A composition for alleviating drug-resistant lung cancer based on peony petals, its preparation method and application

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a composition for alleviating drug-resistant lung cancer based on peony petals, its preparation method, and its application. The composition for alleviating drug-resistant lung cancer based on peony petals includes 1,2,3,4,6-pentagalloglucopyranoside and osimertinib. This invention obtained a polyphenol compound, 1,2,3,4,6-pentagalloglucopyranoside (PGG), from the mass spectrometry analysis of the osimertinib-PP fraction obtained from peony petal (PP) extract. PGG induces ferroptosis in osimertinib-resistant cells and parental lung cancer cells by inhibiting key metabolic pathways, thus improving EGFR-TKI resistance in lung cancer and showing promise as a therapeutic candidate.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a composition for alleviating drug-resistant lung cancer based on peony petals, its preparation method, and its application. Background Technology

[0002] Lung cancer is a leading cause of cancer death worldwide. It is classified into small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with approximately 85% of cases being NSCLC. In NSCLC patients, a significant subgroup carries activating mutations in the epidermal growth factor receptor (EGFR), such as exon 19 deletion (Del19) and L858R substitution in exon 21. These mutations drive tumor progression by constitutively activating EGFR signaling, promoting cell proliferation, survival, and anti-apoptosis. The advent of EGFR tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of EGFR-mutant lung cancer, offering significant clinical benefits compared to conventional chemotherapy.

[0003] Osimertinib (OSI) is a third-generation TKI that irreversibly binds to the cysteine ​​797 (C797) residue of EGFR ATP, leading to sustained inhibition of downstream oncogenic signaling. However, despite initial efficacy, resistance inevitably develops, posing a significant challenge in clinical treatment. The underlying mechanisms of osimertinib resistance are complex and heterogeneous, involving secondary mutations in EGFR (e.g., C797S), activation of alternative signaling pathways (e.g., MET amplification, HER2 expression, KRAS mutations), histological transformation (e.g., epithelial-mesenchymal transition, EMT, small cell lung cancer transformation), and epigenetic modifications that allow tumors to evade treatment-induced stress by altering their bioenergetic dependence.

[0004] Peony petals (PP) possess various biological activities, making them valuable in both traditional medicine and modern pharmacology. Rich in paeoniflorin, flavonoids, polyphenols, tannins, and volatile oils, peony petals exhibit antioxidant, antimicrobial, and immunomodulatory properties. Modern research indicates that peony petal (PP) extracts possess antitumor potential, inducing apoptosis, inhibiting cell proliferation, and regulating key signaling pathways in various cancer models, including lung cancer. However, the specific bioactive components responsible for these anticancer effects remain to be fully elucidated. Summary of the Invention

[0005] The purpose of this invention is to provide a composition for alleviating drug-resistant lung cancer based on peony petals, its preparation method, and its application, thereby overcoming the shortcomings of existing technologies. A specific bioactive component, 1,2,3,4,6-pentagalloylglucose (PGG), is obtained from peony petal extract. PGG can effectively improve the resistance of EGFR-mutant lung cancer patients to osimertinib by inducing ferroptosis and disrupting metabolic pathways. The combined use of PGG and OSI can enhance ferroptosis-related cell death, providing a novel and promising therapeutic strategy for overcoming EGFR-TKI resistance.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a peony petal-based drug composition for alleviating drug-resistant lung cancer, comprising 1,2,3,4,6-pentagalloglucoside and osimertinib, wherein the molar ratio of 1,2,3,4,6-pentagalloglucoside and osimertinib is (5-160):(1-2).

[0008] The lung cancer is human non-small cell lung cancer with acquired resistance to osimertinib.

[0009] 1,2,3,4,6-Pentagalloglucoside (PGG) is a key component of peony petal (PP) extract in this invention. PGG effectively improves resistance to osimertinib in EGFR-mutant lung cancer patients by inducing ferroptosis and disrupting metabolic pathways. The combined use of PGG and OSI enhances ferroptosis-related cell death, providing a novel and promising therapeutic strategy for overcoming resistance to epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs).

[0010] In some other embodiments, the molar ratio of 1,2,3,4,6-pentagalloglucoside to osimertinib is (20-160):(1-2). PGG combined with OSI within this ratio range shows good efficacy.

[0011] In a second aspect, the present invention provides a method for preparing a composition for alleviating drug-resistant lung cancer based on peony petals, which is obtained by mixing 1,2,3,4,6-pentagalloglucoside and osimertinib.

[0012] In some other embodiments, 1,2,3,4,6-pentagalloglucopyranose is selected from peony petal extract, and the preparation method of 1,2,3,4,6-pentagalloglucopyranose is as follows:

[0013] Peony petals were added to a solvent, heated under reflux to extract, and the extracts were combined and concentrated to obtain a concentrated solution.

[0014] The concentrate was enriched by passing it through a macroporous resin column and eluted with an ethanol aqueous solution with a mass concentration of 10%-70%. The eluent with a mass concentration of 40% was collected and concentrated to obtain the crude extract.

[0015] The crude extract was dissolved in a two-phase solvent, purified by high-speed countercurrent chromatography, and dried to obtain the peony petal extract.

[0016] Peony petals possess various biological activities, making them valuable in both traditional medicine and modern pharmacology. Rich in paeoniflorin, flavonoids, polyphenols, tannins, and volatile oils, peony petals exhibit antioxidant, antimicrobial, and immunomodulatory properties. Paeoniflorin, one of the main active ingredients in peony petals, exerts neuroprotective effects by reducing oxidative stress, inhibiting inflammatory cytokines, and regulating neurotransmitter release, making it a potential candidate for treating neurodegenerative diseases. Flavonoids and polyphenols possess strong free radical scavenging capabilities, thus preventing cell damage, slowing aging, and reducing the incidence of chronic diseases. Peony petal extracts also have hepatoprotective effects, reducing liver fibrosis and alleviating liver damage by inhibiting pro-inflammatory pathways. Peony petals also possess cardioprotective properties, improving blood circulation, reducing platelet aggregation, and preventing ischemia-reperfusion injury. Modern research indicates that PP extract has anti-tumor potential; studies have shown it can induce apoptosis, inhibit cell proliferation, and regulate key signaling pathways in various cancer models, including lung cancer. However, the specific bioactive components responsible for these anti-cancer effects still need to be fully elucidated.

[0017] The preparation method of this invention is simple to operate and has low separation cost, enabling the high-value use of peony petals. Using this method, 1,2,3,4,6-pentagalloglucopyranose with high separation and purity can be obtained well from peony petal (PP) extract.

[0018] In some other embodiments, the solvent is one of an aqueous solution of methanol and an aqueous solution of ethanol, wherein the mass concentration of the solvent is 90%-95%.

[0019] The mass ratio of peony petals to solvent is 1:(5-10);

[0020] The temperature for reflux extraction is 50-60℃, the time is 2-4 h, and the number of extractions is 2-4.

[0021] The concentration temperature is 35-45℃.

[0022] For example, the solvent is an aqueous solution of methanol or an aqueous solution of ethanol, with a solvent mass concentration of 90% or 95%; the mass ratio of peony petals to solvent is 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10; the temperature for reflux extraction is 50℃, 55℃, or 60℃; the reflux extraction time is 2 h, 3 h, or 4 h; the number of extractions is 2, 3, or 4; and the concentration temperature is 35℃, 40℃, or 45℃.

[0023] This extraction method can obtain a high crude extract.

[0024] In some other embodiments, the macroporous resin column is one of MCI, HPD-750 and MQ-01;

[0025] The elution conditions were gradient elution with aqueous ethanol solutions of 10%, 20%, 30%, 40%, and 70% by mass, respectively.

[0026] In some other embodiments, the two-phase solvent is n-hexane / ethyl acetate / methanol / water, and the volume ratio of n-hexane / ethyl acetate / methanol / water is (1-2):(8-9):(1-4):(6-9);

[0027] The flow rate of high-speed countercurrent chromatography is 2 mL / min-5 mL / min, the rotation speed is 800 r / min-1000 r / min, and the retention rate is 65%-75%.

[0028] For example, the volume ratio of n-hexane / ethyl acetate / methanol / water is 1:9:1:9, 2:8:4:6, or 2:8:2:8; the flow rate of the high-speed countercurrent chromatography is 2 mL / min, 3 mL / min, 4 mL / min, or 5 mL / min, the rotation speed is 800 r / min, 850 r / min, 900 r / min, 950 r / min, or 1000 r / min, and the retention rate is 65%, 70%, or 75%.

[0029] In high-speed countercurrent chromatography (HSCCC), the choice of solvent system is fundamental, directly determining whether stable stationary and mobile phases can be formed, thus affecting separation efficiency and the recovery rate of target compounds. During their research, the inventors optimized the solvent system to adjust the partition coefficients of the compounds. K D To improve separation efficiency, a high-speed countercurrent chromatography (HSC) system of hexane / ethyl acetate / methanol / water was selected based on factors such as the polarity of the peony petal extract. K D The value represents the solubility and partitioning of the compound between different phases in a two-phase solvent system. K DThe value >2 and the larger the value, the slower the compound elutes in countercurrent chromatography using the above phase as the stationary phase; K D <0.5 indicates that the compound has a short retention time and may be difficult to separate effectively; K D When the value is between 0.5 and 2, separation is relatively reasonable. To determine whether two compounds can be separated, the separation coefficient α needs to be used. 1,2 = K D1 / K D2 ( K D1 > K D2 When α>1.5, it indicates that the compounds can be separated well; when α<1.5, it is necessary to use the cyclic method of countercurrent chromatography for separation.

[0030] In some other embodiments, the volume ratio of n-hexane / ethyl acetate / methanol / water is 2:8:4:6;

[0031] The flow rate of high-speed countercurrent chromatography was 2 mL / min; the main unit speed was 850 r / min; and the retention rate was 70%.

[0032] Under these conditions, high-speed countercurrent chromatography separation has the advantages of simple sample pretreatment, good repeatability, and avoidance of sample adsorption by packing material compared with traditional separation and purification methods.

[0033] Thirdly, the present invention provides the use of the peony petal-based composition for alleviating drug-resistant lung cancer in the preparation of a product for alleviating drug-resistant lung cancer, wherein the lung cancer is human non-small cell lung cancer with acquired resistance to osimertinib.

[0034] In some other embodiments, the product is a drug that includes pharmaceutically acceptable excipients.

[0035] In some other embodiments, the excipients include one of buffers, stabilizers, preservatives, and excipients.

[0036] The beneficial effects of this invention are:

[0037] (1) The present invention 1,2,3,4,6-pentagalloglucoside (PGG) effectively overcomes the resistance of EGFR mutant lung cancer cells to osimertinib (OSI) by inducing ferroptosis and disrupting key metabolic pathways, providing a new therapeutic approach to overcome EGFR-TKI resistance. PGG not only has independent anti-tumor effects, but its combined application with OSI may become a potential strategy to combat lung cancer drug resistance.

[0038] (2) The present invention isolates 1,2,3,4,6-pentagalloglucoside (PGG), a specific bioactive component with strong anti-cancer activity, from peony petal (PP) extract. The operation is simple and the separation cost is low, which can realize the high-value use of peony petals. Attached Figure Description

[0039] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0040] Figure 1 The figure shows the separation and identification results of PGG from peony petals in Example 1 of the present invention, where A is the high performance liquid chromatogram of PGG, B is the purity chromatogram of PGG, and C is the electrospray ionization mass spectrum of PGG.

[0041] Figure 2 This is a graph showing the inhibition of PGG on the proliferation of otinib-resistant cells and parental lung cancer cells in Example 1 of the present invention. A is a graph showing cell viability after treatment with PGG on parental and otinib-resistant lung cancer cells using CCK8 assay; B is a graph showing the effect of PGG dosage on cell survival in PC9 and PC9-OR cells; C is a graph showing the effect of combined PGG and OSI on fractional anisotropy in PC9 and H1975 cells, where C-1 shows the effect on PC9-resistant cells, C-2 shows the effect on H1975-resistant cells; D is a graph showing the effect of PGG on cell survival in PC9, PC9-OR, and PC9-OR-OSI cells in the control group, where D-1 is a microscopic image of cell clonal morphology, and D-2 is the quantitative statistical graph corresponding to D-1.

[0042] Figure 3The diagrams shown in Example 1 of this invention illustrate the inhibition of lung cancer cell migration, invasion, and apoptosis induced by PGG. A represents the inhibition of lung cancer cell migration by PGG, where A-1 is a microscopic observation image, and A-2 is a statistical analysis of the cell count in A-1. B represents the inhibition of lung cancer cell invasion by PGG, where B-1 is a scratch healing experiment image, and B-2 is a quantitative statistical bar chart corresponding to B-1. C represents the induction of apoptosis by PGG, where C-1 is a flow cytometry scatter plot of the blank control group of PC9 cells, and C-2 is a flow cytometry scatter plot of PC9 cells. Scatter plots of flow cytometry using PGG were used. C-3 is the scatter plot of flow cytometry for the blank control group of PC9 resistant cells; C-4 is the scatter plot of flow cytometry for PC9 resistant cells using PGG; C-5 is the scatter plot of flow cytometry for the blank control group of PC9 resistant cells combined with osimertinib; and C-6 is the scatter plot of flow cytometry for PC9 resistant cells combined with osimertinib using PGG. In C-1 to C-6, Q1 represents mechanically damaged cells, Q2 represents late apoptotic cells, Q3 represents early apoptotic cells, and Q4 represents live cells.

[0043] Figure 4 These are the PGG differential gene expression analysis diagram, cluster analysis diagram, and enhanced ferroptosis-related gene expression diagram in Example 1 of the present invention. In this diagram, A represents the differential gene expression analysis diagram, B represents the cluster analysis diagram, and B-1 represents the Kyoto Encyclopedia of Genes and Genomes analysis diagram of the C7 gene cluster, while B-2 represents the gene ontology analysis diagram of the C7 gene cluster.

[0044] B-3 is the Kyoto Encyclopedia of Genes and Genomes analysis diagram of the C3 gene cluster; B-4 is the gene ontology analysis diagram of the C3 gene cluster; C is the expression diagram of genes related to enhanced ferroptosis, including C-1, prion protein gene diagram; C-2, microtubule-associated protein 1 light chain 3β gene diagram; C-3, transferrin receptor 1 gene diagram; C-4, long-chain acyl-CoA synthase 4 gene diagram; C-5, solute carrier family 3 member 2 gene diagram; and C-6, long-chain acyl-CoA synthase 3 gene diagram.

[0045] Figure 5 In Example 1 of this invention, PGG inhibits tumor growth by inducing ferroptosis in ostinib-resistant lung cancer cells; wherein, A is the GSH level map after PGG treatment, B is the MDA level map after PGG treatment, C is the downregulation map of iron poisoning-related proteins GPX4 and SLC7A11 after PGG treatment, and D is the map showing that Fer-1 treatment effectively reversed PGG-induced cell death. Detailed Implementation

[0046] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Specific conditions are not specified in the embodiments; they were performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all components used are commercially available conventional products. Some of the reagents, materials, and equipment used are as follows:

[0047] The organic solvents used in the MCI macroporous resin column purification, including methanol, dimethyl sulfoxide (DMSO), and ethanol, were all analytical grade and purchased from Tianjin Kemei Chemical Reagent Co., Ltd. Acetonitrile and formic acid of chromatographic grade, and ultrapure water were used for high-performance liquid chromatography (HPLC) detection. The HPLC system was equipped with an online degasser, a quaternary pump, a UV detector, and a temperature-controlled column chamber. Electrospray ionization mass spectrometry (ESI / MS) analysis was performed using an MSD trap.

[0048] Example 1

[0049] A method for preparing peony petal extract, comprising the following steps:

[0050] (1) Mix 500 g of peony petals with a 95% ethanol aqueous solution at a ratio of 1:10. w / v The extracts were mixed and boiled twice using an electric heating mantle, each extraction lasting 2 hours, to obtain two extracts. The two extracts were combined and concentrated using a rotary evaporator at 40°C to obtain 20 g of crude peony petal extract.

[0051] (2) 20 g of crude peony petal extract was eluted with gradients of ethanol aqueous solution with mass concentrations of 10%, 20%, 30%, 40% and 70% using an MCI macroporous resin column. The eluted fraction with mass concentration of 40% ethanol aqueous solution was collected and concentrated using a rotary evaporator to obtain 560 mg of crude β-PGG extract.

[0052] (3) The crude β-PGG extract was dissolved in hexane / ethyl acetate / methanol / water (2:8:4:6, v / v), with a flow rate of 2 mL / min and a retention rate of 70%, to separate the crude β-PGG extract. The single injection volume was 50 mg, and the separation time was 110 min-125 min. The compound β-PGG was eluted. The eluted sample was collected, concentrated by rotary evaporation to remove the organic solvent, and lyophilized to obtain 95 mg of the β-PGG monomer with a purity of 99%.

[0053] (5) Electrospray ionization mass spectrometry (ESI / MS) analysis of β-PGG was performed using an MSD trap, with the mass scan range being [missing information]. m / z50-1500, carrier gas temperature 320℃, carrier gas flow rate 8 L / min, sprayer pressure 40 psi, capillary voltage 4000 V, nozzle voltage 1000 V, fragmentation voltage 150 V.

[0054] Comparative Example 1

[0055] A method for preparing peony petal extract differs from Example 1 in that, in step (3), the volume ratios of hexane / ethyl acetate / methanol / water are 1:9:1:9 and 2:8:2:8, respectively, while the other steps are the same as in Example 1.

[0056] Studies have found that this ratio is suitable for solvent systems. K D All values ​​are greater than 2, indicating a longer elution time.

[0057] Comparative Example 2

[0058] A method for preparing peony petal extract differs from Example 1 in that, in step (3), the volume ratio of ethyl acetate / n-butanol / methanol / water is 5:1:0.5:4, while the other steps are the same as in Example 1.

[0059] The study found that the solvent system could not achieve the separation of β-PGG because the separation time was too long (more than 10 hours) and the amount of elution solvent consumed was too large.

[0060] Comparative Example 3

[0061] A method for preparing peony petal extract, differing from Example 1 in that step (3) involves column chromatography for separation and purification, while the other steps are the same as in Example 1. The specific column chromatography separation and purification steps are as follows:

[0062] The crude β-PGG extract was purified using a YMC-Pack ODS-A column (250 mm × 10 mm, 5.0 μm) on a Wooking-K2025 HPLC system. The purification process used mobile phases A and B. Mobile phase A consisted of acetonitrile, and mobile phase B consisted of water containing 0.1% formic acid. The elution program was 13% mobile phase A for 30 minutes at a flow rate of 3.0 mL / min and a detection wavelength of 300 nm. Pure β-PGG was obtained.

[0063] Example 2

[0064] A method for using peony petal extract in combination with osimertinib to alleviate drug-resistant lung cancer includes the following steps:

[0065] 1. Cell culture: PC 9 cells (human non-small cell lung cancer cells PC9) and H1975 cells (human EGFR gene mutation-positive non-small cell lung cancer cells H1975) were used as research subjects. PC 9 and H1975 cells were purchased from the American Type Culture Collection (ATCC).

[0066] The cell culture procedure was as follows: PC 9 and H1975 cells were placed in a humidified incubator at 37°C and 5% CO2 in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco). Cells were passaged using 0.25% trypsin containing EDTA (Gibco), and mycoplasma contamination was checked periodically.

[0067] 2. Preparation of osimertinib-resistant (OR) cells: PC9 and H1975 cells were placed in a culture medium with progressively increasing osimertinib concentrations (25 nM–2 µM) to prepare osimertinib-resistant PC9-OR cells and osimertinib-resistant H1975-OR cells. The specific preparation process is as follows:

[0068] PC9-OR (final concentration of osimertinib 1 µM) was prepared by gradually increasing the osimertinib concentration from 25 nM to 1 µM in PC9 cells. Similarly, H1975-OR (final concentration of osimertinib 2 µM) was prepared by gradually increasing the osimertinib concentration from 25 nM to 2 µM in H1975 cells.

[0069] Resistance was confirmed by measuring the EC adhesion value of the cells. Compared with the parental PC9 and H1975 cells, the EC adhesion values ​​of PC9-OR and H1975-OR cells increased by at least 100-fold, thus confirming the preparation of PC9-OR cells and H1975-OR cells resistant to osimertinib.

[0070] 3. Cell viability assay: Cultured PC9 cells and PC9-OR cells were divided into 5 × 10⁶ cells per well. 3Cells were seeded at a density in 96-well plates and allowed to adhere overnight. PC9 cells (final osimertinib concentration 1 µM), PC9-OR cells (final osimertinib concentration 1 µM), H1975 cells (final osimertinib concentration 2 µM), and H1975-OR cells (final osimertinib concentration 2 µM) were treated for 72 h with 1,2,3,4,6-pentagalloglucopyranoside (PGG) at a concentration ratio of (0-160):1 in PC9 and PC9-OR cells; and in H1975 and H1975-OR cells, the ratio was (0-160):2.

[0071] Following the manufacturer's instructions, the cell viability of PC-9 and H1975 related cell lines subjected to different concentrations of PGG was measured using the CCK-8 assay (Yeasen) (each group was tested three times), and the results are shown in Tables 1 and 2.

[0072] Table 1. Cell viability results of PC-9 related cell lines measured using different concentrations of PGG.

[0073]

[0074] Table 2 shows the cell viability results of H1975-related cell lines measured using different concentrations of PGG.

[0075]

[0076] Tables 1 and 2 show that PGG alone, at a concentration of ≥20 µM, can inhibit the activity of PC9, PC9-OR, H1975, and H1975-OR cells by more than 50%. When PGG is combined with 1 µM osimertinib, PGG can alleviate the resistance of PC9-OR and H1975-OR cells to osimertinib, and adding only 10 µM PPG can achieve a tumor inhibition effect of more than 50%. Further research will be conducted using PC9 and PC9-OR cells as examples.

[0077] 4. Cell colony formation assay

[0078] Cells (500 cells / well) were placed in six-well plates and incubated overnight in an incubator. Cells were cultured with PGG for 14 days, with the culture medium changed every 3 days. Cell clones were fixed with 4% paraformaldehyde (PFA, Biosharp) for 25 minutes, stained with crystal violet (Biosharp) for 20 minutes, washed with PBS, and imaged using an optical microscope (Nikon, Japan). The number of cell clones was quantified using ImageJ software.

[0079] 5. Cell scratch test

[0080] Well-grown cells were seeded into six-well plates and cultured to 90% confluence. Linear wounds were created using the tip of a 200 µL pipette, and cells were washed with PBS to remove debris. Cells were then treated with PGG, and cell scratching was monitored at 0, 24, and 48 hours using a phase-contrast microscope (Nikon TS100). Images were analyzed using ImageJ software, and the cell scratch rate was calculated as [(initial wound area - final wound area) / initial wound area] × 100%.

[0081] 6. Cell invasion assay

[0082] Cell invasion was assessed using a transwell (Corning, Cat#356234) pre-coated with 50 µL Matrigel (Corning, Cat#354480). Cells (2×10⁻⁶) 4 Cells were suspended in serum-free medium and seeded in the upper chamber, while in the lower chamber, intact medium containing 10% FBS was used as a chemical inducer. After 24 hours, non-invasive cells were removed, and invasive cells were fixed with 4% paraformaldehyde, stained with crystal violet, and photographed from five randomly selected fields of view under an inverted microscope.

[0083] 7. Apoptosis assay

[0084] Cells were seeded in six-well plates and treated with PGG for 48 hours. Apoptotic cells were analyzed using the annexin V-FITC / PI apoptosis detection kit (Yeasen). Cells were collected, washed with cold PBS, and resuspended in 100 µL binding buffer containing annexin V-FITC and PI. After incubation at room temperature for 15 minutes, samples were analyzed using a BDFACSCanto II flow cytometer. Data were processed using FlowJo software.

[0085] 8. Large-scale RNA sequencing and bioinformatics analysis

[0086] Total RNA was extracted from PGG-treated and control cells using the RNeasy Mini Kit (Qiagen, Cat# 74104). RNA quality was assessed using an Agilent 2100 bioanalyzer. RNA-seq libraries were prepared using the VAHTS Universal V8 RNA-seqLibrary Prep Kit for MGI (Vazyme) and sequenced on the MGI-seq 2000 platform. Differentially expressed genes (DEGs) were identified using DESeq2, and adjusted RNA was then analyzed. p The cutoff value was <0.05. Pathway enrichment analysis was performed using Gene Set Enrichment Analysis (GSEA) and KEGG annotation.

[0087] 9. Real-time quantitative RT-PCR

[0088] Total RNA was isolated from mouse whole lungs using RNA-easy isolation reagent (Vazyme, Nanjing, China) and reverse transcribed using HiScript III RT SuperMix (Vazyme) according to the manufacturer's protocol. qPCR reactions were performed on an Archimed X instrument using ChamQ Universal SYBR qPCR Master Mix (Vazyme). Expression values ​​were normalized to Gapdh. The gene-specific primers used (forward and reverse) are shown in Table 3.

[0089] Table 3 Gene-specific primers

[0090]

[0091] Note in Table 3: F - forward primer, R - reverse primer

[0092] II. Results Analysis

[0093] All experiments were repeated in triplicate. Data are expressed as mean ± standard deviation (SD). Statistical significance was determined using Student's t-test or one-way ANOVA with Tukey's post-hoc test. p A value <0.05 was considered statistically significant. Data analysis was performed using GraphPad Prism 9.0 software. Absorbance was measured at 450 nm using a microplate reader (Molecular Devices, USA). All experiments were performed in triplicate. IC50 values ​​were calculated from the dose-response curves using GraphPad Prism 9.0.

[0094] 1. Isolation and characterization of bioactive substances in peony petals

[0095] Isolation and identification of PGG from peony petals in Example 1. Figure 1 As can be seen from A in the figure, the method of the present invention can effectively separate bioactive compounds, including PGG. High-speed countercurrent chromatography purified 1,2,3,4,6-penta-O-galloyl-β-D-glucoside (PGG) with a purity of 99%, using a single injection volume of 50 mg. Lyophilization yielded 95 mg of the β-PGG monomer, consistent with its low natural abundance. Figure 1 (As shown in B). ESI-MS structural analysis confirmed that the molecular formula of PGG is C. 41 H 32 O 26 It possesses quasi-molecular ions [MH]. - m / z 939.1125 (calculated value: 939.1196). Fragment analysis shows that in m / z There is a distinct peak at 469.0531, corresponding to C. 20 H 21 O 13 The loss of the fragment further confirmed the chemical formula of PGG ( Figure 1 (as shown in C).

[0096] 2. PGG inhibits the proliferation of parental and osimertinib-resistant lung cancer cells.

[0097] The anticancer potential of isolated compounds was evaluated using CCK8 assays on parental (PC9, H1975) and osimertinib (OSI)-resistant lung cancer cells (PC9-OR, H1975-OR). Both parental (PC9, H1975) and osimertinib (OSI)-resistant lung cancer cells (PC9-OR, H1975-OR) were treated with 40 µM of 1,2,3,4,6-penta-O-galloyl-β-D-glucoside (PGG), and cell viability was measured. PGG significantly reduced the viability of PC9, H1975, and OSI-resistant cells. p <0.001)( Figure 2 As shown in Figure A). Treatment of cells with different concentrations (5-160 µM) of PGG revealed that PGG-induced cytotoxicity was concentration-dependent. Figure 2 As shown in B). The combined use of PGG and OSI synergistically reduced the viability of OSI-resistant cells, indicating that PGG induced these PC9 resistant cells (as shown in B). Figure 2 (as shown in C-1 of C) and H1975 drug-resistant cells ( Figure 2 (As shown in C-2 of the diagram) is more sensitive to OSI treatment. p<0.001). Where CI value >1 indicates an antagonistic reaction, CI value = 1 indicates an additive effect, and CI value <1 indicates a synergistic effect.

[0098] Cell colony formation assays confirmed these findings, as PGG treatment resulted in a significant reduction in the number and size of cell colonies in both parental and resistant cells. Figure 2 (As shown in D-1 of D in the figure). These results collectively demonstrate that PGG can effectively inhibit the proliferation of lung cancer cells and overcome OSI resistance (D in D-1 of the figure). Figure 2 (D in D-2 is shown in the diagram). PGG inhibits migration, invasion, and induces cell death in order to evaluate the effects of PGG on cancer cell motility and invasiveness.

[0099] PGG (40 µM) showed a significant effect in impairing cell migration in both parental and OSI-resistant cells, as shown in the microscopic observation figure (Figure 1). Figure 3 (A-1 in A), Statistical analysis of cell number () Figure 3 As shown in A-2 of A in the diagram), the cell scratches decreased after 48 hours ( p <0.01). A Transwell invasion test was performed, as shown in the scratch healing experiment diagram (…). Figure 3 (B in B-1) and quantitative statistical bar chart ( Figure 3 As shown in B-2 of the data, PGG significantly reduced the number of invasive cells, indicating that metastasis potential was suppressed. Apoptosis was detected by flow cytometry, and the scatter plot of flow cytometry data from the blank control group of PC9 cells (…) Figure 3 (C-1 in C) and its flow cytometry scatter plot ( Figure 3 (C-2 in C), scatter plot of flow cytometry data of blank control group of PC9 drug-resistant cells (C-2 in C), Figure 3 (C-3 in C) and its flow cytometry scatter plot ( Figure 3 Flow cytometry scatter plot of PC9 resistant cells combined with osimertinib in a blank control group (C-4 in C). Figure 3 (C-5 in C) and its flow cytometry scatter plot ( Figure 3 As shown in C-6 of the data, PGG treatment led to a significant increase in Annexin V-positive cells, indicating a significant increase in the number of cells undergoing programmed cell death. p <0.001). The combined use of PGG and OSI further increased the apoptosis rate of drug-resistant cells. These results indicate that in the OSI-resistant model, PGG has the ability to inhibit lung cancer cell migration and invasion while promoting cell death.

[0100] 3. PGG induces ferroptosis through metabolic pathways.

[0101] This study performed extensive RNA sequencing on OSI-treated PC9 parental cells, PC9-OR cells, and PC9-OR cells to gain a deeper understanding of the anticancer ability of PGG. Heatmap analysis of differentially expressed genes among parental (PC9), osimertinib-resistant (PC9-OR), and PGG-treated PC9-OR cells identified 1283 significantly dysregulated genes. Figure 4 As shown in A), C1-C8 are cluster genes related to metabolic processes. The Kyoto Encyclopedia of Genetics and Genomes analysis diagram of the C7 gene cluster in the cluster analysis (…). Figure 4 (as shown in B-1 of B in the diagram) and the gene ontology analysis diagram of the C7 gene cluster ( Figure 4 (As shown in B-2 of B in the figure) it indicates that genes associated with iron sagging and cell death pathways were significantly increased after PGG treatment.

[0102] The major apoptosis-related genes that increase after PGG treatment include: PRNPs (prion proteins) involved in oxidative stress regulation and lipid peroxidation. Figure 4 In C1 (C-1), MAP1LC3B (LC3) is crucial for autophagy flux and promotes ferroptosis by degrading cellular antioxidants. Figure 4 C-2 in C), TFRC (transferrin receptor) enhances iron uptake ( Figure 4 C3 in C promotes iron-dependent lipid peroxidation, ACSL4 ( Figure 4 C in C-4), and ACSL3 ( Figure 4 C-6 in the C group (acyl-CoA synthase long chain family members 4 and 3) is a key enzyme incorporating PUFA into membrane lipids, making them prone to peroxidation. SLC3A2 (solute carrier family 3 member 2) participates in cysteine ​​uptake. Figure 4 The C-5 gene in PGG affects glutathione metabolism and oxidative stress balance. qPCR validation results showed that all of the above genes were significantly upregulated after PGG treatment (p<0.001, extremely significant difference), further confirming that ferroptosis activation is a key mechanism by which PGG exerts its effects.

[0103] Furthermore, combined treatment with PGG and OSI further enhanced the expression of ferroptosis-related genes, suggesting a synergistic effect in overcoming OSI resistance. Conversely, the Kyoto Genome and Genome Encyclopedia analysis diagram of the C3 gene cluster ( Figure 4 The gene ontology analysis diagram of gene cluster B (as shown in B-3 in B) and C3 gene cluster ( Figure 4As shown in B-4 of the data, PGG treatment significantly downregulated the expression of cluster 3 genes (C3 in the RNA-Seq data), which are involved in metabolic processes. Gene enrichment analysis (GSEA) revealed that key metabolic pathways, including carbon metabolism, nucleotide metabolism, arginine and proline metabolism, fatty acid metabolism, glutathione metabolism, pyrimidine metabolism, and β-alanine metabolism, were significantly inhibited. Inhibition of these metabolic pathways may lead to cellular energy stress and oxidative imbalance, thereby increasing cellular sensitivity to ferroptosis and ultimately inducing cell death.

[0104] To further verify the RNA-Seq results, PGG inhibited tumor growth by inducing ferroptosis in ostinib-resistant lung cancer cells (results are shown below). Figure 5 As shown in the figure, we detected biochemical markers associated with iron toxicity in PC 9 and PC 9-OR cell lines, and measured the levels of glutathione (GSH) and malondialdehyde (MDA) in the culture supernatant. The results showed that PGG treatment significantly reduced GSH levels. Figure 5 The increase in A) and MDA levels was not associated with osimertinib treatment. Figure 5 (B in the text). These results indicate that lipid peroxidation is enhanced after PGG exposure. Western blot analysis further showed that iron toxicity-related proteins GPX4 and SLC 7A11 were downregulated after PGG treatment ( Figure 5 (C in the original text). To determine whether the cytotoxic effects of PGG are mediated by ferroptosis, we performed a rescue experiment using the ferroptosis inhibitor ferrostatin-1 (Fer-1). Treatment with Fer-1 effectively reversed PGG-induced cell death (C in the original text). Figure 5 (D in the text). In summary, these findings suggest that PGG inhibits the growth of lung cancer cells, including those with acquired resistance to osimertinib, through a ferrous salt-dependent mechanism.

[0105] Based on the above research, the main mechanism by which PGG exerts its anticancer activity is through inducing iron toxicity—a form of iron-dependent programmed cell death characterized by lipid peroxidation and oxidative stress. Our RNA-seq and qPCR analyses showed that PGG significantly increased key apoptosis-related genes, including PRNP, MAP1LC3B, TFRC, ACSL4, ACSL3, and SLC3A2. These genes play crucial roles in lipid peroxidation, iron metabolism, and autophagy flux, collectively promoting ferroptosis. The combined use of PGG and OSI further enhanced the ferroptosis effect, as evidenced by the increased expression of iron-induced apoptosis-related genes. This synergistic effect suggests that PGG enhances cellular sensitivity to OSI by weakening the antioxidant defenses of drug-resistant cells and promoting iron-catalyzed lipid peroxidation. The dual targeting of the EGFR signaling pathway (via OSI) and the iron sagging pathway (via PGG) provides a novel therapeutic strategy for overcoming OSI resistance.

[0106] In addition to inducing ferroptosis, PGG exerts its anticancer effects by modulating metabolic processes essential for cancer cell survival. RNA-seq data showed that PGG treatment significantly downregulated metabolic pathways, including carbon metabolism, nucleotide metabolism, arginine and proline metabolism, fatty acid metabolism, glutathione metabolism, pyrimidine metabolism, and β-alanine metabolism. Inhibition of these pathways may disrupt the metabolic plasticity of resistant cells, reducing their ability to evade treatment-induced stress. By impairing glutathione metabolism, PGG reduces the availability of the key antioxidant glutathione, thereby increasing oxidative stress and sensitizing cells to ferroptosis. Simultaneously, inhibiting fatty acid metabolism exacerbates lipid peroxidation by preventing cancer cells from repairing lipid peroxidation, promoting cell death and further enhancing ferroptosis.

[0107] This study found that, in addition to its cytotoxic effects, PGG can also inhibit the migration and invasion of EGFR-mutant lung cancer parental cells and OSI-resistant cells. Cell scratch assays and Transwell invasion assays confirmed that PGG treatment significantly reduced cell migration speed and invasive ability, indicating that PGG not only impairs tumor cell survival but also disrupts metastasis. This anti-metastatic effect may be related to its regulation of cytoskeleton remodeling and inhibition of epithelial-mesenchymal transition (EMT) pathways—EMT pathways are typically closely related to OSI resistance and tumor metastasis. Furthermore, Annexin V / PI staining combined with flow cytometry analysis showed that PGG can induce apoptosis and cell death, with a significant increase in both early and late apoptotic cell populations after treatment. This result indicates that PGG promotes programmed cell death through a dual mechanism of ferroptosis and apoptosis, further highlighting its multifaceted anti-cancer activity.

[0108] The ability of PGG to restore OSI sensitivity in drug-resistant lung cancer cells highlights its potential clinical significance. Since OSI resistance is a major barrier to treating EGFR-mutant lung cancer, incorporating PGG or its derivatives into treatment regimens may offer a promising therapeutic strategy. By simultaneously targeting iron depletion and metabolic vulnerability, PGG may overcome multiple resistance mechanisms, thereby improving treatment efficacy and preventing relapse. Furthermore, PGG's natural origin and polyphenolic properties suggest a favorable safety and tolerability profile, making it a viable candidate for future preclinical and clinical studies. Given the growing interest in iron-based therapies, PGG represents a compelling lead compound for developing novel combination strategies aimed at overcoming TKI resistance.

[0109] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a peony petal-based composition for alleviating drug-resistant lung cancer in the preparation of drugs for alleviating drug-resistant lung cancer, characterized in that, The peony petal-based composition for alleviating drug-resistant lung cancer comprises 1,2,3,4,6-pentagalloglucoside and osimertinib, wherein the molar ratio of 1,2,3,4,6-pentagalloglucoside and osimertinib is (20-160):(1-2). The 1,2,3,4,6-pentagalloglucoside was selected from peony petal extract; The lung cancer in question is human non-small cell lung cancer with acquired resistance to osimertinib.

2. The use of the peony petal-based composition for alleviating drug-resistant lung cancer according to claim 1 in the preparation of drugs for alleviating drug-resistant lung cancer, characterized in that, The product is obtained by mixing 1,2,3,4,6-pentagalloglucoside and osimertinib.

3. The application of the peony petal-based composition for alleviating drug-resistant lung cancer according to claim 2 in the preparation of drugs for alleviating drug-resistant lung cancer, characterized in that, The preparation method of the 1,2,3,4,6-pentagalloglucopyranose is as follows: Peony petals were added to a solvent, heated under reflux to extract, and the extracts were combined and concentrated to obtain a concentrated solution. The concentrate was enriched by passing it through a macroporous resin column and eluted with an ethanol aqueous solution with a mass concentration of 10%-70%. The eluent with a mass concentration of 40% was collected and concentrated to obtain the crude extract. The crude extract was dissolved in a two-phase solvent, purified by high-speed countercurrent chromatography, and dried to obtain 1,2,3,4,6-pentagalloglucopyranoside.

4. The application of the peony petal-based composition for alleviating drug-resistant lung cancer according to claim 3 in the preparation of drugs for alleviating drug-resistant lung cancer, characterized in that, The solvent is one of an aqueous solution of methanol and an aqueous solution of ethanol, wherein the mass concentration of the solvent is 90%-95%. The mass ratio of peony petals to solvent is 1:(5-10); The heating reflux extraction is performed at a temperature of 50-60℃ for 2-4 hours, and the extraction is repeated 2-4 times. The concentration temperature is 35-45℃.

5. The application of the peony petal-based composition for alleviating drug-resistant lung cancer according to claim 3 in the preparation of drugs for alleviating drug-resistant lung cancer, characterized in that, The macroporous resin column is one of MCI, HPD-750 and MQ-01; The elution conditions were gradient elution using aqueous ethanol solutions with mass concentrations of 10%, 20%, 30%, 40%, and 70%, respectively.

6. The application of the peony petal-based composition for alleviating drug-resistant lung cancer according to claim 3 in the preparation of drugs for alleviating drug-resistant lung cancer, characterized in that, The two-phase solvent is n-hexane / ethyl acetate / methanol / water, and the volume ratio of n-hexane / ethyl acetate / methanol / water is (1-2):(8-9):(1-4):(6-9); The flow rate of high-speed countercurrent chromatography is 2 mL / min-5 mL / min, the rotation speed is 800 r / min-1000 r / min, and the retention rate is 65%-75%.

7. The application of the peony petal-based composition for alleviating drug-resistant lung cancer according to claim 6 in the preparation of drugs for alleviating drug-resistant lung cancer, characterized in that, The volume ratio of n-hexane / ethyl acetate / methanol / water is 2:8:4:6; The high-speed countercurrent chromatography (HSC) flow rate was 2 mL / min, the rotation speed was 850 r / min, and the retention rate was 70%.

8. The application of the peony petal-based composition for alleviating drug-resistant lung cancer according to claim 1 in the preparation of drugs for alleviating drug-resistant lung cancer, characterized in that, The drug includes pharmaceutically acceptable excipients.

Images