Application of brooklet anemone root active component in preparation of medicine for treating lung cancer

Abstract

The invention provides application of brooklet anemone root active ingredients in preparation of a medicine for treating lung cancer. The active monomer component PCP4 is successfully separated and extracted from the n-butyl alcohol part of brooklet anemone root by adopting an activity-directed separation technology, and it is found that the active monomer component PCP4 remarkably inhibits the proliferation capacity of non-small cell lung cancer A549 cells and can promote autophagy activity in U87 cells. The monomeric compound has the advantages of good pharmacological activity and small toxic and side effects. The compound can be used as a raw material medicine of a medicine preparation for treating lung cancer, especially has the best effect on non-small cell lung cancer, and has a wide market application prospect.

Description

Technical Field

[0001] The application of an active ingredient from *Tiger's Paw Herb* in the preparation of a drug for treating lung cancer belongs to the field of new applications in the extraction, separation, and purification of traditional Chinese medicine. Background Technology

[0002] Lung cancer is a malignant tumor with a high incidence and mortality rate worldwide. In recent years, the incidence and mortality rates of lung cancer have been rising continuously. Globally, approximately 2.5 million people die from lung cancer each year, of which about 2.1 million die from non-small cell lung cancer. Lung cancer accounts for 11.6% of all cancer cases, making it the cancer with the highest incidence rate.

[0003] Lung cancer is divided into two main types: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Epidemiological data shows that small cell lung cancer accounts for approximately 15% of all lung cancer patients, while NSCLC accounts for approximately 85%. NSCLC mainly includes three types: lung adenocarcinoma, large cell undifferentiated carcinoma of the lung, and squamous cell carcinoma of the lung. Factors contributing to NSCLC include smoking, exposure to toxic and harmful chemicals, and lung diseases (such as tuberculosis). The incidence of NSCLC is increasing year by year. Most importantly, patients diagnosed with NSCLC are already in the middle or late stages, with a poor prognosis, including uncontrollable long-term disease and a short 5-year survival rate. The cell models used in this experiment are human lung adenocarcinoma alveolar basal epithelial cells-A549 and humanized NSCLC-H1299 lymph nodes.

[0004] Treatment for NSCLC primarily involves single or combined therapies, including immunotherapy, surgery, chemotherapy, radiotherapy, and targeted therapy. Immunotherapy currently employs two strategies: passive immunotherapy and active immunotherapy. Active immunotherapy stimulates the immune system using drugs such as levamisole or cytokines; however, its effectiveness is limited due to the weak antigenicity of lung cancer tumors. Passive immunotherapy, on the other hand, controls the disease by blocking abnormal signal transduction pathways in cancer cells through the combination of abnormally expressed oncogene products and monoclonal antibodies. Surgical treatment, based on the international TNM staging system which classifies NSCLC into stages O to IV, primarily treats patients with lung cancer at stages IA, IB, IIA, and IIB. Surgery is not recommended for some patients with intermediate or advanced stages; chemotherapy, radiotherapy, and targeted drug delivery are generally used instead. For radiotherapy, fractionation therapy is not currently the first-line treatment. The theory of accelerated reproliferation of malignant tumors during treatment and the movement of the lungs with respiration increase the difficulty of radiotherapy; currently, radiotherapy is mainly used as a major component of combination therapies. However, most patients do not receive early detection or attention for lung cancer, and by the time it is discovered, it is already in an advanced stage. At this point, chemotherapy becomes the main treatment for advanced lung cancer. For stage IIB and IIIA patients who are not candidates for surgery, although treatment regimens such as gemcitabine, paclitaxel, docetaxel, and cisplatin can prolong survival, long-term use of drugs such as cisplatin, docetaxel, gefitinib, and erlotinib, while killing tumor cells, can also damage normal tissues and produce a series of adverse reactions, such as nephrotoxicity, ototoxicity, bone marrow toxicity, and skin and mucous membrane toxicity.

[0005] Therefore, exploring non-platinum-based treatment options has become an important area of ​​clinical research. Currently, specific histological chemotherapy regimens and molecularly targeted drugs have made significant progress in NSCLC patients with specific genomic alterations. Although these regimens offer higher selectivity and fewer side effects compared to chemotherapy, and can improve the prognosis of patients with metastatic lung adenocarcinoma, no new breakthroughs have been achieved in the treatment of NSCLC. Therefore, finding effective treatments for NSCLC is urgently needed.

[0006] Currently, traditional Chinese medicine has received considerable attention in reversing tumor drug resistance and in targeted therapy. Although targeted therapy has developed rapidly, drug resistance can occur during its use. *Tiger's Paw Grass*, a dicotyledonous Ranunculaceae plant, is recorded in books such as *Selected Yunnan Traditional Chinese Medicines* and *Collection of Folk Prescriptions of Guizhou*. The entire plant can be used medicinally, possessing anti-inflammatory, analgesic, liver-soothing, bile-promoting, and muscle-relaxing effects. It is mainly used to treat cirrhosis, chronic hepatitis, stomach pain, laryngitis, toothache, and chronic rheumatoid arthritis. The root of *Tiger's Paw Grass* contains betulinic acid and succinate saponins. However, no research reports on the use of *Tiger's Paw Grass* extract for the treatment of lung cancer have been found in existing patents and journal articles. Therefore, developing a highly effective pharmaceutical preparation based on *Tiger's Paw Grass* extract is particularly important. Summary of the Invention

[0007] This invention provides the application of an active ingredient from *Smilax china* in the preparation of drugs for treating lung cancer. Using activity-directed separation technology, the active monomeric component PCP4 was successfully isolated and extracted from the n-butanol fraction of *Smilax china*. It was found that PCP4 significantly inhibits the proliferation of non-small cell lung cancer (NSCLC) A549 cells while promoting autophagy in U87 cells. This monomeric compound exhibits good pharmacological activity and low toxicity. It can be used as a raw material for lung cancer drug formulations, especially effective against NSCLC, and has broad market application prospects.

[0008] The technical solution provided in this invention patent application is as follows:

[0009] The application of an active ingredient of *Hedyotis diffusa* in the preparation of a drug for treating tumor diseases, characterized in that the active ingredient compound of *Hedyotis diffusa* is PCP4.

[0010] The compound of this invention is used to treat tumor diseases, specifically lung cancer. Preferably, it is used for non-small cell lung cancer.

[0011] Preferably, the tiger paw grass extract is a n-butanol fraction extract.

[0012] Preferably, the preparation method of the tiger paw grass extract includes the following steps:

[0013] (1) Ethanol extraction of Tiger Paw Grass:

[0014] Weigh out the crude powder of Tiger Paw Herb, add 10 times the amount of 75% ethanol, heat and reflux for 2 hours, heat and reflux 3 times, collect and combine the three extracts, and dry under vacuum at 60℃ to obtain the total extract of Tiger Paw Herb.

[0015] (2) Extraction of the n-butanol fraction of *Spatholobus suberectus*

[0016] Take the total extract of *Sedum sarmentosum* from step (1), suspend it in pure water, and extract it from the aqueous suspension of *Sedum sarmentosum* in sequence with petroleum ether, ethyl acetate and n-butanol. Extract each organic solvent 3-5 times, and combine the n-butanol extracts. Dry the extracts under vacuum at 60°C to obtain the n-butanol extract of *Sedum sarmentosum*.

[0017] (3) Column chromatography separation of the n-butanol fraction of *Symplocos cuspidatum*

[0018] Take the n-butanol extract of *Spatholobus suberectus* from step (2) and perform preliminary separation using a normal-phase silica gel (100-200 mesh) column chromatography. The mobile phase is a dichloromethane-methanol gradient elution system, with the elution ratio gradually changing from 85:15 to 65:35, with each gradient change being 10%. The elution volume for each elution ratio is 100 mL. After collecting and combining the eluents, evaporate the collected fractions to dryness to obtain the n-butanol active fraction of *Spatholobus suberectus* for later use.

[0019] (4) Isolation and purification of the active ingredient n-butanol from *Tiger Paw Grass*

[0020] Next, the n-butanol active fraction of *Sedum sarmentosum* from step (3) was taken and subjected to preparative high-performance liquid chromatography (HPLC) with acetonitrile:water = 35:75 as the mobile phase and a flow rate of 4 mL / min. The chemical components of the n-butanol active fraction of *Sedum sarmentosum* were finely separated and purified, and the target active component, namely compound PCP4, was collected at a retention time of 17 min.

[0021] The chemical formula of the PCP4 is: C 46 H 74 O 16 .

[0022] To illustrate the inventive concept of this invention, the results of some experimental screening of the active ingredients of Tiger Paw Grass are summarized below, further demonstrating the innovativeness of the technical solution of this invention.

[0023] 1. Screening of the inhibitory activity of Tiger Paw Herb Extract and its different polar fractions on A549 proliferation.

[0024] Based on previous extraction and liquid-liquid extraction of *Smilax china*, we obtained the total extract (ATE) and four fractions of different polarities: aqueous fraction (AWF), ethyl acetate fraction (AEF), n-butanol fraction (ANF), and petroleum ether fraction (APF). The MTT assay was used to determine the inhibitory effects of different concentrations (6.25, 12.5, 25, 50, 100, 125, 150, 175, and 200 μg / mL) of the total extract and the four fractions on A549 proliferation over 24 h. The results are as follows: Figure 1As shown in Figure A, the IC50 values ​​of ATE, AEF, ANF, and APF for inhibiting A549 cell proliferation were 94.85, 49.65, 48.53, and 66.09 μg / mL, respectively. ANF, which showed the best inhibitory effect on A549 cell proliferation, was separated into nine fractions (F1-F9) by column chromatography, and the MTT assay was used to detect the nine fractions. The results are as follows. Figure 1 As shown in B, the F2, F3, and F4 fractions showed the best inhibitory effect on A549 proliferation.

[0025] 2. Screening of autophagy activation effects of *Spatholobus suberectus* extract and its different polar fractions.

[0026] To screen for the fraction with the best autophagy-activating effect, the total extract of *Smilax china* and the highest working concentration of its four polar fractions were prepared at 200 μg / mL and sequentially diluted 2 / 3 downwards. These fractions were then applied to U87 cells stably transfected with GFP-LC3 for 24 h, and images were acquired using an inverted fluorescence microscope. The percentage of autophagy-positive cells (cells with more than 10 autophagy dots) was counted and plotted in the acquired images. The results are shown below. Figure 2 As shown in Figure A, the minimum concentration of ATE to activate autophagy was 25 μg / mL; the maximum concentration of AWF (200 μg / mL) did not induce autophagy; the minimum concentration of AEF to activate autophagy was 25 μg / mL; the minimum concentration of ANF to activate autophagy was 12.5 μg / mL; and the minimum concentration of APF to activate autophagy was 25 μg / mL. ANF, which showed the best autophagy-inducing effect, was used for column chromatography to separate nine fractions (F1-F9). The highest working concentration of each of the nine fractions was prepared at 200 μg / mL, and each fraction was sequentially diluted 2 / 3 times downwards and treated on U87 cells for 24 h. Images were acquired using an inverted fluorescence microscope. The percentage of autophagy-positive cells in the acquired images was statistically analyzed and plotted. The results are shown below. Figure 2 As shown in B, fractions F2, F3, and F4 exhibited the best autophagy-inducing effects. Their minimum activation concentrations for autophagy were 1.56 μg / mL, 1.56 μg / mL, and 0.78 μg / mL, respectively.

[0027] 3. Isolation and identification of the active ingredient PCP4 in this invention

[0028] This invention utilizes silica gel column chromatography and high-performance liquid chromatography to further separate and collect nine fractions of ANF. In previous experiments, we found that fractions F2, F3, and F4 showed the best inhibitory effect on A549 proliferation and induced autophagy in U87 cells. We further analyzed the components of F2, F3, and F4. Figure 3The total ion chromatogram of C shows that F2-F4 have a main peak with similar elution times between 16 and 20 min. After further separation of F2-F4 by preparative high-performance liquid chromatography (HPLC), the same main peak from the three fractions was collected and further analyzed by LC-MS / MS and NMR. The results indicate that... Figure 3 As shown in Figure D, LC-MS / MS (m / z): 919.05 [M+Na]+, 895.35 [MH]-, 889 [M+Na]+, 865.4 [MH]-, confirming the molecular formula as C46H74O15. The 1H-NMR, 13C-NMR, and NMR data of this compound were compared with literature data, confirming the compound as PCP4, with the molecular structure shown below. Figure 4 As shown. Therefore, the results above suggest that a monomeric component with a relatively high content was isolated from F2-F4, which is PCP4, and its molecular formula and structural formula were determined.

[0029] The beneficial effects of the technical solution of this invention are as follows:

[0030] (1) In this invention, through extraction and liquid-liquid extraction of *Spatholobus suberectus*, we obtained the total extract (ATE) of *Spatholobus suberectus* and four different polar components including water fraction (AWF), ethyl acetate fraction (AEF), n-butanol fraction (ANF), and petroleum ether fraction (APF). Pharmacological activity analysis revealed that the active ingredient in the n-butanol fraction exhibited the best activity against A549 cells. To obtain the most effective and least toxic active monomeric components, this invention employs normal-phase silica gel column chromatography for preliminary separation. The mobile phase is a dichloromethane-methanol gradient elution system (the elution ratio gradually changes from 85:15 to 65:35, with each gradient change being 10%, and the elution volume for each elution ratio is 100 mL). After collecting and combining the eluents, the collected fractions are evaporated to dryness. The active fraction of *Smilax china* n-butanol is then further separated and purified using preparative high-performance liquid chromatography (HPLC) with an acetonitrile:water ratio of 35:75 and a flow rate of 4 mL / min. The target active component, compound PCP4, is collected at a retention time of 17 min.

[0031] (2) The active monomeric component PCP4 of *Smilax china* in this invention was verified by cell experiments. PCP4 significantly inhibited the proliferation of NSCLC cell lines A549 and H1299. PCP4 also significantly inhibited the migration rate of A549 and H1299 cells. PCP4 significantly inhibited NSCLC cell invasion in a concentration-dependent manner. PCP4 also induced mitochondrial damage in A549 and H1299 cells, reduced mitochondrial mass in A549 and H1299 cells, upregulated mitochondrial ROS levels in A549 and H1299 cells in a concentration-dependent manner, significantly increased the apoptosis rate of A549 and H1299 cells in a concentration-dependent manner, reduced cell viability by inducing apoptosis in A549 and H1299 cells, and induced apoptosis in NSCLC cells. Furthermore, PCP4 showed a concentration difference in inducing apoptosis in human embryonic lung cells at 8 μM. At the same concentration and time, the apoptosis rate of MRC-5 cells was lower than that of A549 cells. PCP4 also increases the formation of GFP-LC3 spots in cells in a concentration-dependent manner. The above experimental results indicate that PCP4, the active monomeric component of *Smilax china* in this invention, has anti-tumor effects, especially significant therapeutic and inhibitory effects on non-small cell lung cancer.

[0032] (3) Mitophagy is mainly regulated by PINK1 and Parkin. Downregulation of cyclooxygenase-2 (COX-2) leads to the accumulation of PINK1 and subsequent Parkin mitochondrial translocation, thereby activating mitophagy. In this experiment, A549 and H1299 cells were treated with PCP4 (0, 4, 6, 8, and 10 μM) and CCCP (10 mM), and the expression of mitophagy pathway-related proteins PINK1 / Parkin, COX-2, MFN1, and MFN2 was detected by Western blotting. The results are as follows: Figure 15 As shown, PCP4 significantly downregulated the expression of COX-2, MFN1, and MFN2, and significantly upregulated the expression of PINK1 and p-Parkin. Furthermore, transient transfection of GFP-LC3 into A549 cells followed by AC220 intervention was used to examine the effect of PCP4 on autophagy spots in A549 cells. The results showed that AC220 inhibited PCP4-induced LC3 spot formation in A549 cells. In conclusion, PCP4 activates mitophagy in A549 and H1299 cells through PINK1 / Parkin. This further demonstrates that the active monomeric component PCP4 of *Smilax china* in this invention has the effect of enhancing the body's resistance.

[0033] (4) The PCP4 isolated from *Smilax china* in this invention can induce NSCLC cell death in vitro by activating mitophagy. Furthermore, there is a concentration difference between PCP4 inducing apoptosis in MRC-5 cells and A549 cells. This indicates that PCP4 has low toxicity to normal human lung cells while inducing NSCLC cell death.

[0034] Experimental results showed that PCP4 had a strong anti-proliferative effect on A549 and H1299 cells. We further verified the inhibitory effect of PCP4 on NSCLC growth in A549 tumor-bearing nude mice. The relative weights of the heart, liver, spleen, lungs, and kidneys of the nude mice did not change significantly. These results suggest that PCP4 can effectively inhibit the proliferation of A549 cells in tumor-bearing nude mice without significant toxic side effects. Attached Figure Description

[0035] The accompanying drawings, which are provided to further illustrate the invention and constitute a part of this invention, are illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention.

[0036] Figure 1 (A) Effects of different concentrations of total extract (ATE) and four polar fractions of *Sedum sarmentosum* on the viability of A549 cells; (B) Effects of nine fractions of the n-butanol fraction (ANF) of *Sedum sarmentosum* on the viability of A549 cells;

[0037] Figure 2 (A) Effects of different concentrations of total extract ATE and four polar fractions of *Smilax china* on the formation of GFP-LC3 spots in U87 cells; (B) Effects of nine components isolated from the total extract ANF of *Smilax china* on the formation of GFP-LC3 spots in U87 cells; Note: Compared with the control group, *p<0.05, ***p<0.001;

[0038] Figure 3 Isolation and identification of PCP4: (A) Root and stem of *Sedum sarmentosum*; (B) UV full-wavelength scans of ATE, AWF, AEF, ANF and APF; (C) UV full-wavelength scans of the nine fractions (F1-F9) separated by chromatography from the n-butanol fraction of *Sedum sarmentosum*; (D) Mass-to-charge ratio of PCP4.

[0039] Figure 4 The structural and chemical formulas of Prosapogenin CP4 (PCP4);

[0040] Figure 5Effects of PCP4 on NSCLC cell proliferation and colony formation: (A) Effect of PCP4 on A549 and H1299 cell viability detected by CellTiter-Lumi Plus chemiluminescence assay; (B) Effect of PCP4 on A549 and H1299 cell cytotoxicity detected by LDH assay; (C, D) Effect of PCP4 on A549 and H1299 cell colony formation detected by crystal violet staining assay; (E, F) Effect of PCP4 on A549 and H1299 cell death detected by Calcein-AM-PI cell staining assay. Representative images were acquired under a fluorescence microscope, and statistical graphs indicated the percentage of cell death. Note: Compared with the control group, *p<0.05, ***p<0.001. Scale bar: 200 μm (10×).

[0041] Figure 6 Effects of PCP4 on the migration of A549 (A) and H1299 (B) cells, scale bar: 600 μm (4×);

[0042] Figure 7 Effects of PCP4 on the invasion of A549 and H1299 cells; Note: Compared with the control group, **p<0.01, ***p<0.001, scale bar: 100 μm (20×);

[0043] Figure 8 Mito-Tracker staining was used to detect the effects of PCP4 and CCCP on mitochondrial morphology in A549 and H1299 cells; Note: Compared with the control group, ***p<0.001, scale bar: 5 μm (63×);

[0044] Figure 9 Effect of PCP4 on mitochondrial quality in A549 (A) and H1299 (B) cells was detected by 10-NAO staining method; Note: Compared with the control group, **p<0.01, ***p<0.001, scale bar: 200 μm (10×);

[0045] Figure 10 The effect of PCP4 on mitochondrial ROS levels in A549 (A) and H1299 (B) cells was detected by Mito-SOX-Red staining. Note: Compared with the control group, **p<0.01, ***p<0.001; Scale bar: 200 μm (10×).

[0046] Figure 11 : TMRM staining method was used to detect the effect of PCP4 on mitochondrial membrane potential in A549 (A) and H1299 (B) cells; Note: Compared with the control group, **p<0.01, ***p<0.001, scale bar: 200 μm (10×);

[0047] Figure 12 The effect of PCP4 on apoptosis in A549(A) and H1299(B) cells was detected by flow cytometry using the Annexin V-FITC / PI apoptosis detection kit. Note: Compared with the control group, ***p<0.001;

[0048] Figure 13 Effects of Z-VAD-FMK on the inhibition of PCP4 activity in A549 (A) and H1299 (B) cells;

[0049] Note: Compared with the control group, **p<0.01, ***p<0.001

[0050] Figure 14 Cell viability of MEF BAX- / - and MEF BAX+ / + cells treated with PCP4 for 24 h; Note: Compared with the control group, **p<0.01, ***p<0.001;

[0051] Figure 15 Western blot analysis was performed to detect the effects of PCP4 on the expression of Bax, Bcl2, caspase-3, caspase-9 and GAPDH proteins in A549 (A) and H1299 (B) cells.

[0052] Figure 16 The effect of PCP4 on apoptosis in A549 (A) and MRC-5 (B) cells was detected using the caspase-3 fluorescent probe. Scale bar: 400 μm (10×).

[0053] Figure 17 Western blot analysis was performed to detect the effect of PCP4 on the LC3II / LC3I conversion rate in A549 (A) and H1299 (B) cells. Note: Compared with the control group, **p<0.01, ***p<0.001;

[0054] Figure 18 Effects of PCP4 and Rap on GFP-LC3 spot formation in A549 (A) and H1299 (B) cells transiently transfected with GFP-LC3 plasmid;

[0055] Figure 19 Effects of PCP4 on the viability of A549 (A) and (B)H1299 cells under Baf intervention. Note: Compared with the control group, ***p<0.001;

[0056] Figure 20Effect of PCP4 on cell death in A549 (A) and H1299 (B) cells under Baf intervention. Note: Compared with the control group, ***p<0.001, scale bar: 200 μm (10×);

[0057] Figure 21 Effects of PCP4 on tumor proliferation in A549 tumor-bearing nude mice: (A) Image acquisition of nude mice in different groups 15 days after treatment with PCP4 and Gefi; (B) Image acquisition of tumors in different groups 15 days after treatment with PCP4 and Gefi; (C) Tumor weight 15 days after treatment with PCP4 and Gefi; (D) Tumor volume during PCP4 and Gefi administration; (E) Tumor weight during PCP4 and Gefi administration; (F) Relative organ weight 15 days after PCP4 and Gefi treatment; Note: Compared with the control group, *p<0.05, **p<0.01, ***p<0.001. Detailed Implementation

[0058] To better understand the implementation of this invention, one or more experimental examples are listed below, and the invention will be further illustrated through typical embodiments. It should be noted that unless specific conditions are specified, all results are obtained under conventional conditions, and if the manufacturers of the reagents used are not specified, they are all conventional products available on the market.

[0059] Example 1: Extraction, isolation, identification, and pharmacological activity verification of CP4, the active ingredient in *Smilax china*.

[0060] 1. Experimental materials, reagents and instruments

[0061] 1.1 Tiger Paw Grass

[0062] The rhizome of Tiger Paw Grass was purchased from Chengdu Hehuachi Medicinal Herbs Professional Market (batch number: ARB220609);

[0063] 1.2 Experimental cell lines

[0064] A549 cells: American Type Culture Collection (ATCC, FS-0167); H1299 cells: American Type Culture Collection (ATCC, YB-4806); Stable RFP-GFP-LC3 U87 cells (human glioma cell line): donated by Dr. Xiaoming Zhu from Macau University of Science and Technology, my country; MEF ATG7 + / + Cells (wild-type mouse embryonic fibroblasts): donated by Professor Masaaki Komatsu of Juntendo University, Japan; MEF ATG7 - / - Cells (ATG7 knockout mouse embryonic fibroblasts): Donated by Professor Masaaki Komatsu of Juntendo University, Japan; MEF BAX+ / + Cells (wild-type mouse embryonic fibroblasts): donated by Professor Masaaki Komatsu of Juntendo University, Japan; MEF BAX - / - Cells (knockout BAX mouse embryonic fibroblasts): donated by Professor Masaaki Komatsu of Juntendo University, Japan.

[0065] 1.3 Main reagents for the experiment

[0066] Table 1. Reagents used in the experiment

[0067]

[0068] 2. Experimental Methods

[0069] 2.1.1 Preparation of total extract of Tiger Paw Grass

[0070] Powder the tiger paw grass, weigh an appropriate amount of coarse tiger paw grass powder, add 10 times the amount of 75% ethanol, heat and reflux for 2 hours (repeat three times), collect the three extracts and combine them, then vacuum dry them in a rotary evaporator at 60℃, and store them in a -20℃ refrigerator.

[0071] 2.1.2 Extraction of different polarity fractions of *Spatholobus suberectus*

[0072] The dried total extract of *Smilax china* was suspended in pure water and extracted sequentially with petroleum ether, ethyl acetate, and n-butanol. Each organic solvent was used for extraction 3-5 times (until the extract was colorless and clear). The extracts from the same organic solvent were combined and dried under vacuum in a rotary evaporator at 60°C in a water bath. The dried extracts were then stored at -20°C for later use.

[0073] 2.1.3 Column Chromatography Separation of the n-Butanol Fraction of *Spatholobus suberectus*

[0074] The n-butanol fraction of *Spatholobus suberectus* was added to a silica gel-packed chromatography column using a wet loading method. The sample was eluted sequentially with dichloromethane, ranging from 90% to 0%, and collected in 100 mL vials.

[0075] The dichloromethane-methanol elution ratio was gradually changed from 95:5 to 5:95, with each gradient change being 10%. For sample F1, the dichloromethane-methanol elution ratio was 95:5, and elution volumes were collected in 100 mL bottles. Similarly, for sample F2, the dichloromethane-methanol elution ratio was 85:15, and elution volumes were collected in 100 mL bottles. For sample F3, the dichloromethane-methanol elution ratio was 75:25, and elution volumes were collected in 100 mL bottles. For sample F4, the dichloromethane-methanol elution ratio was 65:35, and elution volumes were collected in 100 mL bottles. The eluent ratio for sample F5 was 55:45 (dichloromethane:methanol), for sample F6 it was 45:35, for sample F7 it was 35:65, for sample F8 it was 25:75, and for sample F9 it was 15:85. Each elution volume was 100 mL, and the eluents were collected in one vial. After collection and combination, nine fractions (F1 to F9) were obtained. These fractions were analyzed by mass spectrometry and combined. The resulting nine fractions were evaporated to dryness and stored at -20°C for later use.

[0076] Based on bioactivity evaluation and preliminary component analysis, we further used preparative high performance liquid chromatography (mobile phase: acetonitrile:water = 35:75, flow rate: 4 mL / min) to finely separate and purify the chemical components in the significantly active fractions F2-F4. The target main peak was collected at a retention time of 17 min, which is compound PCP4.

[0077] 2.1.4 High-performance liquid chromatography (HPLC) preparation and separation of PCP4

[0078] F2-F4 were further separated and purified using rapid high-performance liquid chromatography (Shimadzu) on a reversed-phase YMC-Triart C18 column (250 mm × 10 mm, S-5 μm, YMC). Column temperature: 30°C; mobile phase: methanol and 0.1% formic acid aqueous solution (80:20, v / v); flow rate: 4 mL / min; the main peak corresponding to PCP4 was collected.

[0079] 2.1.5 Mass spectrometry analysis of PCP4

[0080] Sample analysis was performed using an LC-MS-8050 triple quadrupole mass spectrometer. Specific parameters were as follows: Column: Shim-pack GIST column (2.1 mm ID × 50 mm I, 2.0 μm, P / N 227-30001-02); Flow rate: 0.3 mL / min; Column temperature: 40°C; Mobile phase: water with 0.05% ammonia (solvent A) and acetonitrile containing 0.05% ammonia (solvent B); Elution program: isocratic elution with 10% solvent B for 0.5 min, followed by linear gradient elution with 10% ~ 98% solvent B for 1.5 min, held for 1 min, and then equilibrated to initial conditions over 2 min; Sample tube temperature: 10°C; Injection volume: 10 μL.

[0081] Mass spectrometry was used for analysis in negative ion mode. Electrospray ionization parameters were set as follows: ion spray voltage: -3500 V; ion source temperature: 400°C; scan range: m / z 100-2000 Da; desolvation temperature: 200°C; PCP4 collision energy: 51 eV; flow rates of nebulizing gas, heating gas, and drying gas were 2.5 L / min, 8.0 L / min, and 10.0 L / min, respectively. Multiple reaction monitoring (MRM) was used to monitor the transition from precursor ions to product ions.

[0082] 2.1.6 NMR Detection and Analysis of PCP4

[0083] The chemical structure of PCP4 was determined using 1H-NMR and 13C-NMR spectra acquired by a Bruker-400MHz nuclear magnetic resonance spectrometer. Specific parameters are as follows:

[0084] 1H-NMR: Pulse sequence: zg30, temperature: 25℃, spectral width: (SW) 20×10-6, number of scans: 16, center frequency: (01P) 6.2×10-6, scan delay (D1): 1 s, acquisition time (AO) 2.8 s, line broadening factor (LB): 0.3 Hz.

[0085] 13C-NMR: Pulse sequence: 28 pg 30, temperature: 25℃, spectral width (SW): 236 × 10⁻⁶, number of scans: 1024, center frequency (O1P): 100 × 10⁻⁶, scan delay (D1): 2 s, acquisition time (AO): 1 s, line broadening factor (LB): 1 Hz. Measurement results can be found in [reference needed]. Figure 3 See compound structure. Figure 4 .

[0086] 2.2.1 PCP4 inhibits the proliferation of NSCLC cells

[0087] In this study, the antiproliferative effect of PCP4 on A549 and H1299 cells was detected by CellTiter-Lumi Plus chemiluminescence and LDH assays. Figure 5 A. CellTiter-Lumi Plus chemiluminescence assay results showed that PCP4 significantly inhibited the cell viability of A549 and H1299 cells, with IC50 values ​​of 5.644 and 9.665 μg / mL, respectively. Correspondingly, as... Figure 5 B, LDH results showed that PCP4 significantly increased the cytotoxicity of A549 and H1299 cells. Furthermore, as... Figure 5 C and D, PCP4 also significantly inhibited colony formation in A549 and H1299 cells. Subsequently, Calcein-AM-PI cell staining was used to observe cell death in A549 and H1299 cells treated with PCP4 concentration gradients (0, 4, 6, 8, and 10 mM), and the percentage of red blood cells in the total cell count was plotted. Results are as follows: Figure 5 In experiments E and F, we found that PCP4 induced A549 and H1299 cell death in a dose-dependent manner. In conclusion, PCP4 significantly inhibited the proliferation of NSCLC cell lines A549 and H1299.

[0088] 2.2.2 PCP4 inhibits the migration of NSCLC cells

[0089] Cancer cell migration is a crucial process in tumor metastasis. Cell migration assays were used to investigate the inhibition of NSCLC cell migration by PCP4. Results were as follows: Figure 6 As shown, compared with the control group, PCP4 significantly inhibited the migration rate of A549 and H1299 cells.

[0090] 2.2.3 PCP4 inhibits the invasion of NSCLC cells

[0091] To further investigate the inhibitory effect of PCP4 on NSCLC cells, we used a Tranwell cell invasion assay to examine its ability to inhibit NSCLC cell invasion. Tranwell chambers containing A549 and H1299 cells were treated with PCP4 (0, 4, 6, 8, and 10 mM). After 24 h, cells were fixed with 4% PFA for 20 min, washed twice with PBS, and then incubated with 1% crystal violet solution for 30 min. Images were acquired using a bright-field microscope, and the number of cells that penetrated the matrix gel was plotted. The results are shown below. Figure 7 As shown, PCP4 significantly inhibited NSCLC cell invasion in a concentration-dependent manner.

[0092] 2.2.4 PCP4-induced mitochondrial damage in NSCLC cells

[0093] To further investigate the effects of PCP4 on NSCLC cells, we treated A549 and H1299 cells with PCP4 (8 mM) and PCP4 (10 mM), respectively, and used the mitochondrial membrane potential destabilizer CCCP (10 mM) as a positive control to induce mitochondrial damage. After drug treatment, mitochondria were stained with a Mito-Tracker Red fluorescent probe, and morphological changes in mitochondria were observed using confocal microscopy. The results are as follows: Figure 8 As shown, the mitochondria in the CTRL group were mainly irregularly elongated lines; the mitochondria in the CCCP and PCP4 groups were broken and irregularly round. This indicates that PCP4 induces mitochondrial damage in A549 and H1299 cells.

[0094] 10-NAO can specifically bind to cardiolipin in mitochondria and is commonly used to analyze mitochondrial quality. In this experiment, A549 and H1299 cells were treated with PCP4 (0, 4, 6, 8, and 10 μM), and then stained with a 10-NAO fluorescent probe. Images were acquired under a fluorescence microscope. The results are as follows: Figure 9 As shown, PCP4 concentration-dependently reduced the intensity of red fluorescence in cells. This indicates that PCP4 reduces mitochondrial quality in A549 and H1299 cells.

[0095] To investigate the effect of PCP4 on mitochondrial ROS levels in A549 and H1299 cells, we used the Mito-SOX-Red fluorescent probe to detect mitochondrial ROS levels. After treating A549 and H1299 cells with PCP4 (0, 4, 6, 8, and 10 mM), the cells were stained with Mito-SOX-Red, and images were acquired using a fluorescence microscope. The results are as follows: Figure 10 As shown, PCP4 upregulated the red fluorescence intensity of cells in a concentration-dependent manner. This indicates that PCP4 upregulated mitochondrial ROS levels in A549 and H1299 cells in a concentration-dependent manner.

[0096] To further investigate PCP4-induced mitochondrial damage in A549 and H1299 cells, this experiment used TMRM to detect mitochondrial membrane potential. After treating A549 and H1299 cells with PCP4 (0, 4, 6, 8, and 10 mM), the cells were stained with TMRM, and images were acquired using a fluorescence microscope. Results are as follows: Figure 11 As shown, PCP4 downregulates the red fluorescence intensity of cells in a concentration-dependent manner. This indicates that PCP4 reduces the mitochondrial membrane potential of A549 and H1299 cells in a concentration-dependent manner.

[0097] 2.2.5 PCP4-induced apoptosis in NSCLC cells

[0098] Apoptosis, also known as type I programmed cell death, plays a crucial role in inducing cancer cell death. In this experiment, we used the Annexin V-FITC / PI apoptosis detection kit to detect the effect of PCP4 on apoptosis in A549 and H1299 cells by flow cytometry, while using Gefitinib (an epidermal growth factor receptor tyrosine kinase inhibitor, EGFR-TK, Gefi) as a positive control. Figure 12 As shown, PCP4 significantly increased the apoptosis rate of A549 and H1299 cells in a concentration-dependent manner.

[0099] To further verify the role of PCP4 in inducing apoptosis in A549 and H1299 cells, we pretreated A549 and H1299 cells with Z-VAD-FMK (pan-cysteine ​​inhibitor, 10 mM) and then intervened with PCP4 for 24 h. Figure 13 As shown, Z-VAD-FMK attenuated the inhibitory effect of PCP4 on A549 and H1299 cells. This indicates that PCP4 reduces cell viability by inducing apoptosis in A549 and H1299 cells.

[0100] To further investigate whether apoptosis-related genes are involved in PCP4-induced apoptosis, MEFBAX- / - and MEF BAX+ / + cells were used in this study. The effect of different concentrations of PCP4 on the viability of MEF BAX- / - and MEFBAX+ / + cells after 24 h of treatment was detected by the MTT assay. The results are as follows: Figure 14 As shown, compared with MEF BAX- / - cells, 6, 8, and 10 μM PCP4 effectively reduced the viability of MEF BAX+ / + cells. These results suggest that PCP4-dependent BAX-induced apoptosis.

[0101] To further investigate the changes in apoptosis-related proteins induced by PCP4 in A549 and H1299 cells, Western blot was used to detect apoptosis-related proteins B-cell lymphoma / leukemia-2 (Bcl-2), Bcl-2-associated X protein (BAX), cleaved-caspase-9, and cleaved-caspase-3. The results are as follows: Figure 15 As shown, PCP4 increased the expression of Bax / Bcl-2. Simultaneously, PCP4 also upregulated the protein expression of cleaved-caspase-9 and cleaved-caspase-3. In conclusion, PCP4 induces apoptosis in A549 and H1299 cells.

[0102] In previous studies, we found that PCP4 can induce apoptosis in A549 and H1299 cells. To investigate the effect of PCP4 on normal human lung cells MRC-5, we treated A549 and MRC-5 cells with different concentrations of PCP4 and administered caspase-3 dye. Using the Incucyte live-cell imaging system, images were acquired every hour starting from 0 h. The results are as follows: Figure 16 As shown, after 3 hours of drug treatment, PCP4 at 8 μM and 10 μM induced apoptosis in A549 cells to the maximum value, while PCP4 at 8 μM did not induce apoptosis in MRC-5 cells at this time. Furthermore, the apoptosis-inducing effect of PCP4 at 10 μM on MRC-5 cells was half that of A549 cells at the same concentration. After 12 hours, PCP4 at 10 μM induced apoptosis in MRC-5 cells to the maximum value, but this was lower than the maximum value induced by PCP4 at 10 μM on A549 cells. These results indicate that PCP4 induces apoptosis in NSCLC cells, and there is a concentration difference in PCP4 at 8 μM and 10 μM in inducing apoptosis in human embryonic lung cells. At the same concentration and time, the apoptosis rate of MRC-5 cells was lower than that of A549 cells.

[0103] 2.2.6 PCP4 activates mitochondrial autophagy in NSCLC cells

[0104] To further confirm whether PCP4 activates autophagy in A549 and H1299 cells, Western blotting was first used to detect the effect of PCP4 on LC3 protein in A59 and H1299 cells. Figure 17 The results showed that PCP4 significantly increased the LC3II / LC3 ratio in A549 and H1299 cells, suggesting that PCP4 may activate autophagy in A549 and H1299 cells.

[0105] Furthermore, we transiently transfected A549 and H1299 cells with the GFP-LC3 plasmid, treated the cells with PCP4 (0, 4, 6, 8, and 10 mM) and Rap (10 mM), and acquired images using confocal microscopy. The results are as follows: Figure 18 As shown, PCP4 increases the formation of GFP-LC3 spots in cells in a concentration-dependent manner.

[0106] 2.2.7 PCP4 activation and excessive mitophagy induce apoptotic cell death in NSCLC cells

[0107] Mitophagy plays a dual role in tumor cells: moderate mitophagy promotes tumor survival, while excessive mitophagy inhibits tumor growth. To investigate whether PCP4 induces cell death through autophagy, A549 and H1299 cells were pretreated with Baf (a lysosomal inhibitor) for 2 h, followed by PCP4 intervention for 24 h. The MTT assay was used to detect the effect of PCP4 on the viability of A549 and H1299 cells with and without inhibitors. The results are as follows: Figure 19 As shown, the inhibitor Baf attenuated the inhibitory effect of PCP4 on the viability of A549 and H1299 cells. Calcein-AM-PI cell staining was used to detect the effect of PCP4 on cell death in A549 and H1299 cells with and without the inhibitor. Results are as follows... Figure 20 As shown, the inhibitor Baf attenuated PCP4-induced cell death in A549 and H1299 cells. In summary, this suggests that PCP4 induces autophagic cell death in A549 and H1299 cells.

[0108] 2.2.8 PCP4 inhibits tumor growth in A549-bearing nude mice.

[0109] Based on previous experiments, PCP4 exhibits strong anti-proliferative effects against A549 and H1299 cells. We further verified the inhibitory effect of PCP4 on NSCLC growth in A549 tumor-bearing nude mice. Starting from day 10 of tumor formation, nude mice were intraperitoneally injected with PCP4 and Gefi for 15 consecutive days. The mice were weighed and the long and short diameters of the tumors were measured every two days. After drug treatment, we found that compared with the vehicle group, the tumor volume and weight in the PCP4 and Gefi groups were significantly reduced. Figure 21 AD), while the weight of nude mice did not change significantly. Figure 21 E), while the relative weights of the heart, liver, spleen, lungs, and kidneys of nude mice did not change significantly between groups. Figure 21 F). The above results suggest that PCP4 can effectively inhibit the proliferation of A549 cells in tumor-bearing nude mice without significant toxic side effects.

Claims

1. The application of an active ingredient from *Hedyotis diffusa* in the preparation of a drug for treating tumor diseases, characterized in that, The active ingredient compound of the tiger paw grass is PCP4.

2. The application as described in claim 1, characterized in that, The tumor disease mentioned is lung cancer.

3. The application as described in claim 2, characterized in that, The lung cancer mentioned can be: non-small cell lung cancer.

4. The application as described in claim 1, characterized in that, The tiger paw grass extract is an extract of the n-butanol fraction.

5. The application as described in claim 1, characterized in that, The preparation method of the tiger paw grass extract includes the following steps: (1) Ethanol extraction of Tiger Paw Grass: Weigh out the crude powder of Tiger Paw Herb, add 10 times the amount of 75% ethanol, heat and reflux for 2 hours, heat and reflux 3 times, collect and combine the three extracts, and dry under vacuum at 60℃ to obtain the total extract of Tiger Paw Herb. (2) Extraction of the n-butanol fraction of *Spatholobus suberectus* Take the total extract of *Sedum sarmentosum* from step (1), suspend it in pure water, and extract it from the aqueous suspension of *Sedum sarmentosum* in sequence with petroleum ether, ethyl acetate and n-butanol. Extract each organic solvent 3-5 times, and combine the n-butanol extracts. Dry the extracts under vacuum at 60°C to obtain the n-butanol extract of *Sedum sarmentosum*. (3) Column chromatography separation of the n-butanol fraction of *Symplocos cuspidatum* Take the n-butanol extract of *Spatholobus suberectus* from step (2) and perform preliminary separation using a normal-phase silica gel column chromatography system. The mobile phase is a dichloromethane-methanol gradient elution system, with the elution ratio gradually changing from 85:15 to 65:35, with each gradient change being 10%. The elution volume for each elution ratio is 100 mL. After collecting and combining the eluents, evaporate the collected fractions to dryness to obtain the n-butanol active fraction of *Spatholobus suberectus* for later use. (4) Isolation and purification of the active ingredient n-butanol from *Tiger Paw Grass* Next, the n-butanol active fraction of *Sedum sarmentosum* from step (3) was taken and subjected to preparative high-performance liquid chromatography (HPLC) with acetonitrile:water = 35:75 as the mobile phase and a flow rate of 4 mL / min. The chemical components of the n-butanol active fraction of *Sedum sarmentosum* were finely separated and purified, and the target active component, namely compound PCP4, was collected at a retention time of 17 min.

6. The application as described in claim 1, characterized in that, The chemical formula of the PCP4 is: C 46 H 74 O 16 .

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