Fingerprint spectrum and detection methods and applications of key active ingredients in compound tung leaf burn oil

Abstract

The application discloses a detection method and application of a fingerprint spectrum and a mark characteristic effective component of compound tung oil for burns and a detection method of the fingerprint spectrum, and the detection method of the fingerprint spectrum comprises the following steps: preparing a sample solution and a standard control solution of the compound tung oil for burns, and adopting HPLC chromatography to construct the fingerprint spectrum of the compound tung oil for burns; the HPLC chromatography conditions for constructing the fingerprint spectrum of the compound tung oil for burns comprise the following steps: adopting an octadecyl silica water-resistant analysis column chromatographic column, adopting methanol (A) and 0.08-0.12% ammonium acetate (B) as a mobile phase, adopting a flow rate of 0.5-0.7 mL / min, and adopting a sample injection amount of 5-15 muL. The method system is reliable in results and provides a scientific screening method for quality control of the compound tung oil for burns.

Description

Technical Field

[0001] This invention belongs to the field of quality testing technology for traditional Chinese medicine compound prescriptions, and particularly relates to the fingerprint spectrum and detection method and application of the marker characteristic active ingredients of compound tung leaf burn oil. Background Technology

[0002] Chronic refractory wounds (CRW), commonly known as skin ulcers, are internationally defined as wounds that cannot achieve anatomical and functional integrity through a normal, orderly, and timely repair process. CRW places a significant burden on individuals, healthcare systems, and society as a whole, yet it is often underestimated. In my country, the annual demand for wound treatment is approximately 100 million visits, of which 30 million are for CRW, making it a major chronic disease seriously affecting public health. The inability of a wound to heal properly depends on various factors, including wound size, specific causes, individual differences, and the patient's overall health. Generally, a wound that fails to shrink by 50% within one month is considered a CRW.

[0003] Epidemiological surveys show that with the rapid increase in population aging, CRW (traumatic foot ulcer) has shifted from a "traumatic" to a "disease-related" condition, with diabetic ulcer (DU) being the most common. In my country, the incidence of foot ulcers within one year is 8.1% in type 2 diabetic patients over 50 years old, the recurrence rate within one year after healing 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, considering the current development of CRW, especially DU, the prevention and treatment of CRW is urgently needed.

[0004] Modern research confirms that persistent hyperglycemia and inflammatory response are the key factors contributing to the prolonged nature of diabetic ulcerative colitis (DU). Endothelial dysfunction, a critical event in the early stages of DU, has also received increasing attention. During the development of DU, prolonged and excessive inflammation leads to endothelial cell dysfunction and apoptosis, subsequently causing pathological changes in peripheral nerves and blood vessels. Furthermore, long-term exposure to a high-glycemic environment presents the body with multiple challenges, including hemodynamic abnormalities, increased blood viscosity, and mitochondrial dysfunction. These factors exacerbate endothelial damage, making DU wound healing even more difficult. Therefore, repairing endothelial cell function undoubtedly provides a strategy for improving DU.

[0005] Compound Paulownia Leaf Burn Oil is a secret formula medicine developed by the Yao family of the Tujia ethnic group (National Drug Approval Number: Z20063825). The prescription is simple, consisting of only two ingredients: white Paulownia fortunei (Seem.) Hemsl. leaves and sesame oil. It is currently the only innovative medicine with Tujia ethnic characteristics, belonging to the Class III New Traditional Chinese Medicine category. It possesses the functions of "clearing heat and detoxifying, reducing swelling and relieving pain, removing necrotic tissue and promoting tissue regeneration." It is characterized by generally not leaving scars on deep burns and can heal small-area third-degree burns, and is safe with no toxic side effects. Furthermore, this medicine has been clinically tested for CRW (crease wound healing) caused by various reasons, such as diabetic foot, pressure sores, bedsores, and postoperative perianal abscesses, with significant efficacy. Research has found that some active ingredients in Compound Paulownia Leaf Burn Oil can promote wound healing by repairing damage to vascular endothelial cells.

[0006] Currently, the drug registration standard for Compound Paulownia Leaf Burn Oil has been officially included in Volume 77 of the "National Drug Standards for New Drug Approval". This standard uses high-performance liquid chromatography (HPLC) to detect the content of ursolic acid and oleanolic acid, using these as quality control indicators. The filler is octadecylsilane-bonded silica gel; the mobile phase is acetonitrile-methanol-0.5% ammonium acetate solution (61:18:21); the detection wavelength is 210 nm; and the column temperature is 35℃. However, it is worth noting that Compound Paulownia Leaf Burn Oil is carefully formulated from two medicinal materials: white-flowered Paulownia leaves and sesame oil. Ursolic acid and oleanolic acid are only found in white-flowered Paulownia leaves, while the active ingredients in sesame oil are not included in the current detection standard. From the perspective of quality markers for traditional Chinese medicine, marker components should adhere to five core principles: characteristicness, measurability, traceability, relevance to traditional Chinese medicine theory, and effectiveness. Based on the above principles, it is not difficult to see that there is still room for improvement in the existing drug registration standards for Compound Paulownia Leaf Burn Oil.

[0007] Compound Paulownia Leaf Burn Oil, as a traditional Chinese medicine, has the characteristics of multiple components and multiple targets. The following prior art discloses high-performance liquid chromatography (HPLC) methods for the detection of four components in Compound Paulownia Leaf Burn Oil, but these methods cannot be designated as marker components.

[0008] Patent CN115060840A discloses a quality control method for a sesame oil preparation made from white paulownia leaves, which quantitatively analyzes sesamin and sesamolin. The chromatographic column used is a Waters XBridge C18 (4.6 mm × 150 mm, 3.5 μm); gradient elution is performed, with mobile phase A being 5% methanol and mobile phase B being methanol. The proportions of mobile phase B are as follows: HPLC conditions: 0–12 min: 50–100% methanol, 12–25 min: 100–100% methanol, 25–26 min: 100–50% methanol, 26–36 min: 50–50% methanol; column temperature: 30℃; flow rate: 1.0 mL / min; injection volume: 20 μL; detection wavelength: 300 nm. This method can only detect sesamin and sesamolin, which are components of sesame oil and cannot be used as quality markers for compound paulownia leaf burn oil. Summary of the Invention

[0009] To address the above technical problems, the present invention aims to provide a method for detecting the fingerprint spectrum of Compound Paulownia Leaf Burn Oil, which accurately detects the characteristic components of Compound Paulownia Leaf Burn Oil, thereby improving the detection standard of Compound Paulownia Leaf Burn Oil. This method determines its markers from multiple aspects such as characteristicity, measurability, and effectiveness.

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

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

[0012] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

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

[0014] Prepare a sample solution of compound tung leaf burn oil and construct a fingerprint chromatogram of the compound tung leaf burn oil using HPLC.

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

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

[0017] Using the HPLC chromatographic conditions of the fingerprint spectrum in this application, the marker components in white tung oil and sesame oil can be determined simultaneously, and their markers can be identified from multiple aspects such as characteristic, measurable, and effective.

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

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

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

[0021] S1. The total ion chromatogram of the compound tung leaf burn oil sample solution was recorded by UPLC-Q-TOF-MS. The relative molecular mass of each component was analyzed and the chemical composition was inferred. The inferred chemical composition in S1 was prepared as a standard control solution.

[0022] The chromatographic conditions in S1 include: a C18 column, a mobile phase of 0.08–0.12% formic acid water (A)-acetonitrile (B), a flow rate of 0.3–0.5 mL / min, a column temperature of 20–30 °C, and an injection volume of 1.0–4.0 μL. Preferably, the mobile phase uses gradient elution with the following gradient elution parameters: 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). More preferably, the chromatographic column in S1 is an Agilent ZORBAX Eclipse Plus C18.

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

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

[0025] In a preferred embodiment of the present invention, when preparing the test solution of compound tung leaf burn oil, 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.

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

[0027] In a preferred embodiment of the present invention, constructing the fingerprint spectrum of the compound tung leaf burn oil further includes predicting the eigenvalues, contribution rates, and initial factor loading matrices of the components in the compound tung leaf burn oil sample using OPLS-DA, predicting VIP values, and using VIP > 0.8 as a moderate influence to screen out the characteristic components; preferably, the characteristic components are hawthorn acid, corosolic acid, oleanolic acid, ursolic acid, sesamin, linolenic acid, and linoleic acid.

[0028] This invention also discloses a method for detecting the characteristic active ingredients of 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 key active ingredients is calculated by fingerprinting, and these key active ingredients include sesamin, hawthorn acid, corosolic acid, oleanolic acid, and ursolic acid.

[0030] In a preferred embodiment of the present invention, the marker active ingredient is obtained by docking the marker active ingredient in the compound tung leaf burn oil with the target protein through molecular docking technology. The marker active ingredient is obtained by analyzing the complex and clustering analysis of the complex binding energy. Preferably, the target protein is a core target protein in the IL-17 signaling pathway. More preferably, the core target protein in the IL-17 signaling pathway includes IL-17A, NF-κBp65 and AP-1.

[0031] The endothelial cell injury model validated that these components 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] This invention also discloses the application of the marker active ingredient of compound tung leaf burn oil in endothelial cell damage caused by inflammation and high sugar, wherein the marker active ingredient includes one or more of hawthorn acid, corosolic acid, oleanolic acid, ursolic acid and sesamin; preferably, the intervention concentration of the marker active ingredient is 1-100 μM and the intervention time is 18-36 h; more preferably, the intervention concentration is 1-10 μM.

[0033] A model of endothelial dysfunction was established by constructing human microvascular endothelial cells, and the aforementioned key characteristic active ingredients were used for therapeutic intervention.

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

[0035] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0036] This invention provides a fingerprint spectrum and a method and application for detecting the key characteristic active ingredients of compound tung leaf burn oil. It clarifies the differential key characteristic components between different batches of compound tung leaf burn oil. The method is systematic and the results are reliable, providing a scientific screening method for the quality control of compound tung leaf burn oil. Attached Figure Description

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

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

[0039] (A) HPLC fingerprint of 10 batches of compound tung leaf burn oil extract. (B) HPLC chromatograms of mixed standards, sesame oil, and compound tung leaf burn oil; 1. Sesamin; 2. Sesamin; 3. Peak-3; 4. Linoleic acid; 5. Crataegus 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 The binding energy heatmap and cluster analysis of the seven components of compound tung leaf burn oil with characteristic proteins of the IL-17 signaling pathway.

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

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

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

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

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

[0046] (A) Sesamin's effect on the expression of related gene mRNA. (B) Haucacic acid's effect on the expression of related gene mRNA. (C) Corosolic acid's effect on the expression of related gene mRNA. (D) Oleanolic acid's effect on the expression of related gene mRNA. (E) Ursolic acid's effect on the expression of related gene mRNA. Compared with the lipopolysaccharide / high sugar 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's active ingredients promote healing.

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

[0050] Example 1 (S1)

[0051] This step uses UPLC-Q-TOF-MS liquid chromatography-mass spectrometry to perform in-depth qualitative analysis of the compound tung leaf burn oil (see...). Figure 1 The corresponding spectra were analyzed to determine the classification of each compound (see Table 1).

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

[0053] Chromatographic conditions: The column was an Agilent ZORBAX Eclipse Plus C18 (3.0 mm × 100 mm, 1.8 μm), the mobile phase was 0.1% formic acid water (A)-acetonitrile (B), the flow rate was 0.4 mL / min, the column temperature was 25℃, the injection volume was 2.0 μL, and the elution gradient of the mobile phase was 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 ionization (ESI) was used for detection in both positive and negative ion modes. Before sample injection and analysis, Agilent standard tuning solution ESI-L Low Concentration Tuning Mix (G1969-85000) was used for standard mass number calibration. Primary mass spectrometry scanning range: m / z 100-1700, using nitrogen as the desiccant, temperature 325℃, flow rate 6.8 L / min, sheath gas temperature 350℃, capillary voltage 4.0 kV, and fragmentation voltage 150 V.

[0055] Data Analysis: Molecular formulas were deduced from the precise relative molecular masses obtained by liquid chromatography-mass spectrometry (LC-MS / MS) within the possible elemental composition range. The fragmentation patterns of each compound were analyzed by referring to relevant literature and the TCMSP database, along with reference standards, characteristic multi-stage mass spectrometry fragmentation data, and relative abundance information, to further confirm the chemical composition. Simultaneously, the classification of each compound was determined by referring to relevant literature and the 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] Example 2 (S2)

[0063] This step first used HPLC technology to construct fingerprint profiles for 10 batches of compound tung leaf burn oil samples (see...). Figure 2 A) Peak attribution is identified through comparison with standard samples (see...) Figure 2 B). Further analysis using chemometrics (see...) Figure 2 C-E) screened out important components with VIP > 0.8, and finally molecular docking technology (see Figure 3 Candidate marker components of compound tung leaf burn oil were screened out.

[0064] Preparation of the test solution: Weigh 1.0 g of the compound tung leaf burn oil sample and extract it three times with 20 times its volume of methanol at room temperature. Prepare sesame oil using the same method. Collect the methanol and concentrate it to 10 mL, centrifuge at 12000 r / min, and set aside. Accurately weigh 5 mg of each of the following eight standards: sesamin, sesamin, linoleic acid, crataegolic acid, corosolic acid, linolenic acid, oleanolic acid, and ursolic acid. Add them to a 10 mL volumetric flask and dissolve them in methanol to obtain a 0.5 mg / mL standard control solution. High performance liquid chromatography (Agilent 1260) was used for comparative analysis of the test sample and the standard controls.

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

[0066] Chemometric multivariate analysis: Similarity analysis was performed on 10 batches of compound tung leaf burn oil samples using the Traditional Chinese Medicine Fingerprint Similarity Evaluation Software (Version 2012A). Origin 2021 software was used for in-depth analysis of all generated data matrices. Two unsupervised pattern recognition methods, cluster analysis and principal component analysis (PCA), were employed to identify differences in relative peak areas among different batches. Then, the importance of feature components was screened using the threshold variable of projection values ​​(VIP≥0.8) in the orthogonal partial least squares discriminant analysis (OPLS-DA) model.

[0067] Fingerprint analysis: Characteristic fingerprint peaks were identified based on retention time, and the HPLC chromatograms of compound paulownia leaf burn oil and sesame oil were compared and analyzed, identifying a total of 11 common peaks. Among them, peaks 1, 2, 3, 4, 7, 8, and 11 originated from sesame oil components, while peaks 5, 6, 9, and 10 originated from paulownia leaf components (see...). Figure 2 B). After comparison with the standard, eight components were identified, in the following order: sesamin (peak 1), sesamin (peak 2), linoleic acid (peak 4), crataegolic 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 Paulownia Leaf Burn Oil was calculated to be in the range of 0.955–0.997, indicating little difference between 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 assigned to another group (see...). Figure 2 C). This phenomenon suggests that the compound tung leaf burn oil may have subtle effects on the composition due to time factors.

[0068] Multivariate analysis results: Combining the eigenvalues, contribution rates, and initial factor loading matrices of 10 batches of compound tung leaf burn oil (see Tables 2 & 3), and referring to the VIP values ​​of 11 components predicted by OPLS-DA, with VIP > 0.8 considered as moderate influence, 10 characteristic components were screened out, including sesamin, peak-3, linoleic acid, hawthorn acid, corosolic acid, linolenic acid, peak-8, oleanolic acid, ursolic acid, and peak-11 (see Tables 2 & 3). Figure 2 D). Based on the above analysis, ursolic acid, corosolic acid, oleanolic acid and ursolic acid (triterpenoids 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 2.10 Characteristic values ​​and contribution rates of compound tung leaf burn oil

[0070]

[0071] Table 3.10 Initial factor loading matrix of compound tung leaf burn oil

[0072]

[0073]

[0074] Molecular docking analysis: Based on the multivariate analysis results of the chemical components of Compound Paulownia Leaf Burn Oil, molecular docking technology was used to explore the interaction mode and efficiency between the known components of Compound Paulownia Leaf Burn Oil and the core target proteins in the IL-17 signaling pathway, including IL-17A, NF-κB p65 and AP-1.

[0075] Cluster analysis of the binding energies of the complexes revealed that the complexes formed by linolenic acid and linoleic acid with the target protein were grouped together, and their affinity and stability were significantly inferior to the other five complexes. Figure 3 This indicates that these two components have low efficiency in facilitating interactions between target proteins in the IL-17 signaling pathway.

[0076] Regarding the analysis of the complex, five components—sesamin, crataegolic acid, corosolic acid, oleanolic acid, and ursolic acid—exhibited significant binding abilities with IL-17A, NF-κB p65, and AP-1 (see [link to analysis]). Figure 4 The results suggest that sesamin, hawthorn acid, corosolic acid, oleanolic acid, and ursolic acid are more suitable as candidate marker components for compound tung leaf burn oil.

[0077] Example 3 (S3-1)

[0078] This step uses human microvascular endothelial cells (HMEC-1) to establish an endothelial function injury model, and uses RT-qPCR to screen out the marker components with anti-inflammatory effects in 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 alanylglutamine, 1 μg / mL hydrocortisone, and 1% P / S (v / v). Cells were incubated at 37°C in a humidified atmosphere of 5% CO2.

[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). The Plus All-in-One-1st Strand cDNA Reverse Transcription Kit (Novoprotein, Shanghai, China) was used to reverse transcribe RNA to obtain cDNA. mRNA-specific primers were designed using NCBI Primer Blast. Real-time quantitative PCR (qPCR) was employed. Quantitative analysis was performed using SYBR qPCR Supermix Plus. Transcriptional levels were normalized to GAPDH using the 2-ΔΔCq method. Primer information used in this study is shown in Table 4.

[0081] Table 4. Primers for RT-qPCR amplification

[0082]

[0083]

[0084] Establishment of an endothelial dysfunction model: Based on the understanding that high glucose and inflammatory response are key factors leading to endothelial injury in duodenal endothelial cells (DU), human microvascular endothelial cells (HMEC-1) were treated for 24 hours 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) to investigate their potential effects on the expression levels of downstream genes in the IL-17 signaling pathway (see [link to study]). Figure 5This study simulated a combined model of inflammation and endothelial cell damage in a high-glucose microenvironment. By comprehensively considering the expression levels of multiple pathway proteins and inflammatory markers, the optimal concentration combination for constructing the lipopolysaccharide / high-glucose combined endothelial cell damage model was selected: lipopolysaccharide 2 μg / mL and glucose 25 mM. Under this combination, the IL-17-mediated NF-κB / AP-1 signaling pathway was highly activated. In this state, downstream inflammatory factors, including but not limited to TNF-α, IL-6, and IL-1β, were overexpressed, further exacerbating endothelial cell dysfunction and damage.

[0085] Screening of active ingredients: A 24-hour treatment intervention was conducted using different concentrations of the active ingredients from compound tung leaf burn oil on a constructed endothelial cell injury model to explore the effective regulatory ability of the active ingredients of compound tung leaf burn oil on the downstream inflammatory cascade of the IL-17 signaling pathway. Based on previous CCK8 detection results, the investigation concentrations of crataegolic acid, corosolic acid, oleanolic acid, and ursolic acid were determined to be 1 μM and 5 μM, respectively. For IC50... 10 Larger amounts of sesamin were examined at concentrations of 2.5 μM and 10 μM. Following this, the mRNA expression levels of four classic inflammatory markers—IL-6, TNF-α, TLR4, and IL-1β—were systematically detected. The mRNA expression of core proteins in the IL-17 signaling pathway, TRAF6, NF-κB, MAPK1, and c-Fos, was also investigated (see [link to study]). Figure 6 The results showed that the active ingredients of the compound tung leaf burn oil generally exhibited significant inhibitory effects on gene expression downstream of the IL-17 signaling pathway. Among them, only corosolic acid showed relatively low sensitivity in regulating c-Fos protein.

[0086] Example 4 (S3-2)

[0087] This step uses human microvascular endothelial cells (HMEC-1) to establish an endothelial function damage model, and uses scratch experiments to screen for marker components in compound tung leaf burn oil that promote healing.

[0088] Scratch test procedure: Place 2×10 6 HMEC-1 cells were seeded in 6-well plates and allowed to adhere for 24 hours. Afterward, wounds were created at the bottom of the wells using the tip of a 200 μL pipette. The culture medium was then replaced with basal medium (5.5 mM glucose), and cells were treated with different interventions for 24 hours. High-resolution images of cell migration were captured immediately after scratching (0 hours) and 24 hours after scratching. ImageJ was used to quantify the migration amount of each group of cells.

[0089] Screening of scratch assay conditions: To ensure the experiment comprehensively reflects cell migration behavior under different pathophysiological conditions, multiple control groups were set up, including a lipopolysaccharide (LPS)-only group, a high-glucose group, and a LPS / high-glucose group receiving both LPS and high-glucose stimulation, to comprehensively assess the potential impact of inflammation and high-glucose environment on cell migration. A blank control group (5.5 mM glucose) was used. The treatment group was set as the LPS / high-glucose + active ingredient group, which introduced the active ingredient of compound tung oil on top of LPS / high-glucose treatment, aiming to observe the specific effects of the active ingredient of compound tung oil on cell migration. Analysis of the scratch assay results showed that the scratch width in the high-glucose group and the LPS / high-glucose combined group was significantly wider than that in the blank control group and the LPS group after 24 hours of treatment (P < 0.05), while no significant difference in scratch width was observed between the LPS group and the blank control group (see...). Figure 7 (A&B) This finding strongly supports the view that a high-sugar microenvironment is one of the key factors contributing to slow wound healing.

[0090] Screening of active ingredients promoting healing: Further, it was observed that the addition of 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 significantly increased cell migration rate. This fully demonstrates that the active ingredients of the compound tung leaf burn oil do indeed have the ability to promote cell migration (see...). Figure 7 (C&D). Unfortunately, while crataegolic acid also showed some promoting effect, 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 in a 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. Statistical analysis was performed using GraphPad Prism 8.0.2 software. Analysis of variance (ANOVA) was used to analyze differences between groups. P < 0.05 was considered statistically significant.

Claims

1. A detection method of a fingerprint of compound tung oil for burns, characterized in that The method comprises the following steps: The sample solution of the compound leaf of Aegiceras corniculatum Burn oil is prepared, and a fingerprint of the compound leaf of Aegiceras corniculatum Burn oil is constructed by HPLC; The HPLC chromatographic conditions for constructing the fingerprint of the compound leaf of Aegiceras corniculatum Burn oil comprise: an octadecyl silica water-resistant analytical column, a mobile phase A of methanol, a mobile phase B of 0.08-0.12% ammonium acetate, a flow rate of 0.5-0.7 mL / min, and a sample injection amount of 5-15 μL; The HPLC chromatography for constructing the fingerprint of the compound leaf of Aegiceras corniculatum Burn oil adopts gradient elution, and the parameters of the gradient elution are as follows: 0-5 min, 80-85% of the mobile phase A; 5-20 min, 85-90% of the mobile phase A; 20-35 min, 85-90% of the mobile phase A; 35-36 min, 90-85% of the mobile phase A; and 36-40 min, 85-80% of the mobile phase A; The preparation method for preparing the sample solution of the compound leaf of Aegiceras corniculatum Burn oil comprises the following steps: the compound leaf of Aegiceras corniculatum Burn oil sample is extracted with methanol, concentrated, diluted to a constant volume, and centrifuged. When the sample solution of the compound leaf of Aegiceras corniculatum Burn oil is prepared, the volume of the methanol is 10-30 times the volume of the compound leaf of Aegiceras corniculatum Burn oil sample, and the methanol extraction is performed for 2-5 times.

2. The method according to claim 1, wherein, The detection wavelength of the HPLC chromatography in S2 is as follows: 0-12 min, λmax is 240-270 nm; and 12-40 min, λmax is 205-215 nm.

3. The method according to claim 1, wherein the method is characterized by, The chromatographic column used for constructing the fingerprint of the compound leaf of Aegiceras corniculatum Burn oil is a Supersil AQ-C18 chromatographic column.

4. The method according to claim 3, wherein, The mass of the compound leaf of Aegiceras corniculatum Burn oil sample is 0.5-1.5 g, the volume of the constant volume is 5-15 mL, and the centrifugal speed is 10000-14000 r / min.

5. A method for detecting the characteristic active ingredient of compound tung leaf burn oil, characterized in that, The detection method of the fingerprint of the compound leaf of Aegiceras corniculatum Burn oil according to any one of claims 1-4 further comprises the following steps: The content of the marker characteristic effective component is calculated by the fingerprint, and the marker characteristic effective component comprises sesamin, maslinic acid, corosolic acid, oleanolic acid and ursolic acid.

6. The method for detecting the marker components of the compound leaf of Azadirachta indica A. Juss burn oil according to claim 5, characterized in that, The marker characteristic effective component is obtained by performing molecular docking between the marker characteristic component in the compound leaf of Aegiceras corniculatum Burn oil and a target protein, analyzing the complex, and performing cluster analysis on the binding energy of the complex.

7. The method for detecting the characteristic active ingredient of compound tung leaf burn oil according to claim 6, characterized in that, The target protein is a core target protein in an IL-17 signaling pathway.

8. The method for detecting the characteristic active ingredient of compound tung leaf burn oil according to claim 7, characterized in that, The core target protein in the IL-17 signaling pathway comprises IL-17A, NF-κB p65 and AP-1.

9. The application of a characteristic active ingredient of a compound tung leaf burn oil in endothelial cell damage induced by inflammation and high glucose, characterized in that, The marker characteristic effective component comprises one or more of maslinic acid, corosolic acid, oleanolic acid, ursolic acid and sesamin.

10. Use according to claim 9, characterized in that, The intervention concentration of the marker characteristic effective component is 1-100 μM, and the intervention time is 18-36 h.

11. Use according to claim 10, characterized in that, The intervention concentration is 1-10 μM.

12. The use according to claim 10, characterized in that, The construction steps of the endothelial function damage model of human microvascular endothelial cells include: scratching the human microvascular endothelial cells, and then respectively inducing the scratched human microvascular endothelial cells by combining lipopolysaccharide 1-3 μg / mL with glucose 20-30 mM, and the combined induction time is 18-36 h.

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