Use of an indole alkaloid for the preparation of a medicament for the treatment of non-small cell lung cancer

Abstract

The application discloses application of an indole alkaloid with a chemical structure shown in the following formula in preparation of a drug for treating non-small cell lung cancer; the indole alkaloid demethoxygardmultine (DMGM) can significantly inhibit growth, proliferation and migration of PC-9 cells, and with the increase of the DMGM concentration, the apoptosis rate of the PC-9 cells gradually increases, and the proliferation and migration abilities of the PC-9 cells gradually weaken; the DMGM can induce excessive accumulation of ROS in the PC-9 cells, trigger cell oxidative stress and kill targeted drug-resistant non-small cell lung cancer cells in a concentration-dependent manner; the DMGM provided by the application has the effect of interfering with the targeted drug-resistant non-small cell lung cancer, provides a new direction for the targeted treatment of lung cancer and provides a new idea for the research of small molecule drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of an indole alkaloid in the preparation of a drug for treating non-small cell lung cancer. Background Technology

[0002] Non-small cell lung cancer (NSCLC) is one of the most common malignant tumors, accounting for approximately 80% of all lung cancers. It is a leading cause of high morbidity and mortality in lung cancer and a leading cause of cancer-related deaths worldwide. Despite numerous treatment advances over the past decade, NSCLC remains an incurable disease for most patients. While chemotherapy, targeted therapy, and immunotherapy have significantly improved NSCLC treatment, the vast majority of advanced NSCLC cases develop resistance to current treatments, leading to relapse and a lack of cure. Modern small molecule drugs typically work by blocking specific molecular pathways that play crucial roles in cancer cell differentiation, proliferation, and metastasis, enabling targeted treatment. Therefore, the search for new small molecule drugs may be an effective approach to treating NSCLC.

[0003] Compared to traditional therapies, modern small-molecule drugs typically work by blocking specific molecular pathways that play a crucial role in the proliferation, survival, and metastatic potential of cancer cells. These drugs offer a targeted approach to treating NSCLC, often resulting in more effective and personalized treatment with fewer side effects. Vincristine, a compound with a complex molecular structure, is administered intravenously and inhibits tumor cell division by binding to tubulin and inhibiting microtubule assembly, thereby blocking mitotic spindle formation. Cisplatin, a widely used anti-NSCLC drug, exerts its toxic effects by binding to DNA and forming intra- and inter-strand crosslinks, ultimately leading to impaired DNA synthesis, cell cycle arrest, and cell death in cancer cells. However, the severe nephrotoxicity caused by cisplatin significantly limits its use and raises considerable questions about its efficacy. Docetaxel, a taxane agent, binds to β-tubulin and stabilizes microtubules, causing mitotic arrest at the G2 / M transition, thus inducing apoptosis and cell death. For decades, cytotoxic drugs such as docetaxel, vincristine, and cisplatin have been used clinically to treat NSCLC patients. However, with the long-term use of cytotoxic drugs, drug resistance and genetic alterations in cancer cells have increased over the past decade, limiting their use.

[0004] Overcoming drug resistance could significantly extend the survival of NSCLC patients and alleviate their financial burden. Currently, there is little research or drug development focused on delaying, reversing, or killing drug-resistant tumor cells. Due to the continuous development and evolution of tumors, and their adaptation to drug selectivity, treatment methods for NSCLC patients after drug resistance has become an ongoing research challenge. Therefore, there is an urgent need to find new drugs for non-small cell lung cancer with better resistance, and the development of new drugs to combat drug-resistant NSCLC remains a significant potential area for research. Summary of the Invention

[0005] This invention provides an indole alkaloid and its application in the preparation of drugs for treating non-small cell lung cancer, in order to solve the problems of easy drug resistance and insufficient efficacy of existing drugs for treating non-small cell lung cancer.

[0006] The chemical structural formula of the indole alkaloid demethoxygardmultine (DMGM) is as follows: .

[0007] This invention, through in vitro cell experiments, found that indole alkaloids can significantly inhibit the growth, proliferation, and migration of PC-9 cells. Furthermore, with increasing indole alkaloid concentration, the apoptosis rate of PC-9 cells gradually increases, while their migration and proliferation abilities gradually weaken, exhibiting a concentration-dependent effect. This compound can also promote the accumulation of reactive oxygen species within non-small cell lung cancer cells. This provides a new direction for the treatment of drug-resistant NSCLC and offers insights for the research of new anti-tumor small molecule drugs.

[0008] The indole alkaloids of this invention are derived from the plant *Potentilla anserina*, ensuring a safe source. Indole alkaloids are abundant in the leaves of *Potentilla anserina*, and were previously isolated and purified in the laboratory with a purity exceeding 90%. Therefore, indole alkaloids show promising application potential in the preparation of drugs for treating non-small cell lung cancer.

[0009] The application described in this invention uses indole alkaloids as the active ingredient, and may also include one or more pharmaceutically acceptable excipients, such as fillers, diluents, binders, excipients, absorption enhancers, surfactants, and stabilizers commonly used in the pharmaceutical field. Flavoring agents, colorings, and sweeteners may also be added when necessary. In addition to being made into capsules, it can also be made into various forms such as pills, powders, tablets, granules, oral liquids, and injections.

[0010] Advantages and technical effects of the present invention: (1) In vitro cytotoxicity: The indole alkaloid of this invention has a significant inhibitory effect on the proliferation of human non-small cell lung cancer cell line PC-9, IC50. 50 The value is 1.31 μM; (2) Anti-migration effect: Scratch test showed that indole alkaloids could significantly inhibit the lateral migration ability of PC-9 cells; (3) Induction of apoptosis: Calcein AM fluorescence staining and flow cytometry (Annexin V-FITC / PI staining) confirmed that indole alkaloids can induce apoptosis in PC-9 cells in a dose-dependent manner, accompanied by a significant decrease in mitochondrial membrane potential; (4) Oxidative stress: Intracellular ROS measurements showed that indole alkaloids could significantly increase the level of reactive oxygen species in PC-9 cells, which is one of the mechanisms by which they induce apoptosis. (5) It has both killing and anti-metastasis effects: It simultaneously inhibits proliferation, induces apoptosis, and blocks migration, achieving the triple effects of "inhibiting proliferation, promoting apoptosis, and resisting invasion"; (6) Natural source and low toxicity: It is derived from the willow leaf of the kudzu, the raw materials are readily available, the extraction process is mature, the purity is controllable, and the safety is better than that of synthetic cytotoxic drugs. Attached Figure Description

[0011] Figure 1 The graph shows the proliferation inhibition curve of DMGM on PC-9 cells (CCK-8 assay). Figure 2 Figure showing the effect of DMGM on PC-9 cell migration as determined by a scratch assay; Figure 3 A diagram of PC-9 viable cell status obtained by Calcein AM staining; Figure 4 Scatter plot for detecting apoptosis in PC-9 cells by flow cytometry; Figure 5 This is a statistical chart of flow cytometry results. Figure 6 Figure showing the results of flow cytometry analysis of ROS levels in PC-9 cells; Figure 7 The figure shows the statistical results of ROS levels in PC-9 cells detected by flow cytometry. Detailed Implementation

[0012] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0013] The human lung adenocarcinoma PC-9 cells (drug-resistant non-small cell lung cancer cell line, catalog number: CX0102) used in the examples were purchased from Wuhan Boster Biological Engineering Co., Ltd.; the indole alkaloid demethoxygardmultine (DMGM) was derived from the plant *Potentilla anserina*, which was previously isolated and purified in the laboratory, with a purity of over 90%; fetal bovine serum (Gibco, USA), RPMI-1640 culture medium (Gibco, USA), phosphate buffer (Beyotime, Shanghai), penicillin antibody (HyClone, USA), DMSO (Sigma, USA), CCK-8 (Sigma, USA), alkaloids were prepared with DMSO, Annexin V-FITC / PI kit (Beijing Sizhengbai Biotechnology Co., Ltd.), and DCFH-DA (Corning, USA).

[0014] Example 1: Extraction and separation of indole alkaloids (DMGM) 12 kg of willow-leafed kudzu stems and leaves were air-dried, pulverized, and then extracted three times with a 90% methanol aqueous solution, each extraction lasting 24 hours, with a material-to-liquid ratio of kg:L = 1:4. The mixture was refluxed for 4 hours, and the extract was collected. The solvent was concentrated to obtain a crude extract. The crude extract was soluble in water, and the pH was adjusted to 2-3 with 0.5% hydrochloric acid. Ethyl acetate was then extracted three times to remove water-soluble non-alkaline substances. The acidic aqueous layer was then adjusted to pH 9-10 with a 10% ammonia solution. Ethyl acetate was extracted four times, and the ethyl acetate phase was collected, concentrated, and dried to obtain a total alkaloid extract (120 g). The extract was mixed with 180 g of silica gel and coarsely separated using a silica gel column (100-200 mesh particle size). Chloroform-methanol was used as the elution solvent (1:0, 50:1, 40:1, 30:1, 20:1, 10:1, 5:1, 1:1, 0:1). TLC was used for tracking and detection, and fractions with similar characteristics were combined, resulting in four fractions: Fr.1-Fr.4. Fr.1 (50 g) of total alkaloids was subjected to polar coarse fractionation using silica gel column chromatography with a gradient elution of chloroform:methanol (1:0-1:1). Thin-layer chromatography (TLC, GF254 plate) was used for process monitoring. Based on colorimetric characteristics, similar fractions were combined, ultimately yielding six fractions (Fr.1.1-Fr.1.6). The crude components were then finely separated using a multi-system approach including normal-phase silica gel chromatography (CC), medium-pressure chromatography (MPLC), dextran gel column chromatography (Sephadex LH-20), and semi-preparative chromatography (HPLC). Fr.1.4 (15 g) was mixed with reverse-phase packing material C18 and then eluted using a methanol / water gradient (20:80-60:40, v:v) by MPLC to obtain two fractions, Fr.1.4.1 and Fr.1.4.2. Fr.1.4.2 was then eluted using a CC petroleum ether / acetone gradient (3:1-1:1, v:v) to obtain compound DMGM (32 mg).

[0015] The results of the identification are as follows: Demethoxygardmultine: White powder. HR-ESI-MS m / z: 781.3787 [M+H]+, molecular formula is C 44 H 52N4O9; 1HNMR (500 MHz, CDCl3) δH: 7.37 (1H, s, N1-H), 3.59 (1H, d, J =12.9 Hz, H-3), 2.98 (1H, dd, J = 13.8, 5.9 Hz, H-5), 3.56 (2H, m, H-17), 1.58(3H, d, J = 6.6 Hz, H-18), 5.03 (1H, q, J = 6.9 Hz, H-19), 3.87 (3H, s, 10-OMe), 3.82 (3H, s, 9-OMe), 3.82 (1H, overlap, 12-OMe), 0.87 (3H, d, J = 5 Hz,H-18′), 4.95 (1H, q, J = 6.8 Hz, H-19′), 3.56 (2H, m, H-21′), 3.79 (s, 9-OMe′), 3.77 (s, 10-OMe′), 3.76 (s, 12-OMe′); 13C NMR (125 MHz, CDCl3) δC:179.1(s, C-2), 68.0(d, C-3), 68.6(d, C-5), 32.9 (t, C-6), 58.2(s, C-7), 122.4(s, C-8), 148.8(s, C-9), 124.5(s, C-10), 99.3 (d, C-11), 149.9(s, C-12),125.4(s, C-13), 22.6 (t, C-14), 35.5(d, C-15),43.0(d, C-16), 53.3 (t, C-17),11.7 (q, C-18), 112.6(d, C-19), 141.9(s, C-20), 49.7 (t, C-21), 57.3(q, 9-OMe), 57.0(q, 10-OMe), 57.3(q, 12-OMe),84.2 (s, C-2′), 68.0 (d, C-3′), 65.2(d, C-5′), 31.6(t, C-6′), 60.1(s, C-7′), 129.4(s, C-8′), 132.5(s, C-9′),138.6(s, C-10′), 98.3 (d, C-11′), 142.7 (s, C-12′), 138.9 (s, C-13′), 29.7(t, C-14′), 30.6 (d, C-15′), 42.The spectroscopic data of this indole alkaloid were compared with specific data from the literature, and finally, the indole alkaloid was identified as demethoxygardmultine. (89(d, C-16′), 66.1 (t, C-17′), 12.6(q, C-18′), 111.3(d, C-19′), 140.6 (s, C-20′), 49.3(t, C-21′), 61.7(q, 9′-OMe), 56.3(q, 10′-OMe), 61.0 (q, 12′-OMe).

[0016] Example 2: Effects of indole alkaloids (DMGM) on PC-9 cell growth Cell culture: PC-9 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin antibiotics and passaged in a cell culture incubator at 37°C and 5% CO2 until the logarithmic growth phase.

[0017] Sample preparation: Weigh the monomer compound (DMGM) and dissolve it in cell-grade DMSO to prepare a stock solution with a concentration of 40 μM. Store the stock solution at -20°C in the dark for later use. Before the experiment, dilute the prepared stock solution to the required concentration using RPMI-1640 complete medium. The concentration of DMSO in the medium should not exceed 0.1%.

[0018] PC-9 cells were uniformly seeded into 96-well plates and cultured for 24 h. After the culture medium was discarded, a blank group (RPMI-1640 medium only, no cells), compound treatment groups (different concentrations of DMGM + PC-9 cells), and a control group (RPMI-1640 medium + PC-9 cells) were set up. The compound treatment groups were added 100 μL of RPMI-1640 medium containing 5 μM, 10 μM, and 20 μM DMGM, respectively, with 3 replicates per group. Incubation continued for 24 h. Then, 10 μL of CCK-8 solution was added to each well under dark conditions and incubated for 3 h. The absorbance (OD) value was measured at 450 nm, cell viability was calculated, and IC50 was fitted. 50 Calculate cell viability and inhibition rate using the following formulas:

[0019] Experimental data OD values ​​are expressed as "mean ± standard deviation", and statistical analysis and variance analysis were performed using Origin software.

[0020] The results are as follows Figure 1 As shown in the figure, the inhibition rate of PC-9 cells increases with increasing DMGM concentration. The half-maximal inhibitory concentration (IC50) of DMGM in PC-9 cells was calculated. 50The concentration of DMGM was 1.31 μg / mL, indicating that DMGM could significantly inhibit the growth and proliferation of PC-9 cells.

[0021] Example 3: Effects of DMGM on PC-9 cell migration PC-9 cells in the logarithmic growth phase were digested with 0.25% trypsin solution and then gently pipetted into a single-cell suspension using RPMI-1640 complete culture medium. Cell counting was performed, and the cell density was adjusted. Subsequently, the cells were seeded into 6-well cell culture plates at a density of 3 × 10⁶ cells per well. 5 Cells per well. To ensure uniform cell adhesion and distribution, gently shake the culture plate horizontally after inoculation, then incubate at 37°C with 5% CO2. When the cells in the culture plate reach 90%-100% confluence, perform a scratch treatment. Using a 200μL sterile pipette tip, perpendicular to the bottom of the plate, apply even pressure and quickly draw a straight line in the center of each well. Keep the pipette tip vertical during the scratching process to avoid uneven scratch width due to tilting. After scratching, discard the old culture medium and gently rinse the cells 2-3 times with sterile PBS buffer to remove cell debris and suspended cells. Then, add RPMI-1640 medium containing different concentrations (5μM, 10μM, 20μM) of DMGM, 1% serum, and 1% penicillin-dextrose antibody for continued culture. Samples are usually taken and photographed at specific time points such as 0h, 12h, and 24h after scratching. The culture medium is changed and the cells are rinsed with PBS before each photograph, and drug administration is continued. A blank control group was also set up (0 μM: RPMI-1640 culture medium without DMGM). The results are as follows Figure 2 As shown, at a concentration of 5 μM, the scratch healing rate was slower than the control group after 12 hours, and a clear blank zone remained in the scratched area. Although the scratches partially healed at 24 hours, the degree of healing was significantly lower than the control group, and cell migration was mildly inhibited. At a concentration of 10 μM, the scratch healing process was further delayed; at 12 hours, cell filling in the scratched area was sparse, and a clear scratch band was still visible at 24 hours, indicating a significant inhibitory effect on cell migration. At a concentration of 20 μM, a very strong cell migration inhibitory effect was observed; at both 12 and 24 hours, there was almost no cell migration filling in the scratched area, the scratch morphology remained largely intact, and cell migration was almost completely blocked. This indicates that DMGM has significant cell migration regulatory activity and can be used to prepare drugs that inhibit cell migration and intervene in tumor metastasis and related diseases.

[0022] Example 4: Effects of DMGM on the viable state of PC-9 cells PC-9 cells in the logarithmic growth phase were digested with 0.25% trypsin solution and then gently pipetted into a single-cell suspension using RPMI-1640 complete culture medium. After cell counting, the cell density was adjusted, and the cells were seeded into 12-well cell culture plates at a density of 1 × 10⁶ cells per well.5 Cells were cultured overnight at 37°C with 5% CO2 to allow them to adhere. When cell confluence reached 70%-80%, the original culture medium was discarded and replaced with RPMI-1640 medium containing different concentrations (5μM, 10μM, 20μM) of DMGM. A blank control group (0μM: RPMI-1640 complete culture medium without DMGM) was also set up. Culture was continued for 24 hours. After the intervention, the drug-containing medium in the 12-well plate was discarded, and the cells were gently washed 1-2 times with warm PBS buffer to remove serum and residual compounds. Then, an appropriate amount of prepared Calcein-AM staining working solution was added to each well, and the plate was incubated in the dark at 37°C with 5% CO2 for 15-30 minutes. After incubation, the staining solution was discarded, and the plate was gently rinsed with PBS to remove background dye. The 12-well plate was immediately observed under an inverted fluorescence microscope. Calcein-AM was excited at 490 nm and emitted at 515 nm, producing bright green fluorescence in live cells under the microscope. At least three fields of view were randomly selected from each group for photographing.

[0023] The results are as follows Figure 3 As shown, compared with the control group, treatment with 5 μM DMGM slightly reduced the number of viable cells, and the cells were round or polygonal in shape with clear outlines and intact structures. The 10 μM treatment group showed a significant decrease in viable cell density, with some cells shrinking in size and having blurred outlines, and a few exhibiting shrunken appearance. The 20 μM treatment group showed an extremely significant decrease in the number of viable cells, with only a few scattered cells visible in the field of view. The remaining cells were mostly severely shrunken and irregular in shape, with uneven fluorescence distribution and significantly impaired cell structural integrity. In conclusion, DMGM exhibits concentration-dependent cytotoxicity, effectively inhibiting cell survival and disrupting cell morphology.

[0024] Example 5: Effect of DMGM on PC-9 cell apoptosis PC-9 cells in the logarithmic growth phase were harvested and their density adjusted using complete medium containing RPMI-1640. Cells were grown at a density of 1 × 10⁶ cells per well. 5Cells were seeded at a density of 1,000 cells / well in 6-well plates and incubated overnight at 37°C with 5% CO2. When cell confluence reached 70%-80%, the old culture medium was discarded. The control group received an equal volume of RPMI-1640 complete medium, while the compound treatment groups received RPMI-1640 medium containing different concentrations (2.5 μM, 5 μM, 10 μM) of DMGM. Different concentrations of DMGM were added to the corresponding wells, and the cells were incubated for another 24 hours. After drug treatment, the cell culture supernatant from each well was collected into a pre-labeled 5 mL centrifuge tube. A small amount (0.3 mL) of EDTA-free trypsin was added to the wells, gently shaken, and incubated at 37°C for an appropriate digestion time. When the intercellular spaces increased and the cells became rounded but not completely floating, the serum-containing old culture medium collected in the first step was added to stop the digestion. The cell suspension was collected and transferred to the corresponding centrifuge tubes, centrifuged at 1000-1500 rpm for 5 minutes, and the supernatant was discarded. Add 1 mL of pre-chilled PBS phosphate buffer to the cell pellet, gently resuspend the cells, centrifuge again under the same conditions, discard the supernatant, and repeat this washing step once. The effect of compound DMGM on PC-9 cell apoptosis was detected using the Annexin V-FITC / PI apoptosis detection kit. Add 300 µL of prepared staining working solution to each tube of cell pellet, resuspend the cells, and incubate at room temperature in the dark for 15-20 minutes. After incubation, add 400-500 µL of pre-chilled PBS to each tube and mix gently. Place the samples on ice and analyze cell apoptosis using flow cytometry within 1 hour.

[0025] The results are as follows Figure 4 , 5 As shown, the compound DMGM significantly induced apoptosis in PC-9 cells in vitro. Compared with the control group, the proportion of viable cells in the treatment group was reduced, while the proportion of apoptotic cells was significantly increased. The apoptosis-inducing effect of DMGM on PC-9 cells showed a clear concentration-dependent effect, meaning that the higher the concentration, the stronger the pro-apoptotic effect. Within the concentration range set in this experiment, high concentrations of DMGM exhibited the strongest pro-apoptotic activity. DMGM has a significant, concentration-dependent pro-apoptotic effect on PC-9 cells and is a potential active compound for inhibiting PC-9 cell proliferation, with the most prominent pro-apoptotic effect observed at a concentration of 10 μM.

[0026] Example 6: Effect of DMGM on intracellular ROS levels in PC-9 cells PC-9 cells in the logarithmic growth phase were harvested and their density adjusted using complete medium containing RPMI-1640. Cells were grown at a density of 2 × 10⁶ cells per well. 5Cells were seeded at a density of 1000 g / cm³ in 6-well plates and incubated at 37°C with 5% CO₂ for 24 hours. The old culture medium was then discarded. The blank control group received an equal volume of RPMI-1640 complete medium, while the compound treatment groups received RPMI-1640 medium containing different concentrations (2.5 μM, 5 μM, 10 μM) of DMGM. Different concentrations of DMGM were added to the corresponding wells, and the cells were incubated for another 24 hours. After drug treatment, the cell culture supernatant from each well was collected into a pre-labeled 5 mL centrifuge tube. The tube was washed once with pre-cooled PBS phosphate-buffered saline, and then 0.3 mL of EDTA-free trypsin was added to the well. After gentle shaking, the tube was incubated at 37°C for an appropriate digestion time. When the intercellular spaces increased and the cells became rounded but not completely floating, the old culture medium containing serum collected in the first step was added to stop the digestion. The collected cell suspension was centrifuged at 2000 rpm for 8 minutes at 4°C, and the supernatant was discarded. Add 1 mL of pre-chilled PBS phosphate buffer to the cell pellet, gently resuspend the cells, and centrifuge again under the same conditions, discarding the supernatant. Dilute the DCFH-DA probe with PBS phosphate buffer to a final concentration of 10 μM according to the ROS detection kit (DCFH-DA) instructions. Add 500 µL of the prepared staining working solution to each cell pellet, resuspend the cells, and incubate at 37°C in the dark for 30 minutes. After incubation, wash twice with pre-chilled PBS, add 400 µL of pre-chilled PBS, mix gently, and place on ice. Measure the intensity of green fluorescence in the cells using a flow cytometer within 2 hours.

[0027] The results are as follows Figure 6 , 7 As shown, compared with the control group, the green fluorescence peaks of cells treated with 2.5 μM, 5 μM, and 10 μM DMGM all shifted significantly towards higher fluorescence intensity, suggesting that DMGM can significantly increase the ROS level in PC-9 cells. Quantitative statistical results further confirmed that the relative ROS intensity of each DMGM treatment group was significantly higher than that of the control group (P < 0.0001), and the effect increased in a concentration-dependent manner; among them, the relative ROS intensity of the 10 μM DMGM group was significantly higher than that of other concentration groups, reaching the highest level among all groups. In summary, DMGM can induce excessive ROS accumulation in PC-9 cells in a concentration-dependent manner, triggering cellular oxidative stress, and this effect is one of the key mechanisms by which it exerts its anti-PC-9 cell proliferation activity.

[0028] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. The application of an indole alkaloid in the preparation of a drug for treating non-small cell lung cancer, characterized in that: The chemical structural formula of indole alkaloids is as follows: 。

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