucose and high glucose on IL-17 downstream characteristic genes in vitro (n=4).

[0044] (A) Effects of different concentrations of lipopolysaccharide on IL-17A downstream genes. (B) Effects of different concentrations of glucose on IL-17A downstream genes. All statistical results were compared with the drug-loaded group (glucose content 5.5 mM), *P<0.05, ****P<0.0001.

[0045] Figure 6 . Effect of TYY active ingredients on mRNA expression of IL-17 and its marker genes in an in vitro cell model (n=4).

[0046] (A) Sesamin on the expression of related gene mRNA. (B) Maslinic acid on the expression of related gene mRNA. (C) Corosolic acid on the expression of related gene mRNA. (D) Oleanolic acid on the expression of related gene mRNA; (E) Ursolic acid on the expression of related gene mRNA; Compared with the LPS / high glucose group, *P<0.05, **P<0.01, ***P<0.001,

[0047] ****P<0.0001; compared with the blank control group, #P<0.05, ##P<0.01, ###P<0.001, ####P<0.0001.

[0048] Figure 7 .TYY active ingredients promote healing effects.

[0049] (A&B) Effects of multiple control groups on the average scratch migration distance after the 24-hour intervention period and semi-quantitative analysis. Note: vehicle group (glucose 5.5mM), lipopolysaccharide group (lipopolysaccharide 2μg / mL), high glucose group (glucose 25mM), lipopolysaccharide / high glucose group (simultaneous intervention of glucose 25mM and lipopolysaccharide 2μg / mL). (C&D) Effects of TYY active ingredients on the average scratch migration distance after the 24-hour intervention period and semi-quantitative analysis. Compared with the lipopolysaccharide / high glucose group, *P<0.05, ***P<0.001, ****P<0.0001. (Microscope magnification, ×40). DETAILED DESCRIPTION

[0050] Example 1 (S1)

[0051] In this step, UPLC-Q-TOF-MS liquid chromatography-mass spectrometry was used to conduct in-depth qualitative analysis of the compound tung leaf burn oil (see Figure 1 ) and perform corresponding spectrum analysis to determine the identity of each compound (see Table 1).

[0052] Preparation of test solution: Weigh 1.0 g of compound tung leaf burn oil sample and extract it three times with 20 volumes of methanol at room temperature. Prepare sesame oil in the same way. Collect methanol and concentrate it to 10 mL, centrifuge it at 12000 r / min, and set aside.

[0053] Chromatographic conditions: the chromatographic column is Agilent ZORBAX Eclipse Plus C18 (3.0 mm×100 mm, 1.8 μm), the mobile phase is 0.1% formic acid water (A)-acetonitrile (B), flow rate: 0.4 mL / min, column temperature: 25°C, injection volume: 2.0 μL, the mobile phase elution gradient is as follows: 0-10 min, 95% (A); 10-20 min, 85-65% (A); 20-30 min, 65-45% (A); 30-40 min, 45-15% (A); 40-50 min, 15-5% (A).

[0054] Mass spectrometry conditions: Electrospray positive and negative ion modes were used for detection, and Agilent standard tuning solution ESI-L Low Concentration Tuning Mix (G1969-85000) was used to calibrate the standard mass number before sample injection analysis. The primary mass spectrometry scanning range was m / z 100-1700, nitrogen was used as the solution drying gas, the temperature was 325°C, the flow rate was 6.8L / min, the sheath gas temperature was 350°C, the capillary voltage was 4.0kV, and the fragmentation voltage was 150V.

[0055] Data analysis: The molecular formula is inferred within the possible elemental composition range through the precise relative molecular mass in the data obtained by LC-MS. The pyrolysis rules of each compound are analyzed by referring to relevant literature and TCMSP database and combining with the reference substance, characteristic multi-stage mass spectrometry fragmentation and relative abundance information to further confirm the chemical composition. At the same time, the attribution of each compound is determined by referring to relevant literature and TCMSP database.

[0056] Table 1. Identification of chemical components in compound tung leaf burn oil by UPLC-Q-TOF-MS (1)

[0057]

[0058] Table 1. Identification of chemical components in compound tung leaf burn oil by UPLC-Q-TOF-MS (2)

[0059]

[0060] Table 1. Identification of chemical components in compound tung leaf burn oil by UPLC-Q-TOF-MS (3)

[0061]

[0062] Embodiment 2 (S2)

[0063] In this step, HPLC technology was first used to construct 10 batches of compound tung leaf burn oil samples to establish fingerprints (see Figure 2 A), identify the peaks by comparing with the standard (see Figure 2 B). Then through chemometric analysis (see Figure 2 C-E) screened out the important components with VIP>0.8, and finally used molecular docking technology (see Figure 3 )Screen out the candidate characteristic components of compound tung leaf burn oil.

[0064] Preparation of test solution: Weigh 1.0g of compound tung leaf burn oil sample and extract it three times with 20 times volume of methanol at room temperature. Prepare sesame oil in the same way. Collect methanol and concentrate it to 10mL, centrifuge it at 12000r / min and set aside. Accurately weigh 5mg of 8 kinds of standard products such as sesamin, sesamelin, linoleic acid, hawthorn acid, corosolic acid, linolenic acid, oleanolic acid, ursolic acid, etc., add them to a 10ml volumetric flask, dissolve them with methanol, and obtain a 0.5mg / mL standard control solution. Use high performance liquid chromatography (Agilent1260) to compare and analyze the test sample and standard reference product.

[0065] HPLC chromatographic conditions: Supersil AQ-C18 column (4.6×250mm, 5μm, Elite). The mobile phase was methanol (A)–0.1% ammonium acetate (B), the flow rate was 0.6mL / min, and the injection volume was 10μL. Detection wavelength: 0-12min, λmax=254nm; 12-40min, λmax=210nm. Gradient program: 0-5min, 85% (A); 5-20min, 85-90% (A); 20-35min, 90% (A); 35-36min, 90-85% (A); 36-40min, 85% (A).

[0066] Chemometric multivariate analysis method: The similarity analysis of 10 batches of Fufang Tongye burn oil samples was performed using the Chinese medicine fingerprint similarity evaluation software (version 2012A). All generated data matrices were analyzed in depth using Origin 2021 software. Two unsupervised pattern recognition methods, cluster analysis and principal component analysis (PCA), were used to identify the differences in relative peak areas between batches. Then, the threshold variable importance of the projection value (VIP ≥ 0.8) in the orthogonal partial least squares discriminant analysis (OPLS-DA) model was used to screen the characteristic components.

[0067] Fingerprint analysis: The characteristic fingerprint peaks were calibrated according to the retention time, and the HPLC spectra of the compound tung leaf burn oil and sesame oil were compared and analyzed, and a total of 11 common peaks were identified. Among them, peaks 1, 2, 3, 4, 7, 8, and 11 are derived from the components of sesame oil, and peaks 5, 6, 9, and 10 are derived from the components of paulownia leaves (see Figure 2 B). After comparison with the standard, a total of 8 components were identified, namely: sesamin (peak 1), sesamelin (peak 2), linoleic acid (peak 4), hawthorn acid (peak 5), corosolic acid (peak 6), linolenic acid (peak 7), oleanolic acid (peak 9) and ursolic acid (peak 10). Then, using the Chinese medicine fingerprint similarity evaluation software (2012A version), the similarity index between batches of compound tung leaf burn oil was calculated to range from 0.955 to 0.997, indicating that there was little difference between the batches. However, from the PCA scoring results, it can be observed that the two earliest produced batches, S202220403 and S20221002, were clustered into the same category, while the remaining batches were classified into another group (see Figure 2 C). This phenomenon suggests that the compound tung leaf burn oil may have a slight influence on the ingredients due to time factors.

[0068] Multivariate analysis results: Combining the characteristic values, contribution rates and initial factor loading matrices of the 10 batches of Tongye burn oil (see Tables 2 & 3), and referring to the VIP values ​​of the 11 components predicted by OPLS-DA, VIP>0.8 was considered a medium impact, and 10 characteristic components were screened out, including sesamin, Feng-3, linoleic acid, hawthorn acid, corosolic acid, linolenic acid, Feng-8, oleanolic acid, ursolic acid and Feng-11 (see Figure 2 D). Based on the above analysis, maslinic acid, corosolic acid, oleanolic acid and ursolic acid (triterpenes in Paulownia leaves), sesamin (lignans in sesame oil), linolenic acid and linoleic acid (fatty acids in sesame oil) were selected as candidate substances for subsequent experiments.

[0069] Table 2. Characteristic values ​​and contribution rates of 10 batches of compound tung leaf burn oil

[0070]

[0071] Table 3. Initial factor loading matrix of 10 batches of Tongye burn oil

[0072]

[0073]

[0074] Molecular docking analysis: Based on the multivariate analysis results of the chemical components of the compound tung leaf burn oil, molecular docking technology was used to explore the interaction pattern and efficiency between the known components in the compound tung leaf burn oil and the core target proteins in the IL-17 signaling pathway, including IL-17A, NF-κB p65 and AP-1.

[0075] By clustering the binding energy of the complexes, it was found that the complexes formed by linolenic acid and linoleic acid with the target protein were grouped together. The affinity and stability of the two complexes were significantly inferior to those of the other five complexes ( Figure 3 ), indicating that the interaction between these two components on the target proteins of the IL-17 signaling pathway is less efficient.

[0076] The analysis of the complex showed that the five components, sesamin, maslinic acid, corosolic acid, oleanolic acid and ursolic acid, showed significant binding ability with IL-17A, NF-κB p65 and AP-1 (see Figure 4 The results suggest that sesamin, maslinic acid, corosolic acid, oleanolic acid and ursolic acid are more suitable as candidate signature components of compound tung leaf burn oil.

[0077] Example 3 (S3-1)

[0078] In this step, human microvascular endothelial cells (HMEC-1) were used to establish an endothelial function injury model, and the RT-qPCR method was used to screen out the characteristic components with anti-inflammatory effects in the compound tung leaf burn oil.

[0079] Cell culture: HMEC-1 cells were cultured in MCDB-131 medium supplemented with 10% fetal bovine serum (v / v), 10 ng / mL human epidermal growth factor, 2 mM / L alanyl-glutamine, 1 μg / mL hydrocortisone, and 1% P / S (v / v). Cells were incubated at 37°C with 5% CO 2 Incubate in humidified atmosphere.

[0080] RT-qPCR detection method: Total RNA was extracted from tissues or cells according to the instructions of the total RNA extraction kit (Simgen, Hangzhou, China). The purified RNA was quantified using an Epoch microplate spectrophotometer (BioTek). RNA was reverse transcribed to obtain cDNA using the Plus All-in-One-1st Strand cDNA Reverse Transcription Kit (Novoprotein, Shanghai, China). mRNA-specific primers were designed using NCBI Primer Blast. Real-time fluorescence quantitative PCR (qPCR) was used to SYBR qPCR Supermix Plus was used for quantitative analysis. Transcript levels were normalized to GAPDH using the 2-ΔΔCq method. The primer information used in this study is shown in Table 4.

[0081] Table 4. RT-qPCR amplification primers

[0082]

[0083]

[0084] Establishment of endothelial function injury model: Based on the fact that high glucose and inflammatory response are the key factors leading to DU endothelial injury, human microvascular endothelial cells (HMEC-1) were treated with different concentrations of lipopolysaccharide (0, 2, 4, 6, 8, 10 μg / mL) and different concentrations of glucose (5.5, 15, 25, 35, 50 mM) for 24 hours to investigate their potential effects on the expression levels of downstream genes of the IL-17 signaling pathway (see Figure 5). This simulates a complex model of endothelial cell injury under an inflammation and high-glucose microenvironment. By comprehensively considering the expression levels of multiple pathway proteins and inflammatory indicators, the optimal concentration combination for constructing a lipopolysaccharide / high-glucose combined induced endothelial cell injury model was screened out, namely lipopolysaccharide 2μg / mL and glucose 25mM. Under this combination, the NF-κB / AP-1 signaling pathway mediated by IL-17 is in a highly activated state. In this state, downstream inflammatory factors, including but not limited to TNF-α, IL-6 and IL-1β, are overexpressed, further aggravating the dysfunction and damage of endothelial cells.

[0085] Screening of active ingredients: The constructed endothelial cell injury model was treated for 24 hours using different concentrations of active ingredients of compound tung leaf burn oil to explore the effective regulatory ability of active ingredients of compound tung leaf burn oil on the downstream inflammatory cascade of IL-17 signaling pathway. Based on the previous CCK8 test results, the concentrations of hawthorn acid, corosolic acid, oleanolic acid and ursolic acid were determined to be 1μM and 5μM, and for IC 10 The concentrations of sesamin were 2.5 μM and 10 μM. Then, the mRNA expression levels of four classic inflammatory markers, IL-6, TNF-α, TLR4 and IL-1β, were systematically detected. The mRNA expression of TRAF6, NF-κB, MAPK1 and c-Fos, the core proteins in the IL-17 signaling pathway, was also examined (see Figure 6 ). The results showed that the active ingredients of the compound tung leaf burn oil generally showed a significant inhibitory effect on the expression of genes downstream of the IL-17 signaling pathway. Among them, only corosolic acid had a relatively low sensitivity in regulating c-Fos protein.

[0086] Example 4 (S3-2)

[0087] In this step, human microvascular endothelial cells (HMEC-1) were used to establish an endothelial function injury model, and the characteristic components in the compound tung leaf burn oil that have the effect of promoting healing were screened through a scratch test.

[0088] Scratch test steps: 2×10 6 HMEC-1 cells were plated in a 6-well plate, and after 24 hours of attachment, a 200 μL pipette tip was used to make a wound at the bottom of the well. The culture medium was then replaced with a basal medium (glucose content 5.5 mM), and the cells were treated with different interventions for 24 hours. The migration of cells in each group was recorded in high definition immediately after the scratch (0 hours) and after the scratch (24 hours), and the migration of cells in each group was quantified using Image J.

[0089] Screening of scratch experiment conditions: In order to ensure that the experiment can fully reflect the cell migration behavior under different pathophysiological conditions, multiple control groups were set up in the experiment, including the LPS-only group, the high sugar group, and the LPS / high sugar group that received both LPS and high sugar stimulation, in order to comprehensively evaluate the potential effects of inflammation and high sugar environment on cell migration. The blank group (glucose content 5.5mM) served as the control. The treatment group was set as the LPS / high sugar + active ingredient group. This group introduced the active ingredients of the compound tung leaf burn oil on the basis of the LPS / high sugar treatment, aiming to observe the specific effects of the active ingredients of the compound tung leaf burn oil on cell migration. Through the analysis of the results of the scratch experiment, it was found that the scratch width of the high sugar group and the LPS / high sugar combined group was significantly wider than that of the blank group and the LPS group after 24 hours of treatment (P < 0.05), while there was no significant difference in the scratch width between the LPS group and the blank group (see Figure 7 A&B), this finding strongly supports that a high-sugar microenvironment is one of the key factors leading to slow wound healing.

[0090] Screening of active ingredients that promote healing: Further, it was observed that the cell migration rate was significantly increased by adding corosolic acid (P < 0.001), oleanolic acid (P < 0.001), ursolic acid (P < 0.0001) and sesamin (P < 0.05) to the lipopolysaccharide / high sugar treatment, which fully demonstrated that the active ingredients of the compound tung leaf burn oil do have the ability to promote cell migration (see Figure 7 C&D). Unfortunately, hawthorn acid also showed a certain promoting effect, but the difference did not reach a statistically significant level. In summary, these data not only verify the promoting effect of the active ingredients of compound tung leaf burn oil on cell migration under high sugar and inflammatory microenvironment, but also provide favorable evidence for understanding its mechanism of accelerating wound healing.

[0091] Statistical analysis: Data are expressed as mean ± SEM. GraphPad Prism 8.0.2 software was used for statistical analysis. Analysis of variance (ANOVA) was used to analyze the differences between groups. P < 0.05 was considered statistically significant.

Claims

1. A method for detecting the fingerprint of compound tung leaf burn oil, characterized in that The following steps are involved: A sample solution of the compound tung leaf burn oil was prepared, and a fingerprint of the compound tung leaf burn oil was constructed by HPLC chromatography; The HPLC chromatographic conditions for constructing the fingerprint of the compound tung leaf burn oil include: using an octadecyl pure water-resistant analytical column chromatographic column, the mobile phase is methanol (A)-0.08-0.12% ammonium acetate (B), the flow rate is 0.5-0.7mL / min, and the injection volume is: 5-15μL; preferably, the detection wavelength of the HPLC chromatogram in S2 is: 0-12min, λmax is 240-270nm; 12-40min, λmax=205-215nm.

2. The method for detecting the fingerprint of the compound tung leaf burn oil according to claim 1, characterized in that: The HPLC chromatogram for constructing the fingerprint of the compound tung leaf burn oil adopts gradient elution, and the parameters of the gradient elution are: 0-5min, 80-85% (A); 5-20min, 85-90% (A); 20-35min, 85-90% (A); 35-36min, 90-85% (A); 36-40min, 85-80% (A).

3. The method for detecting the fingerprint of the compound tung leaf burn oil according to claim 1, characterized in that: The chromatographic column used in constructing the fingerprint of the compound tung leaf burn oil is a SupersilAQ-C18 chromatographic column.

4. The method for detecting the fingerprint of the compound tung leaf burn oil according to claim 1, characterized in that: The preparation method of the compound tung leaf burn oil sample test solution includes the following preparation method: taking the compound tung leaf burn oil sample, extracting it with methanol, concentrating it, fixing the volume, and centrifuging it.

5. The method for detecting the fingerprint of the compound tung leaf burn oil according to claim 4, characterized in that: When preparing the compound tung leaf burn oil sample test solution, the volume of the methanol is 10 to 30 times the volume of the compound tung leaf burn oil sample, and the number of methanol extractions is 2 to 5 times.

6. The method for detecting the fingerprint of the compound tung leaf burn oil according to claim 4, characterized in that: The mass of the compound tung leaf burn oil sample is 0.5-1.5 g, the volume of the fixed volume is 5-15 mL, and the centrifugal speed is 10000-14000 r / min.

7. A method for detecting the characteristic active ingredients of compound tung leaf burn oil, characterized in that: The method for detecting the fingerprint spectrum of the compound tung leaf burn oil further comprises the following steps: The content of the characteristic active ingredients is calculated by fingerprint spectrum, and the characteristic active ingredients include sesamin, maslinic acid, corosolic acid, oleanolic acid and ursolic acid.

8. The method for detecting the characteristic active ingredients of the compound tung leaf burn oil according to claim 7, characterized in that: The characteristic active ingredients are docked with target proteins by molecular docking technology, and the characteristic active ingredients are obtained by analyzing the complex and clustering the binding energy of the complex; preferably, the target protein is a core target protein in the IL-17 signaling pathway, and further preferably, the core target proteins in the IL-17 signaling pathway include IL-17A, NF-κB p65 and AP-1.

9. Application of a characteristic active ingredient of a compound tung leaf burn oil in endothelial cell damage caused by inflammation and high sugar, characterized in that: The characteristic active ingredients include one or more of maslinic acid, corosolic acid, oleanolic acid, ursolic acid and sesamin; preferably, the intervention concentration of the characteristic active ingredients is 1 to 100 μM, and the intervention time is 18 to 36 h; further preferably, the intervention concentration is 1 to 10 μM.

10. The use according to claim 9, characterized in that: The steps of constructing the human microvascular endothelial cell endothelial function injury model include: scratching the human microvascular endothelial cells, and then co-inducing the scratched human microvascular endothelial cells with 1-3 μg / mL lipopolysaccharide and 20-30 mM glucose, respectively, and the combined induction time is 18-36 hours.

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