Composition of paeonol compounds and nobiletin and application thereof

Abstract

This invention relates to the composition of paeoniflorin compounds and nobiletin, and their applications. Using human non-small cell lung cancer A549 and human melanoma A375 cells as cancer cell models, this invention studies the effects of paeoniflorin compounds synergistically with nobiletin on the proliferation or migration of A549 and A375 tumor cell lines, providing data for the development of combined treatment of tumors with natural drug extracts. Results show that paeoniflorin compounds significantly inhibit the proliferation of human non-small cell lung cancer A549 and human melanoma A375 cells and affect their migration ability. Synergistic effects of paeoniflorin compounds with nobiletin on A549 and A375 cells exhibit a synergistic effect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the composition of paeoniflorin compounds and nobiletin and their applications. Background Technology

[0002] Cancer, or malignant tumor, is a group of malignantly growing cells originating from the epithelium. Due to its disordered cellularity, it can grow uncontrollably and is currently the leading cause of death among humans. [1] Traditional Chinese medicine (TCM) has unique advantages in treating cancer and is currently an important means of improving cancer treatment. TCM theory holds that qi stagnation, blood stasis, and meridian obstruction are the pathogenesis of tumors. Blood stasis often accompanies tumors; therefore, promoting blood circulation and removing blood stasis is the main method and approach of TCM in treating cancer.

[0003] The vast majority of lung cancer patients have small cell lung cancer, which has a high incidence and mortality rate that is increasing year by year. [2] Western medicine primarily treats non-small cell lung cancer using methods such as surgical resection, radiotherapy, chemotherapy, and targeted drug therapy. While significant progress has been made, the side effects are quite pronounced. For example, classic treatments using platinum-based doublet chemotherapy or chemotherapy combined with targeted therapy can cause leukopenia, thrombocytopenia, anemia, nausea, vomiting, and severe hair loss, severely increasing the patient's physical and psychological burden. [3] Natural medicines have advantages such as low drug resistance, minimal toxicity and side effects, and high safety. In recent years, many natural medicine extracts have been found to have certain inhibitory effects on non-small cell lung cancer. For example, different concentrations of astragaloside A can significantly inhibit the proliferation of A549 cells in a concentration-dependent manner. [4] Stimulation of A549 cells with different concentrations of curcumin revealed that curcumin could reduce β-catenin gene expression, inhibit the formation of angiogenic mimicry in lung cancer cells, and thus suppress their proliferation. [5] After treatment with crotonol, the Nrf2-Notch1 signaling axis of A549 cells can be inhibited, thereby inhibiting the growth of A549 cells. [6] In addition, bufotalin, turmeric alcohol, Hedyotis diffusa injection, coix seed extract, salvia miltiorrhiza extract, and ginseng extract can all be used to treat non-small cell lung cancer. [7] .

[0004] Malignant melanoma (MM) is a cancerous tumor that develops from melanocytes. It can occur in any location containing melanocytes and is the fastest-growing cancer in terms of incidence. [8], currently, the main Western medical treatment methods include immunotherapy, such as using interleukin, interferon, vaccines, and lymphocytes for treatment; inhibitors such as BEAF / MEK can also be used for treatment, such as drugs like vemurafenib and dabrafenib; and combined treatment of immunotherapy and inhibitors, but none of them have a recognized definite treatment effect [9] .

[0005] Melanoma mostly occurs in the skin and is also the cancer with the highest malignancy and fatality rate among skin cancers. It is recorded in "Plain Questions" that "the lung governs the body's skin and hair". According to traditional Chinese medicine theory, the health of the skin and hair depends on the essence qi input by the lung. The root cause of skin diseases lies in the lung. Therefore, skin diseases and lung diseases are treated together, which means taking both the root cause and the symptoms into account. In ancient China, skin cancer was called "fanhua chuang", "ai chuang", "shi ju" etc. Among the traditional Chinese medicine prescriptions for treating skin cancer in the Ming and Qing dynasties, in addition to strengthening the healthy qi and tonifying deficiency, the Chinese medicine with the highest application frequency is red peony root

[10] , therefore, it can be considered to use the active ingredients in blood-activating and stasis-removing drugs to improve the treatment of non-small cell lung cancer and melanoma.

[0006] Red peony root is the dried root of Paeonia lactiflora Pall. Paeonia lactiflora Pall or Paeonia veitchii Lynch Paeonia veitchii Lynch ). As a traditional Chinese medicine, red peony root belongs to the category of heat-clearing and blood-cooling drugs, with the functions of clearing heat and cooling blood, dissipating stasis and relieving pain, and can treat blood stasis syndrome. According to traditional Chinese medicine theory, red peony root can be applied to cancer. Modern research has found that red peony root can exert various pharmacological effects, such as protecting nerve cells and antidepressant

[11] , anti-atherosclerosis

[12] , lowering blood sugar and blood lipids, treating kidney injury

[13] , anti-tumor and other effects

[14] , it has now been confirmed that the anti-tumor substance basis is total glucosides of red peony root

[15] , including monoterpene glycosides such as paeoniflorin, benzoylpaeoniflorin, paeonolide glycoside, oxypaeoniflorin, triterpenoids such as oleanolic acid, betulinic acid, β-sitosterol, flavonoids such as kaempferol, etc. In addition, there are also a small amount of tannins, phenolic acids, sugars, volatile oils, and alkaloids

[16] .

[0007] Paeoniflorin is one of the main active ingredients in red peony root and belongs to monoterpene glycoside compounds. Modern research has found that paeoniflorin has significant anti-tumor effects. For example, when A549 cells are treated with 5 - 40 μmol·L -1 paeoniflorin, its viability can be weakened. The anti-tumor mechanism may be related to promoting the expression of Caspase-3 and Bax proteins and inhibiting the expression of Bcl-xl protein and NF-κBpp65 protein

[17] . When treated with 20 μmol·L -1Paeoniflorin was found to reduce PD-L1 expression in HepG2 cells, thereby inhibiting tumor growth.

[18] The proliferation rate of Panc-1 cells decreased with increasing paeoniflorin concentration, and its anti-tumor mechanism may be related to promoting Caspase-3 and inhibiting Bcl-2 protein expression.

[19] Use 160 μg·mL -1 80 μg·mL -1 Paeoniflorin was found to have a strong tumor-suppressive effect on SW480 colon cancer cells, achieving half-maximal inhibitory effects at 8 h and 72 h.

[20] Cell scratch assays revealed that paeoniflorin concentrations greater than 10 μmol·L⁻¹ were observed. -1 It can significantly inhibit the migration of HepG2 liver cancer cells.

[21] .

[0008] Oxypaeoniflorin is one of the main active components of Paeonia lactiflora, belonging to the monoterpenoid class of compounds. Its structural formula is shown below. Figure 1 Studies have shown that oxidized paeoniflorin can exert anti-tumor and anti-inflammatory effects in vitro. For example, 4-oxy-methylpaeoniflorin and 9-ethylpaeoniflorin A can inhibit the proliferation of oral cancer KB cells and human lung adenocarcinoma A549 cells.

[22] When paeoniflorin was used to treat THP-1 macrophages, the concentration was greater than 200 μg / mL. -1 It can be toxic to macrophages, at 150 μg / mL. -1 and 200 μg·mL -1 Oxidized paeoniflorin can cause cholesterol to leak from foam cells.

[23] Paeoniflorin can inhibit the secretion of inflammatory cytokines and the expression of corresponding genes, and regulate the TLR signaling pathway in RAW264.7 cells, thereby exerting an anti-inflammatory effect.

[24] .

[0009] Nobiletin is a polymethyl flavonoid derived from the peel of citrus fruits; its structural formula is shown below. Figure 2 Citrus peels and other citrus-based traditional Chinese medicines often have the function of regulating qi, and can treat symptoms caused by qi stagnation and blood stasis. Current research indicates that tangeretin and its derivatives can be used as targeted therapeutic drugs to inhibit tumor cell proliferation and arrest the cell cycle, exhibiting significant anti-tumor effects.

[25] Studies have found that it can inhibit the proliferation of Hep G2 liver cancer, A549 lung adenocarcinoma, and HeLa cervical cancer cells in a concentration-dependent manner.

[26] Non-small cell lung cancer can be treated by inhibiting the TGF-β1 / smad3 pathway.

[27] It can also affect cyclin D through the Wnt signaling pathway, thereby altering the cell cycle and inhibiting melanoma.

[28] 160 μg·mL -1 At concentrations of 100% and above, norepinephrine significantly reduced the in vitro migration ability of Cal-27 cells after 48 hours.

[29] .

[0010] Natural medicines have advantages in treating tumors, such as multiple targets, low drug resistance, and few toxic side effects. The use of natural drug extracts to treat cancer is an important approach to improving cancer treatment methods.

[0011] There are currently no reports of combining paeoniflorin compounds with nobiletin. Summary of the Invention

[0012] In view of this, the object of the present invention is to provide a composition of paeoniflorin compounds and nobiletin and its application.

[0013] To achieve the above objectives, the present invention provides the following technical solution: 1. A combination of paeoniflorin compounds and nobiletin.

[0014] Preferably, the paeoniflorin compound is paeoniflorin or oxypaeoniflorin.

[0015] Preferably, the molar ratio of paeoniflorin compounds to nobiletin is 1:1 / 3 to 3.

[0016] 2. The preparation, the active ingredient of which is the aforementioned composition.

[0017] 3. Application of the aforementioned compositions or preparations in the preparation of anticancer drugs.

[0018] Preferably, the anticancer drug is a treatment for lung cancer, and the molar ratio of paeoniflorin to noriheptacortin is 1:1 / 3 to 3, more preferably 1:1.

[0019] Further preferred is non-small cell lung cancer, and even more preferred is lung adenocarcinoma.

[0020] Preferably, the anticancer drug is a treatment for melanoma, and the molar ratio of paeoniflorin to noriheptacortin is 1:2.

[0021] 4. An anticancer drug whose active ingredients are paeoniflorin compounds and nobiletin.

[0022] Preferably, the anticancer drug is a treatment for lung cancer or melanoma.

[0023] Preferably, the paeoniflorin compound is paeoniflorin or oxypaeoniflorin.

[0024] Preferably, the molar ratio of paeoniflorin compounds to nobiletin is 1:1 / 3 to 3.

[0025] The beneficial effects of this invention are as follows: This invention uses human non-small cell lung cancer (NSCLC) A549 and human melanoma cells A375 as cancer cell models to study the effects of paeoniflorin compounds synergistically with hesperidin on the proliferation or migration of A549 and A375 tumor cell lines, providing data for the development of combined treatment of tumors with natural drug extracts. Specifically, the methods are as follows: A high-content cell image analysis system was used to detect the effects of paeoniflorin compounds on human NSCLC A549 and human melanoma cells A375. The MTT assay was used to detect the effect of paeoniflorin compounds synergistically with hesperidin on the proliferation of human NSCLC A549 and human melanoma cells A375, and the cell proliferation rate was calculated [proliferation rate (%) = (mean OD of this group - mean OD of the zero-adjustment group) / (mean OD of the untreated group - mean OD of the zero-adjustment group) × 100%]. A scratch assay was used to detect cell migration ability. Immunofluorescence assays were used to detect the effects of paeoniflorin compounds combined with hesperidin on the proliferation, apoptosis, and necrosis of human NSCLC A549 and human melanoma cells A375. Results: Paeoniflorin compounds significantly inhibited the proliferation of human non-small cell lung cancer A549 and human melanoma A375 cells and affected their migration ability. When combined with nobiletin, they had a synergistic effect on A549 and A375 cells. Attached Figure Description

[0026] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 paeoniflorin (C) 23 H 28 O 12 The chemical structural formula of ). Figure 2 For the use of tangeretin (C 21 H 22 The chemical structural formula of O8); Figure 3 The inhibitory effect of paeoniflorin synergistically with nobiletin on A549 cells ( p ** <0.01, p *** <0.001); Figure 4 The inhibitory effect of paeoniflorin synergistically with nobiletin on A549 cells ( p * <0.05, p ** <0.01, p *** <0.001); Figure 5The inhibitory effect of paeoniflorin synergistically with nobiletin on A375 cells ( p * <0.05, p ** <0.01, p *** <0.001); Figure 6 The inhibitory effect of paeoniflorin synergistically with nobiletin on A375 cells ( p * <0.05, p *** <0.001); Figure 7 The effect of paeoniflorin, oxypaeoniflorin and synergistic nobiletin on the migration inhibition rate of A549 cells ( p * <0.001); Figure 8 The effects of paeoniflorin, oxypaeoniflorin, and synergistic nobiletin on the migration ability of A549 cells were investigated; groups A, B, C, D, E, F, G, H, I, J, K, and L were the control group, 30 μmol·L⁻¹, and 30 μmol·L⁻¹, respectively. -1 PF group, 30 μmol·L -1 OPF group, 30 μmol·L -1 NOB group, 30 μmol·L -1 PF + 30 μmol·L -1 NOB group, 30 μmol·L -1 OPF + 30 μmol·L -1 NOB group, 15 μmol·L -1 PF + 15 μmol·L -1 NOB group, 15 μmol·L -1 OPF + 15 μmol·L -1 NOB group, 7.5 μmol·L -1 PF + 22.5 μmol·L -1 NOB group, 22.5 μmol·L -1 PF + 7.5 μmol·L -1 NOB group, 22.5 μmol·L -1 OPF + 7.5 μmol·L -1 NOB group, 7.5 μmol·L -1 OPF + 22.5 μmol·L -1 NOB Group; Figure 9 The effects of paeoniflorin and oxypaeoniflorin synergistically on the morphology of A549 cells; Figure 10 The effects of paeoniflorin and oxypaeoniflorin synergistically on the morphology of A375 cells. Detailed Implementation

[0027] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0028] Table 1 Tumor cell information

[0029] Table 2 Drug and Reagent Information

[0030] Table 3. Instrument and Equipment Information

[0031] Both human lung adenocarcinoma A549 cells and melanoma A375 cells are adherent cells. In a BIOBASE clean bench, human lung adenocarcinoma A549 cell lines and melanoma A375 cell lines, frozen in liquid nitrogen, were seeded into culture dishes. A549 and A375 cells were cultured in 8-10 mL of DMEM / F-12 (containing 10% FBS, 1% penicillin and streptomycin) and DMEM high-glucose (containing 10% FBS, 1% penicillin and streptomycin), respectively. Cells were cultured at 37°C in a 5% CO2 incubator, with the culture medium changed every 1-2 days.

[0032] When the cells reached near confluence with the culture dish, the culture medium was aspirated. The cells were washed twice with PBS (0.01M, pH 7.4), 1×, and the mixture was shaken well to remove impurities and dead cells. 1 mL of trypsin-EDTA solution was added, and the mixture was shaken well before digestion. After three minutes, 1 mL of the original culture medium was added to stop the digestion. The cell suspension was aspirated and placed in a 10 mL centrifuge tube, centrifuged at 800 rpm. -1 Centrifuge for 5 minutes, discard the supernatant, break up the cell pellet, add fresh culture medium and mix well, then passage at a 1:3 ratio. Perform experiments when the cells are in good condition and in the logarithmic growth phase.

[0033] For cell experiments, excess cultured cells should be cryopreserved to conserve culture medium and for future use in related experiments. Remove the gradient cooling box and allow it to return to room temperature. If used more than three times, the isopropanol solution in the box should be replaced. Digest the cells from the culture dish wall according to the passage procedure, at 800 r·min. -1 Centrifuge for 5 min, discard the supernatant, mix the cell pellet, add 1 mL of cryopreservation solution (by volume percentage, containing 10% DMSO, 40% FBS, and 50% culture medium), place in a cryovial, put in a gradient cooling box, and store temporarily in a -80°C freezer or in liquid nitrogen for long-term storage.

[0034] First, use a blank 96-well plate to calibrate the instrument, and set the program and parameters. Take A549 and A375 tumor cell lines that have been passaged for 1 to 2 days and seed them into the 96-well plate (preferably selecting wells away from the edge), with approximately 3 × 10⁶ cells per well. 3 Cells were collected, and after the cells adhered, paeoniflorin was added to each well at concentrations of 0, 2.5, 5, 10, 20, 40, and 80 μmol·L⁻¹. -1 The concentrations of paeoniflorin oxide were 0, 10, 20, 40, and 80 μmol·L⁻¹. -1 0.2 mL of F-12 or high-glucose culture medium was used to observe the morphology and number of A549 and A375 cells at 12h, 24h and 48h, respectively, and the data were recorded.

[0035] A375 and A549 tumor cell lines passaged for 1-2 days were collected. After obtaining cell suspension according to the cell passage procedure, 10 μL of the cell suspension was added to 1 mL of PBS 1× and mixed well. The cell count was performed 6 times in parallel using a hemocytometer, and the average value was taken. The cell count was calculated as n / 4 × dilution factor × 10. 4 pcs·mL -1 Calculate the cell count and seed them in 96-well plates, with approximately 3 × 10⁶ cells per well. 3 Each cell line was individually prepared, and a zeroing well was provided. After adhesion, the culture medium was aspirated, and paeoniflorin concentrations of 0, 15, 30, and 60 μmol·L⁻¹ were added to each tumor cell line. -1 The concentrations of paeoniflorin oxide were 0, 10, 20, and 40 μmol·L⁻¹. -1 0.2 mL of culture medium was added to each well at 12 h, 24 h, 48 h, and 72 h. The culture medium was removed from the wells, and the adherent cells were carefully removed without poking them. 10 μL of a 5 mg·mL⁻¹ culture medium was added to each well. -1 Add 90 μL of MTT solution and 90 μL of culture medium, ensuring no air bubbles are generated. Incubate in a dark incubator for 5 h. After 5 h, terminate the culture, discard the solution in the wells, and do not lose the crystals. Add 120 μL of LDMSO to each well, and shake at low speed in a water bath for 10 min to fully dissolve the crystals. Measure the absorbance of each well at 492 nm using a full-wavelength microplate reader, process the data, and determine the IC50 value.

[0036] A375 and A549 tumor cell lines passaged for 1-2 days were seeded into 96-well plates. The following groups were set up: a blank control group, a paeoniflorin group, an oxidized paeoniflorin group, a noriquot group, and groups treated with either drug in combination with noriquot, and groups treated with half the combined drug dosage. Approximately 3 × 10⁶ cells were seeded per well. 3 After the cells adhered, A549 cells were added with a solution containing 30 μmol·L⁻¹. -1 paeoniflorin, 30 μmol·L -1paeoniflorin oxide, 30 μmol·L -1 Noriheptacortin and 30 μmol·L -1 Paeoniflorin synergistic 30 μmol·L -1 Noriheptacorlin, 30 μmol·L -1 Oxidized paeoniflorin synergistic 30 μmol·L -1 Noriheptacorlin, 15 μmol·L -1 Paeoniflorin synergistic 15 μmol·L -1 Noriheptacorlin, 15 μmol·L -1 Oxidized paeoniflorin synergistic 15 μmol·L -1 The F-12 culture medium containing hesperidin was used to treat A375 cells, and A375 cells were added with 20 μmol·L⁻¹ of the medium. -1 paeoniflorin, 20 μmol·L -1 Oxidized paeoniflorin, 40 μmol·L -1 Noriheptacorlin, 20 μmol·L -1 Paeoniflorin synergistic 40 μmol·L -1 Noriheptacorlin, 20 μmol·L -1 Oxidized paeoniflorin synergistic 40 μmol·L -1 Noriheptacorlin, 10 μmol·L -1 Paeoniflorin synergistic 20 μmol·L -1 Noriheptacorlin, 10 μmol·L -1 Oxidized paeoniflorin synergistic 20 μmol·L -1 The culture medium containing hesperidin was used for treatment at high sugar levels for 12 h, 24 h, 48 h, and 72 h. After treatment, the culture medium was aspirated, and 10 μL of a 5 mg·mL⁻¹ solution was added to each well. -1 Add 90 μL of MTT solution and 90 μL of culture medium, and react in the dark for 5 h. Carefully aspirate the solution from the wells, taking care not to lose the crystals. Add 120 μL of LDMSO to each well, and place in a water bath with a shaker at low speed for 10 min to fully dissolve the crystals. Measure the absorbance of each well at 492 nm using a full-wavelength microplate reader.

[0037] A549 tumor cell lines passaged for 1-2 days were used to obtain cell suspensions according to passage methods. These suspensions were then seeded into 6-well plates, with the cell count per well controlled at 1.2 × 10⁶ cells / well. 6Approximately 100 cells were incubated overnight in an incubator. After cell adhesion, a clean 1000 μL pipette tip was used to make a vertical scratch from top to bottom of the plate, repeating three times per well. The cells were washed three times with 1× PBS. FBS-free and antibiotic-free culture medium were added to each well. The following groups were set up: control group, paeoniflorin group, oxidized paeoniflorin group, norihesperidin group, paeoniflorin combined with norihesperidin at different concentrations, and oxidized paeoniflorin combined with norihesperidin at different concentrations. The scratch width was photographed and measured under a fluorescence microscope 0 h after drug addition. The scratch width was photographed and measured again 24 h later. The scratch healing rate was calculated as [healing rate = (scratch width at 0 h - scratch width at 24 h) / scratch width at 0 h]. Data analysis was performed.

[0038] A375 and A549 tumor cell lines, passaged for 1-2 days, were seeded into 96-well plates. Three replicates were prepared for each well, including a control group, a paeoniflorin group, an oxidized paeoniflorin group, a noriheptacorlins group, and groups treated with either drug in combination with noriheptacorlins, and groups treated with half the combined drug. After cell adhesion, the drugs were added to each well. After 24 hours, the culture medium was aspirated, washed twice with PBS (1×), and 35 μL of immunostaining fixative was added to each well for fixation at room temperature for 15 min. The cells were then washed three times with PBS (1×), incubated with strong immunostaining permeabilization buffer for 5 min at room temperature, washed three times with PBS (1×), and aspirated. The cells were pre-treated with a solution containing 50 mg / mL paeoniflorin. -1 Tubulin-Tracker Red was diluted 100-fold with BSA and 0.1% Triton PBS 1×. 80 μL of Tubulin-Tracker Red dilution was added to each well, and staining was carried out at room temperature in the dark for 60 min. The cells were washed four times with 0.1% Triton PBS 1×, 5 min each time. An appropriate volume of YP1 / RNase staining solution was added to each well to cover the cells, and the reaction was carried out at 37℃ in the dark for 20 min. The cells were washed three times with PBS 1×, and fluorescence was observed on a high-content platform.

[0039] Data analysis was performed using IBM SPSS Statistics 26 and GraphPad Prism software. The results of high-content experiments and MTT assays were analyzed using two-way ANOVA, while the cell migration assays were analyzed using one-way ANOVA.

[0040] The combined use of paeoniflorin, oxidized paeoniflorin, and nobiletin can greatly enhance the inhibitory effect on lung cancer cells. For example, after 12 hours of drug treatment, the proliferation rates of A549 cells in the paeoniflorin group, oxidized paeoniflorin group, nobiletin group, paeoniflorin combined with nobiletin and its half-concentration group, and oxidized paeoniflorin combined with nobiletin and its half-concentration group were (90.04±11.26)%, (91.28±6.03)%, (66.58±6.82)%, (55.90±5.40)%, (68.02±16.59)%, (70.05±7.76)%, and (82.44±16.81)%, respectively. After 24 hours of treatment, the values ​​were (59.44±10.42)%, (75.48±10.77)%, (62.32±17.37)%, (33.63±7.41)%, (41.01±8.41)%, (32.67±7.02)%, and (49.36±5.54)%. After 48 hours of treatment, the values ​​were (41.14±5.54)%, (47.28±5.54)%, (46.86±6.93)%, (17.63±3.56)%, (37.26±3.87)%, (15.92±3.73)%, and (42.16±3.32)%. After 72 hours of treatment, the values ​​were (21.96±2.46)%, (25.88±2.52)%, (28.78±3.43)%, (1.34±0.90)%, (13.99±4.11)%, (3.25±0.90)%, and (12.86±5.45)%. See details below. Figure 3 and Figure 4 After 24 h, 48 h, and 72 h of treatment, the cell proliferation rate in the combined drug group was significantly different from that in the control group and the single drug group. p <0.001; The combined drug halving group showed stronger cell inhibition at 24 h, 48 h, and 72 h than the single drug group, and the difference was statistically significant compared with the control group.

[0041] The combined use of paeoniflorin, oxidized paeoniflorin, and nobiletin significantly enhanced the inhibitory effect on melanoma cells. For example, after 12 hours of treatment, the proliferation rates of A375 cells in the paeoniflorin group, oxidized paeoniflorin group, nobiletin group, paeoniflorin combined with nobiletin and its halved concentration group, and oxidized paeoniflorin combined with nobiletin and its halved concentration group were (93.56±8.90)%, (80.60±8.16)%, (87.73±6.99)%, (73.09±9.76)%, (85.23±7.56)%, (68.34±7.46)%, and (78.10±7.64)%, respectively. After 24 hours of treatment... The values ​​after h were (86.05±7.56)%, (78.40±7.45)%, (67.24±8.86)%, (70.37±5.85)%, (78.40±7.45)%, (35.88±6.32)%, and (68.11±7.59)%. After 48 hours of treatment, the values ​​were (51.52±5.19)%, (47.09±5.81)%, (45.72±5.65)%, (14.36±2.64)%, (39.05±3.30)%, (14.85±3.66)%, and (40.53±3.50)%. After 72 hours of treatment, the values ​​were (23.13±3.37)%, (25.64±4.05)%, (22.07±4.10)%, (3.78±0.80)%, (17.65±4.37)%, (5.13±1.07)%, and (22.22±2.09)%. See the detailed results below. Figure 5 and Figure 6 After 12 h, 24 h, 48 h, and 72 h of treatment, the cell proliferation rate in the combined drug group was significantly different from that in the control group and the single drug group. p <0.001, the combined drug halving group showed stronger cell inhibition at 24 h, 48 h, and 72 h than the single drug group, and the difference was statistically significant compared with the control group.

[0042] After 24 hours of treatment, the mean scratch healing rates in the paeoniflorin group, the combined paeoniflorin and hesperidin groups, and the combined oxidized paeoniflorin and hesperidin groups were 4.30%, 0.81%, 5.41%, 1.59%, 0.88%, 2.55%, 1.09%, 0.72%, and 0.44%, respectively. These rates were statistically significantly different from the mean scratch healing rate of 12.94% in the control group. (See details below.) Figure 7 and Figure 8 Paeoniflorin, the combination of paeoniflorin and noriheptacorlin, and the combination of oxidized paeoniflorin and noriheptacorlin all inhibited the migration of A549 cells.

[0043] After staining with Tubulin and YP1 solutions, A549 and A375 cells, treated with paeoniflorin, oxypaeoniflorin, nobiletin, and their combination for 24 hours, showed cell shrinkage or swelling compared to the control group, indicating programmed cell death or necrosis under drug stimulation. In the YP1-only staining group, cell nuclei shrank, divided, and extravasated after drug treatment, even undergoing exocytosis. In the Tubulin-only staining group, changes in the cytoskeleton were observed, with blurred edges and partial disappearance of the cytoskeleton, indicating severe drug-induced cell morphology. See below for specific cell morphology details. Figure 9 , Figure 10 .

[0044] This invention demonstrates that the combined use of paeoniflorin, oxypaeoniflorin, and nobiletin can effectively enhance their in vitro inhibitory effect on tumor cells and inhibit their migration. Observations using a high-content platform and cellular immunofluorescence experiments revealed a corresponding decrease in tumor cell status. Therefore, the use of nobiletin in combination with paeoniflorin and oxypaeoniflorin to treat tumors could be considered to overcome the limitations of drug resistance in non-small cell lung cancer treatment, provide new insights for non-surgical treatment of melanoma, and ultimately provide a scientific basis for further developing combined therapies of traditional Chinese medicine extracts for cancer treatment.

[0045] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

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Claims

1. The application of a combination of paeoniflorin and nobiletin in the preparation of anticancer drugs, characterized in that, The paeoniflorin compound is paeoniflorin or oxidized paeoniflorin; the anticancer drug is a treatment for lung cancer, and the molar ratio of paeoniflorin compound to nobiletin is 1:1 / 3 to 3; or the anticancer drug is a treatment for melanoma, and the molar ratio of paeoniflorin compound to nobiletin is 1:2.

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