Use of a baiyou extract in the preparation of a medicament for treating a neoplastic disease

Abstract

The present application relates to provide a kind of application of Baimei extract in preparation of treating tumor disease drug, wherein, pharmacological and pharmacodynamic results show that Baimei extract has significant inhibitory effect on A549 cell proliferation, and the cytotoxicity of normal lung cell BEAS-2B is lower, which shows that Baimei extract has good anti-non-small cell lung cancer disease potential.The present application also surprisingly found that Baimei extract has obvious effect in treating breast cancer, pancreatic cancer, to further expand the new indication of the drug in clinic.

Description

Technical Field

[0001] This invention relates to the application of a bamboo shoot extract in the preparation of drugs for treating tumor diseases, which belongs to the field of new uses of pharmaceuticals. Background Technology

[0002] Globally, cancer incidence and mortality rates are rising, with lung cancer being one of the most common forms of cancer. Lung cancer is a malignant tumor with a high incidence and mortality rate, remaining a leading cause of cancer death worldwide. In recent years, the incidence and mortality rates of lung cancer have been steadily increasing, with approximately 2.5 million new cases of lung cancer diagnosed globally each year, and about 2.1 million deaths from it. Approximately 80% of lung cancer deaths are caused by smoking, but other risk factors include radon, asbestos, long-term and cumulative exposure to air pollution, especially polycyclic aromatic hydrocarbon (PAH) emissions, and a personal or family history of lung cancer.

[0003] Lung cancer is divided into two main categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), with approximately 85% of lung cancers being NSCLC. Histologically, NSCLC is mainly classified into adenocarcinoma, squamous cell carcinoma, and large cell carcinoma, with adenocarcinoma being common in both smokers and non-smokers. Early-stage NSCLC often presents with no obvious symptoms, but as it progresses, it can manifest as cough, hemoptysis, and chest pain. The prognosis for lung cancer patients is very poor because more than half of cases present as metastatic disease, and recurrence is common in patients with early-stage disease. Most patients do not experience early detection or awareness of lung cancer, leading to disease progression and missed opportunities for optimal treatment. The cell models used in this experiment are humanized NSCLC-H1299 alveolar basal epithelial cells from human lung adenocarcinoma and humanized NSCLC cells from lymph nodes.

[0004] Current Status of NSCLC Treatment: Currently, NSCLC treatment methods are based on stage and molecular characteristics, including surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. Despite some progress in treatment, the prognosis for advanced-stage patients remains poor, making early diagnosis crucial. In recent years, with the discovery of biomarkers and the rapid development of targeted therapy, early-stage patients primarily undergo surgical resection, supplemented by adjuvant chemotherapy to reduce the risk of recurrence. Locally advanced patients typically receive concurrent chemoradiotherapy, and some may undergo surgery after neoadjuvant therapy. Advanced or metastatic patients mainly rely on systemic therapy. Despite the continuous enrichment and optimization of treatment methods, long-term survival rates for patients with advanced NSCLC remain low; therefore, improving early diagnosis rates is essential. In recent years, with the deepening of molecular biology research, NSCLC treatment strategies have made significant progress due to the rapid development of targeted therapy technologies. Targeted therapies against specific molecular characteristics such as epidermal growth factor receptor (EGFR) mutations, anaplastic lymphoma kinase (ALK) rearrangements, and ROS1 gene rearrangements, such as erlotinib, afatinib, osimertinib, and crizotinib, have significantly improved the survival and quality of life of these patients. In particular, third-generation EGFR-TKIs (such as osimertinib) have not only demonstrated good clinical efficacy in patients with brain metastases but have also significantly prolonged progression-free survival (PFS) and overall survival (OS). However, the effectiveness of targeted therapy is often limited by tumor heterogeneity and acquired resistance, making the study of resistance mechanisms and the development of next-generation targeted drugs a hot research topic. Furthermore, inhibitors targeting programmed death receptor-1 (PD-1) and its ligand (PD-L1), such as pembrolizumab and nivolumab, have not only significantly prolonged the survival of patients with advanced NSCLC but have also shown relatively durable therapeutic effects. However, immunotherapy also has significant limitations, such as some patients failing to benefit, immune-related adverse events (irAEs), and drug resistance. In addition to the above treatment methods, the discovery and application of biomarkers such as liquid biopsy provide important means for dynamic monitoring of treatment response and drug resistance mechanisms

[26] . For example, the detection of circulating tumor DNA (ctDNA) can not only assist in molecular subtyping, but also monitor the generation of drug resistance mutations, such as EGFR T790M mutation, and guide subsequent treatment decisions. In general, although the treatment of NSCLC is moving from traditional single therapy to comprehensive therapy, various limitations and adverse reactions still exist widely. Therefore, it is urgent to find effective drugs for the treatment of NSCLC.

[0005] Apoptosis is a programmed cell death process and a key pathway regulating cell growth rate, proliferation, and the development of cancers such as lung cancer. Lung cancer is a result of impaired cell function or inhibited apoptosis. It is mainly activated through two pathways: the exogenous pathway and the endogenous pathway. The exogenous pathway is initiated by the activation of death receptors such as FAS, tumor necrosis factor receptor (TNFR), and TNF-related apoptosis-inducing ligand receptor (TIAR). Ultimately, it is initiated by the recruitment of caspase-8, a downstream factor and an important mediator in the exogenous pathway. Apoptosis begins when caspase-3 is activated by caspase-8. In the endogenous pathway, pro-apoptotic signal transduction leads to the release of cytochrome C into the cytoplasm, forming an apoptosis complex with activator of apoptosis protease 1 (APAF1) and an inactive form of caspase-9. This activates caspase-9, which in turn activates caspases 3, 6, and 7, leading to apoptosis. Autophagy can play both positive and negative roles in promoting apoptosis in NSCLC cells. Chemotherapy drugs and ionizing radiation often induce autophagy in lung cancer cells. While inducing autophagy has a protective effect on lung cancer cells, inhibiting autophagy can enhance the sensitivity of lung cancer cells to chemotherapy and radiotherapy. Therefore, autophagy inhibitors can be used in combination with chemotherapy or radiotherapy to treat lung cancer.

[0006] Furthermore, apoptotic proteins such as caspase-3 and caspase-9 can participate in the regulation of autophagy by targeting autophagy proteins. The mTOR signaling pathway plays a crucial role in the mechanisms of apoptosis and autophagy; inhibiting mTOR expression can promote apoptosis and autophagic cell death, while inhibition of the KRAS / RAF / MEK / ERK pathway can significantly promote apoptosis. Therefore, autophagy and apoptosis play important roles in the development, proliferation, and treatment of lung cancer.

[0007] Traditional Chinese medicine (TCM) is one of the treasure troves of medicine in our country, and it plays a vital role in the treatment of cancer. Studies have confirmed that combining TCM with radiotherapy and chemotherapy can effectively alleviate gastrointestinal reactions, fatigue, and other side effects of radiotherapy and chemotherapy, reduce the risk of tumor recurrence and metastasis, and significantly improve patients' quality of life and prolong their survival. *Disporum cantoniense* (Lour.) Merr.(DE) is a plant belonging to the genus *Disporum* in the family Liliaceae. The root of *Disporum cantoniense* is used medicinally. This plant is used to treat emphysema, nausea and vomiting, cough, and to stop bleeding. Its pharmacological effects, when administered intravenously to anesthetized frogs and rabbits, include a significant cardiotonic effect.

[0008] According to the applicant's review of existing patent literature related to *Baiweisun*, patent publication number CN113230317A describes a transdermal drug delivery preparation for treating knee osteoarthritis and its preparation method. This preparation is a microemulsion made from 20 parts of raw aconite, 15 parts of white dragon's beard (*Baiweisun*), 30 parts of black bone vine, 50 parts of *Clematis chinensis*, and 50 parts of *Spatholobus suberectus*. It is used for knee osteoarthritis, solving the defects of inaccuracy and strong pain sensation of traditional drugs, and improving the clinical treatment effect. Patent CN112741854A discloses a traditional Chinese medicine composition for treating peripheral vascular diseases and its preparation method. The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: Codonopsis pilosula 5-10 parts, Spatholobus suberectus 2-5 parts, Corydalis yanhusuo 1-3 parts, Bupleurum chinense 3-7 parts, Artemisia argyi 2-4 parts, Salvia miltiorrhiza 5-15 parts, Smilax china (white dragon beard) 10-20 parts, Artemisia argyi 2-4 parts, Pheretima aspergillum 3-5 parts, Euonymus alatus 1-5 parts, and Pueraria lobata 1-3 parts.

[0009] Baiwei bamboo shoots are a type of bamboo belonging to the genus *Dendrobium* in the family Liliaceae. Currently, there are few reports on Baiwei bamboo shoots, and their pharmacological effects mainly include antitussive and expectorant properties, lung moisturizing, anti-inflammatory, and antioxidant effects. No existing research reports have been found regarding the use of Baiwei bamboo shoots in the treatment of tumors. To further explore the antitumor activity of Baiwei bamboo shoot extracts, it is particularly important to develop this drug into a formulation with better clinical efficacy, providing more treatment options for clinical patients. Summary of the Invention

[0010] This invention provides the application of *Symplocos buergeriana* extract in the preparation of drugs for treating tumor diseases. Exploratory experimental studies have found that crude extract of *Symplocos buergeriana* has a significant inhibitory effect on the proliferation of A549 cells and low cytotoxicity to normal lung cells BEAS-2B, indicating that *Symplocos buergeriana* extract has good potential for treating non-small cell lung cancer.

[0011] Experimental results showed that the petroleum ether extract of *Ligustrum lucidum* exhibited the strongest anti-proliferative activity against A549 and H1299 cells. The results also indicated that *Ligustrum lucidum* petroleum ether extract significantly inhibited the migration, invasion, and colony formation abilities of A549 and H1299 cells. Mass spectrometry analysis and transcriptome sequencing revealed that the anti-tumor effect of DPF may be related to the synergistic effect of autophagy and apoptosis pathways. Further validation experiments showed that DPF can promote NSCLC cell death by activating autophagy and apoptosis pathways. The anti-proliferative effect of DPF on A549 and H1299 cells was significantly weakened after the addition of the ULK1 inhibitor and the pan-caspase inhibitor Z-VAD-FMK.

[0012] This invention also surprisingly discovered that the extract of baiji bamboo shoots has a significant inhibitory effect on the treatment of breast cancer and pancreatic cancer, which will further expand the new indications of this drug in clinical practice.

[0013] The technical solution provided in this invention patent application is as follows:

[0014] Application of a bamboo shoot extract in the preparation of drugs for treating tumor diseases.

[0015] Preferably, the tumor disease is non-small cell lung cancer.

[0016] Preferably, the tumor disease can be breast cancer.

[0017] Preferably, the tumor disease may also be pancreatic cancer.

[0018] Preferably, the effective dosage of the basil extract is 40 mg / kg-80 mg / kg.

[0019] Preferably, the bamboo shoot extract is: bamboo shoot petroleum ether extract.

[0020] Preferably, the preparation method of the bamboo shoot extract includes the following steps:

[0021] (1) After crushing the bamboo shoots, weigh the coarse powder, add 10 times the volume of 75% ethanol solution, heat in a water bath and reflux for extraction, heat for 2 hours, repeat this process three times. After each extraction, collect the bamboo shoot extract. Combine the three extracts and then evaporate the bamboo shoot extract to obtain the bamboo shoot extract, which is stored at -20 ℃.

[0022] (2) Take the total extract of bamboo shoots from step (1), suspend it in pure water, and then extract the aqueous suspension with petroleum ether, ethyl acetate and n-butanol in sequence. Repeat the extraction operation of each organic solvent 3 to 5 times until the extract solution is colorless and clear. Combine all petroleum ether extracts and vacuum dry them in a water bath at 60 ℃ to finally obtain the petroleum ether extract of bamboo shoots.

[0023] In this study, after screening a large number of traditional Chinese medicine compounds using A549 and BEAS-2B cell models, we obtained the crude extract DE from *Bambusa textilis*. Preliminary experimental results showed that DE significantly inhibited the proliferation of A549 cells and had no significant cytotoxicity to normal human lung cells (BEAS-2B) at high concentrations. To further explore the activity of DE, we continued to isolate DE, extracting it from water-soluble DE using petroleum ether, ethyl acetate, and n-butanol in order of increasing polarity. This yielded four fractions with different polarities: DPF, DEF, DNF, and DWF. By measuring their inhibitory activity against NSCLC cells, we found that DPF exhibited the strongest anti-NSCLC activity. Therefore, we selected DPF as the subject of further investigation in this study.

[0024] Migration and invasion are key steps in cancer metastasis, and the inhibition of migration and invasion is an important indicator for evaluating the efficacy of anti-tumor drugs. Therefore, this is crucial for DPF to become an effective treatment for NSCLC. Through experiments involving cell colony formation, cell migration, and cell invasion, we found that DPF can inhibit the migration, invasion, and cloning of A549 and H1299 cells, indicating that DPF has potential in inhibiting NSCLC cell proliferation. Therefore, DPF could be used as an anti-NSCLC drug. To further elucidate the molecular mechanism of action of DPF, we combined mass spectrometry and transcriptome sequencing to find that the anti-tumor effect of DPF may be achieved by regulating autophagy, apoptosis, and antioxidant pathways. The role of autophagy in tumor cells is complex; it can act as a cell protection mechanism, helping cancer cells survive under stress conditions, and can also promote cancer cell death under certain conditions. Apoptosis can promptly eliminate harmful, damaged, and non-functional cells. Through the combined action of both, the probability of cancer cell death can be increased, improving the therapeutic effect. Furthermore, the tumor microenvironment is usually a high-oxidative-stress environment; therefore, antioxidants are particularly important in tumor treatment.

[0025] In this experiment, to verify this hypothesis, we first selected autophagy inhibitors, apoptosis inhibitors, and ERK inhibitors for intervention. The results showed that the addition of ULK1 and the pan-casapse inhibitor Z-VAD-FMK significantly reduced the inhibitory effect of DPF on A549 and H1299 cells, while the inhibitory effects of 3-MA and CC were relatively weak. These results suggest that DPF may induce NSCLC cell death through autophagy and apoptosis, a finding further supported by flow cytometry and Western blotting. Flow cytometry results showed that A549 and H1299 cells underwent significant apoptosis after DPF administration. Furthermore, Western blotting showed that DPF upregulated the Bax / Bcl-2 ratio and upregulated the protein expression of caspase 3 and caspase 9. Therefore, these results indicate that DPF promotes NSCLC cell apoptosis through the Bax / Bcl-2 / caspase pathway. Furthermore, when DPF was applied to U87 cells stably transfected with GFP-LC3, it produced obvious fluorescent spots, and Western blot results also showed a significant increase in the LC3II / LC3I conversion rate in A549 and H1299 cells. This indicates that DPF can induce autophagy in NSCLC cells.

[0026] To further confirm the role of DPF in autophagy, we transfected A549 and H1299 cells with GFP-LC3 and observed that the number of GFP-LC3 spots increased with increasing DPF concentration. Notably, intervention with 3-MA, CC, and ULK1 significantly reduced the formation of GFP-LC3 spots, and A549 cell death was also inhibited. These results indicate that DPF can induce NSCLC cell death by activating autophagy. Furthermore, the mechanism of action of DPF may involve the MAPK signaling pathway, particularly the ERK pathway. ERK is an important member of the MAPK signaling pathway; intervention with the ERK inhibitor SCH showed that SCH inhibited the inhibitory effect of DPF on tumor proliferation, suggesting that DPF may exert its anti-tumor effect by activating the ERK pathway.

[0027] Therefore, Western blot analysis showed that DPF promotes the phosphorylation of MEK and ERK in the MEK / ERK pathway, confirming that DPF functions through this pathway. Given that ERK pathway activation may affect the AMPK pathway, and that CC and ULK1 are inhibitors of the AMPK pathway, and considering that CC and ULK1 can inhibit the anti-NSCLC effect of DPF (as mentioned earlier), we hypothesize that DPF exerts its anti-tumor effect by activating the MEK / ERK pathway and then affecting the AMPK / mTOR / ULK1 signaling pathway. In cell biology, AMPK, as a core regulator of cellular energy and metabolism, can effectively induce the crucial process of autophagy through precise regulation of multiple downstream signaling molecules. Meanwhile, the key role of mTOR in autophagy has also been confirmed. Therefore, based on Western blotting experiments, we found that DPF can promote autophagy by inhibiting the phosphorylation sites of mTOR and ULK1 at S757. Simultaneously, DPF also activated the phosphorylation of AMPK and ULK1 at S555. These results indicate that DPF induces autophagy through a pathway dependent on the AMPK / mTOR / ULK1 signaling pathway. In conclusion, DPF exerts its anti-tumor effect on NSCLC cells through the synergistic effects of the MEK / ERK, AMPK / mTOR / ULK1, and Bax / Bcl-2 / caspase pathways.

[0028] While we have preliminarily revealed that DPF functions through the MEK / ERK, AMPK / mTOR / ULK1, and Bax / Bcl-2 / caspase pathways, the specific regulatory relationship between ERK and AMPK remains unclear. For example, whether ERK directly phosphorylates AMPK or indirectly regulates the AMPK / mTOR / ULK1 pathway through other intermediate molecules requires further investigation. Therefore, future research needs to further investigate the phosphorylation levels of AMPK, mTOR, and ULK1 under DPF treatment combined with SCH intervention, and verify the integrity of autophagic flux using the RFP-GFP-LC3 dual fluorescence system to clarify whether MEK / ERK regulates autophagy through the AMPK / mTOR / ULK1 pathway.

[0029] In the field of contemporary oncology treatment, the treatment of non-SCLC still faces severe challenges. Current clinical treatment mainly relies on chemotherapy drugs (such as platinum-based and taxane-based drugs) and targeted therapies (such as EGFR-TKIs), but these therapies have significant limitations. Chemotherapy drugs, represented by cisplatin, are often accompanied by dose-limiting toxicities (such as nephrotoxicity and myelosuppression), while targeted drugs such as gefitinib are prone to causing skin and mucous membrane toxicity, and long-term use easily leads to acquired resistance, resulting in limited improvement in patient prognosis. It is worth noting that single-target apoptosis or autophagy treatment strategies have inherent defects: simple apoptosis induction is easily affected by apoptosis escape mechanisms (such as Bcl-2 family protein overexpression and caspase activity inhibition); while single-target autophagy activation may, due to autophagy-dependent survival mechanisms, enhance the tumor cells' ability to adapt to treatment stress. This study discovered that DPF exhibits a unique dual-mechanism—overcoming tumor cells' resistance to a single mode of death by synergistically activating apoptosis and autophagy pathways, thus improving clinical treatment efficacy. From a clinical application perspective, a deeper understanding of the mechanisms by which DPF induces autophagy and apoptosis is crucial for optimizing lung cancer treatment strategies. Future research could explore how to combine other drugs and gene editing technologies to enhance DPF-induced autophagy and apoptosis in NSCLC cells while avoiding drug resistance in cancer cells, thereby optimizing lung cancer treatment strategies.

[0030] The beneficial effects of the technical solution of this invention are as follows:

[0031] (1) This invention obtained four polar components of the total extract of *Bambusa textilis*: DPF, DEF, DNF, and DWF. The effects of these different polar components on the proliferation of A549 and BEAS-2B cells were detected using the MTT assay. The results showed that DNF and DWF components had almost no inhibitory effect on the proliferation of A549 cells, while petroleum ether and ethyl acetate components showed strong inhibitory effects. To further screen for active components, we conducted repeated proliferation experiments in the H1299 cell model. The results showed that the petroleum ether component had a significant inhibitory effect on both A549 and H1299 cells, while the ethyl acetate component had a relatively weak inhibitory effect on H1299 cells.

[0032] (2) Pharmacological experiments showed that: ① DPF significantly inhibited colony formation in A549 and H1299 cells, as demonstrated by Hoechst / PI double staining. The petroleum ether extract of *Scutellaria baicalensis* significantly increased the proportion of PI-positive cells in A549 cells, indicating a significant pro-cell death effect. Similar changes were observed in H1299 cells, further demonstrating the cytotoxic effect of DPF on these two NSCLC cell types. Furthermore, we evaluated the anti-proliferative effects of different concentrations (200, 100, 50, 25 μg / mL) of DPF on A549 and H1299 cells using real-time cell viability monitoring. The results showed that DPF inhibited the viability of A549 and H1299 cells at different concentrations, indicating that DPF significantly reduced cell proliferation. These results combined demonstrate that DPF can effectively inhibit the proliferation of A549 and H1299 cells with good safety.

[0033] ② The diffusion rate of A549 and H1299 cells in the DPF-treated group was significantly slowed, and the mass migration of cells was significantly inhibited. This indicates that DPF can effectively inhibit the migration ability of NSCLC cells. Since ERK inhibitors can significantly reduce the effect of DPF, this suggests that activation of the ERK signaling pathway plays a key role in the process of DPF inhibiting NSCLC cell proliferation. In addition, ERK activation may further inhibit mTOR activity by phosphorylating AMPK or regulating upstream molecules of mTOR (such as TSC2), thereby relieving its negative regulatory effect on autophagy and promoting the initiation of autophagy and the formation of autophagosomes. Combining the results previously confirmed that DPF can induce autophagy in NSCLC cells, we speculate that DPF may first exert its effect by activating the MEK / ERK signaling pathway, then regulating the AMPK / mTOR / ULK1 signaling pathway, and finally inducing autophagy.

[0034] ③ DPF treatment significantly upregulated the protein expression of MEK and ERK in A549 and H1299 cells. This result preliminarily supports the possibility that DPF activates the MEK / ERK signaling pathway, thereby initiating the downstream AMPK / mTOR / ULK1 pathway and inducing autophagy. However, future research needs to further verify whether ERK directly regulates the activity of AMPK or mTOR to clarify its upstream regulatory mechanism. These results indicate the antitumor mechanism of the *Bambusa textilis* extract of this invention.

[0035] DPF can induce autophagy through the AMPK / mTOR / ULK1 signaling pathway. Furthermore, apoptosis also plays a crucial role in cancer cell death. We treated A549 and H1299 cells with the pan-cysteine ​​inhibitor Z-VAD-FMK (10 μM) and DPF (200 μg / mL). In our experiments, we first pretreated the cells with the inhibitor, and then assessed cell viability using the MTT assay 24 hours after treatment. The results showed that DPF could induce apoptosis in both A549 and H1299 cells.

[0036] (3) These results indicate that DPF can effectively inhibit the proliferation of A549 cells in tumor-bearing nude mice. To further verify the inhibitory effect of DPF on tumor growth in A549-bearing nude mice, we performed H&E staining and TUNEL assays on tumor tissues. The experimental results are as follows: Figure 11 As shown, tissue sections treated with DPF and Gefi exhibited inhibited tumor cell proliferation and a significant increase in the number of apoptotic cells. These results indicate that PCP4 has a significant inhibitory effect on tumor growth in A549 tumor-bearing nude mice. Immunohistochemical analysis was performed on tumor tissues to detect the protein expression of LC3, ERK, mTOR, and Bax. The results showed that DPF upregulated the expression levels of these proteins in a concentration-dependent manner. This study demonstrates that the mechanism of action of DPF is consistent with the in vitro results, inhibiting tumor growth by promoting autophagy and inducing apoptosis. These results also suggest that *Bambusa textilis* extract has certain inhibitory and therapeutic effects on breast cancer and pancreatic cancer. Attached Figure Description

[0037] 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.

[0038] Figure 1(A) Preliminary screening of drugs that inhibit A549 proliferation. (B) Preliminary screening of drugs that inhibit BEAS-2B proliferation. (C, D) Effects of bamboo shoots on the activity of A549 and BEAS-2B. *p<0.05, **p<0.01, ***p<0.001;

[0039] Figure 2 (AD) Effects of the four polar sites on the viability of A549 and BEAS-2B cells; (E) Effect of DPF on the viability of H1299 cells. Note: Compared with the control group, *p<0.05, **p<0.01, ***p<0.001.

[0040] Figure 3 DPF can inhibit the proliferation and colony formation of NSCLC cells; (A, B) the effect of DPF on the colony formation of A549 and H1299 cells; (CE) after 24 h of DPF treatment, the death of H1299 and A549 cells was observed using Hoechst / PI cell staining, and images were acquired using a fluorescence microscope, followed by statistical graphs to present the specific percentage of cell death; (F, G) real-time monitoring of the effect of DPF on the activity of A549 and H1299 cells;

[0041] Figure 4 Effects of DPF on the migration of A549 (A) and H1299 (B) cells. Note: Compared with the control group, *p<0.05, **p<0.01, ***p<0.001, scale bar: 200μm (10´);

[0042] Figure 5 SCH reduced the inhibitory effect of DPF on A549. Note: Compared with the control group, *p<0.05, **p<0.01, ***p<0.001;

[0043] Figure 6 Effects of DPF on the expression of MEK / ERK pathway-related proteins in A549 and H1299 cells;

[0044] Figure 7 Effects of DPF on LC3 spot formation in H1299 cells under CC, ULK101, and SCH intervention; Note: ***p<0.001 compared with the control group, scale bar: 5 μm (63´);

[0045] Figure 8 Flow cytometry showed that DPF induced apoptosis in A549 and H1299 cells in a concentration-dependent manner. Note: Compared with the control group, ***p<0.001;

[0046] Figure 9Effects of Z-VAD-FMK on DPF's inhibition of NSCLC cell activity; Effects of Z-VAD-FMK on DPF's inhibition of A549 activity;

[0047] Figure 10 DPF and Gefi inhibited tumor growth in A549-bearing nude mice; (A) Tumor weight measurements showing the effect of 15-day DPF and Gefi treatment; (B) Tumor volume changes monitored during DPF and Gefi administration; (C) Tumor weight measurements during DPF and Gefi treatment; (D) Relative organ weight distribution in nude mice measured 15 days after treatment.

[0048] Figure 11 TUNEL and H&E staining of tumor tissue from A549 tumor-bearing nude mice, scale bar: 200 μm (10×);

[0049] Figure 12 Effects of DPF on the expression of LC3, ERK, mTOR and BAX in tumor tissues of A549 tumor-bearing nude mice; Note: Compared with the control group, *p<0.05, **p<0.01, ***p<0.001, scale bar: 100 μm (20×). Detailed Implementation

[0050] To better understand the implementation of this invention and to enable those skilled in the art to comprehend it, the relevant pronouns involved in this invention are explained below. Specifically, 3-MA represents 3-methyladenine, 4%PFA represents 4% paraformaldehyde, DPF represents *Scutellaria baicalensis* petroleum ether extract, DE represents *Scutellaria baicalensis* crude extract, DEF represents *Scutellaria baicalensis* ethyl acetate extract, DNF represents *Scutellaria baicalensis* n-butanol extract, DWF represents *Scutellaria baicalensis* water extract, Gefi represents gefitinib, H&E represents hematoxylin and eosin staining, NSCLC represents non-small cell lung cancer, PI represents propidium iodide, WB represents Western blotting, and TdT represents terminal deoxyribonucleotide transferase. An experimental example is given below, which is only a typical embodiment to further illustrate this invention. It should be noted that unless specific experimental conditions are specified, all results can be obtained under conventional conditions. If the manufacturer of the reagents used is not specified, they are products that can be obtained from the conventional market.

[0051] Example 1: The effect of DPF on inducing apoptosis and autophagy, and inhibiting NSCLC cell proliferation.

[0052] 1. Experimental materials, reagents and instruments

[0053] 1.1 Bamboo shoots, purchased from Chengdu Hehuachi Medicinal Herbs Market

[0054] 1.2 Experimental cell lines

[0055] A549 cells, H1299 cells: American Type Culture Collection (ATCC), MEF BAX + / + Cells (wild-type mouse embryonic fibroblasts): Professor Masaaki Komatsu, Juntendo University, Japan, MEF BAX - / - Cells (wild-type mouse embryonic fibroblasts): donated by Professor Masaaki Komatsu of Juntendo University, Japan.

[0056] 1.3 Main reagents for the experiment

[0057] 1640 medium, Gibco, DMEM, Gibco, fetal bovine serum, Pronosex, trypsin cell digestion solution, Guangzhou Shuopu Biotechnology, penicillin-streptomycin-gentamicin solution (100×), Beyotime, phosphate buffer (PBS), Seville, DMSO, Seville, Hoechst 33342, Aladdin, propidium iodide (PI), Solarbio, RIPA buffer (10×), Solarbio, CellTiter-Lumi Plus, Beyotime, Rapamycin, Compound C, Taoshu, Striping buffe, Beijing Sizhengbai Biotechnology, 4% paraformaldehyde fixative, Guangzhou Shuopu Biotechnology, primary antibody dilution buffer, secondary antibody dilution buffer, Beyotime, GAPDH, Caspase-3, caspase-9, Bax, Bcl-2, LC3, p-ERK, ERK, Wuhan Sanying, p-AMPK, AMPK, p-ULK1 (S555), ULK1, p-MEK, CST, trichrome pre-stained marker, Bradford protein assay solution, crystal violet staining solution, 10X electrophoresis buffer, CellTiter-Lumi Plus, one-step PAGE gel rapid preparation kit (12.5%, 10%), YARN, protein buffer loading buffer, sodium dodecyl sulfate (SDS), tris(hydroxymethyl)aminomethane (Tris), glycine, Tween 20, Solarbio, Opti-MEM, Gibco, Lipo3000, Invitrogen, PVDF membrane, Merck Milipor, Annexin VAlexa Fluor488 / PI apoptosis detection kit, Beijing Sizhengbo Biotechnology Co., Ltd.

[0058] 2 Experimental Methods

[0059] 2.1 Isolation and Identification of DPF

[0060] 2.1.1 Preparation of DPF extract

[0061] After crushing the bamboo shoots, weigh out an appropriate amount of coarse powder and add 10 times its volume of 75% ethanol solution. Extract the solution using a water bath heating and reflux method, continuing heating for 2 hours. Repeat this process three times, collecting the liquid in the same evaporating dish after each extraction. After completing three extractions, place the evaporating dish in a constant temperature water bath until the solvent is completely evaporated. Then, scrape the extract from the evaporating dish, transfer it to an EP tube, and store it in a -20 ℃ freezer.

[0062] 2.1.2 Extraction of different polar fractions of bamboo shoots

[0063] A certain amount of the total extract of dried bamboo shoots was taken, suspended in pure water, and then extracted sequentially with petroleum ether, ethyl acetate, and n-butanol. The extraction process was repeated 3 to 5 times for each organic solvent until the extract was colorless and clear. All extracts for each solvent were combined and vacuum dried using a rotary evaporator in a 60 °C water bath to obtain four fractions. All fractions should be stored separately at -20 °C for later use.

[0064] 2.2 Cell Experiments

[0065] 2.2.1 Cell Culture

[0066] Cell resuscitation: Remove the well-preserved cell samples from the liquid nitrogen container and quickly thaw them in a 37°C water bath for approximately 1 minute. Add the thawed cell suspension to a centrifuge tube containing pre-prepared complete culture medium, mix well, and centrifuge at 800g for 3 minutes, discarding the supernatant. Then, add an appropriate amount of complete culture medium to resuspend the cells, mix thoroughly, and transfer to a culture flask. Incubate at 37°C with 5% CO2 for further culture.

[0067] Cell culture medium change: Remove the cell culture flask from the 37 ℃, 5% CO2 incubator and carefully discard the culture supernatant. Then, gently wash the cells twice with an appropriate amount of sterile PBS to ensure the removal of any residual old culture medium. After washing, add fresh complete culture medium. Finally, return the culture flask to the 37 ℃, 5% CO2 incubator for continued culture.

[0068] Cell passage: When the cell density in the culture flask reaches approximately 80%, remove the flask from the 37°C, 5% CO2 incubator, discard the culture supernatant, and wash the cells twice with sterile PBS. Next, add an appropriate amount of trypsin to digest the cells. After the cells have completely detached, add double the volume of complete culture medium to terminate the digestion reaction. Transfer the cell suspension to a 15mL centrifuge tube, centrifuge at 800g for 3 minutes, and discard the supernatant. Then, resuspend the cells in an appropriate amount of complete culture medium and aliquot them into 2-3 new culture flasks as needed for continued culture.

[0069] Cell cryopreservation: Remove healthy cells from a 37 ℃, 5% CO2 incubator, digest with trypsin, centrifuge, and discard the supernatant. Add an appropriate amount of cryopreservation solution and quickly mix the cell suspension. Aliquot the treated cell suspension into cryovials. Freeze the cryovials overnight at -80 ℃, then transfer them to liquid nitrogen for long-term storage.

[0070] 2.2.2 Cell Seeding Plate

[0071] Prepare the necessary materials for the experiment in a clean bench, including pipettes, 15 mL centrifuge tubes, cell culture flasks, well plates, counting chambers, and glass slides. Remove the cell culture flasks from the 37 ℃, 5% CO2 incubator and observe whether the cell density has reached 80%. If it has, digest the cells, centrifuge, and discard the supernatant. Then, resuspend the cells in complete culture medium, mix well, and take 10 μL of the cell suspension for counting with a counting chamber. Seed the cells into the plate according to the experimental requirements. The routine seeding conditions are as follows: 96-well plate: Add 100 μL of complete culture medium to each well, and seed 3000-5000 cells per well. 12-well plate: Add 1 mL of complete culture medium to each well, and seed 50,000-100,000 cells per well. 6-well plate: Add 2 mL of complete culture medium to each well, and seed 150,000-200,000 cells per well. If cell spread assays are required, place the glass slides at the bottom of the wells before seeding, and then add the cell suspension for culture.

[0072] 2.2.3 Cell transfection

[0073] When cells have fully adhered to the plate 24 hours after seeding and are observed to be in good condition with 60-70% confluence under a microscope, transfection can be performed. Using a 12-well plate as an example, Lipofectamine 3000 (Thermo Fisher Scientific, USA) is used for transfection. Prepare two sterile EP tubes (labeled A and B) in a clean bench, adding 300 μL of Opti-MEM medium to each tube. Add an appropriate amount of plasmid (1 μg per well) and p3000 reagent (2 μL per well) to tube A, and add Lipofectamine 3000 reagent (1 μL per well) to tube B. Gently tap both tubes to mix and let stand for 10 minutes. Transfer the solution from tube B to tube A, mix gently, and let stand for another 15 minutes. After the mixture has settled, add 50 μL of the solution from tube A to each well of a plate containing 500 μL of complete medium. Eight hours after transfection, replace the medium with fresh complete medium and continue culturing.

[0074] 2.2.4 Reagent Preparation

[0075] The drug solutions were prepared as follows: the total extract of *Scutellaria baicalensis* and the extracts of its four polar fractions were added to DMSO to prepare a stock solution of 100 mg / mL; Rapamycin (Rap) was added to DMSO to prepare a stock solution of 10 mM; SCH was added to DMSO to prepare a stock solution of 10 μM; Chloroquine (CC) was added to DMSO to prepare a stock solution of 10 mM; and Z-VAD-FMK (ZVF) was added to DMSO to prepare a stock solution of 10 mM.

[0076] The complete culture medium formula is as follows: 20% fetal bovine serum, 2% penicillin-streptomycin-gentamicin solution (100×), and 78% basal culture medium.

[0077] The preparation method for PBST is as follows: Add one packet of PBS powder to 2 L of pure water, then add 2 mL of Tween 20, and mix thoroughly by sonication until completely dissolved. MTT powder: The ratio of powder to pure water is 5 mg : 1 mL. 1× Electrophoresis buffer: Add 900 mL of pure water to 100 mL of 10× electrophoresis buffer. 10× Transfer buffer: Dilute 30.3 g of Tris and 151.1 g of glycine to 1000 mL of pure water and sonicate thoroughly. 1× Transfer buffer: Add 200 mL of methanol and 700 mL of pure water to 100 mL of 10× electrophoresis buffer.

[0078] 2.2.5 MTT test method

[0079] The MTT assay is a commonly used method for detecting cell proliferation and cytotoxicity, based on the activity of mitochondrial dehydrogenases within cells. This method involves the reduction of MTT reagent within cells to form deep purple formazan crystals, which can be completely dissolved in a specific solvent. The absorbance is then measured at 570 nm using a microplate reader; the absorbance value is directly proportional to cell viability. The specific procedure is as follows: After drug treatment, add 10 μL of 5 mg / mL MTT solution to each well in a 96-well plate and incubate for 2-4 hours. During this time, mitochondrial dehydrogenases in the living cells reduce MTT to purple formazan. After discarding the culture medium, add 100 μL of DMSO to each well to dissolve the formazan, forming a solution for absorbance measurement. Finally, measure the absorbance (OD value) of each well at 570 nm using a microplate reader. The absorbance value reflects the cell proliferation level or viability.

[0080] 2.2.6 Hoechst / PI staining method

[0081] Hoechst 33342: A cell membrane-permeable fluorescent dye that can enter both live and apoptotic cells, specifically binding to DNA and emitting blue fluorescence. Apoptotic cells show enhanced nuclear staining intensity and exhibit typical morphology such as nuclear fragmentation and chromatin condensation. PI: A cell membrane-insoluble fluorescent dye that cannot enter intact live cells, but can enter necrotic cells with impaired membrane integrity or late-stage apoptotic cells, emitting red fluorescence after binding to DNA. Cells are seeded in culture plates, and after cell adhesion, drugs are added for intervention or treatment according to the experimental design. Hoechst dye working solution (final concentration 5-10 μg / mL) and PI dye working solution (final concentration 1-5 μg / mL) are prepared, ensuring the dyes are freshly prepared. The culture medium is discarded, and the cells are gently washed once with PBS. An appropriate amount of staining working solution containing Hoechst and PI is added to each well. The cells are incubated in the dark for 10-15 minutes (37 ℃). Cells are observed using a fluorescence microscope.

[0082] 2.2.7 Cell colony formation experiment

[0083] Seed A549 and H1299 cells into culture dishes, 500-1000 cells per well or dish. Add sufficient complete culture medium (e.g., 2 mL per well in a 6-well plate), gently mix to ensure uniform cell distribution. After cell attachment (usually 24 hours), treat with the drug. Then, place the culture dishes in a drug-free 37°C, 5% CO2 incubator and continue culturing for 7-14 days until visible cell colonies (generally larger than 50 μm in diameter) form. After culturing, discard the culture medium and gently wash the cells 1-2 times with PBS. Fix the cells with 4% paraformaldehyde and incubate at room temperature for 15 minutes. Subsequently, stain with 1% crystal violet solution for 30 minutes. After washing the cells with PBS and air-drying, image the entire culture dish for analysis.

[0084] 2.2.8 Cell migration experiment

[0085] In this method, a scratch is made in a cell monolayer, and the migration of cells to the wound area is observed to calculate the wound healing rate. This is a commonly used in vitro experiment to assess the migration ability of cells under specific conditions. First, the cells to be tested are seeded in 6-well plates, and the cell density is adjusted to form a dense monolayer (confluence greater than 90%) within 24 hours. Then, a straight line is vertically scratched in the cell monolayer using a sterile pipette tip or scratcher. The cells are gently washed with PBS to remove cells detached from the scratch. The culture medium is replaced with a drug-containing medium (serum-free or low-serum medium can be used to avoid excessive cell proliferation interfering with the results). Initial images of the scratched area (0 hours) are taken using a microscope, and the culture plate is then placed in a 37°C, 5% CO2 incubator for further culture. The scratched area is photographed again after 24 hours and 48 hours to observe cell migration.

[0086] 2.2.9 Transwell cell invasion assay

[0087] The Transwell cell invasion assay is a classic in vitro method used to assess the ability of cells to invade through matrix gel or similar barriers. In a Transwell chamber, the upper surface of the membrane is covered with a substance mimicking the extracellular matrix (such as Matrigel). Cells must break down the matrix and pass through the porous membrane to migrate to the lower chamber. The invasive ability of cells is reflected by counting the number of cells that cross the matrix to reach the lower membrane surface. In this experiment, we used a 12-well plate as an example. First, the serum-free medium was pre-cooled, and Matrigel was diluted 1:6. 20 μL of diluted Matrigel was added to each well, and the Transwell chamber was placed in a sterile 12-well plate and incubated at 37°C for 2 hours to ensure Matrigel solidification. Next, the 12-well plate was removed and hydrated with serum-free medium for 10 minutes. Subsequently, A549 and H1299 cells were seeded into the upper chamber at a density of 50,000 per well, while serum-containing medium and diluted drugs were added to the lower chamber. After 24 hours, when the cells migrated to the lower chamber, the matrix gel and cells in the upper chamber were gently wiped away with a cotton swab. The chamber was then air-dried at room temperature, followed by fixation with 4% paraformaldehyde (PFA) for 20 minutes, and then washed twice with PBS. Afterward, the cells were incubated with 1% crystal violet solution for 30 minutes, and excess crystal violet was removed with PBS. Finally, images of the Transwell chamber were acquired using a TS2R inverted microscope.

[0088] 2.2.10 Flow cytometry

[0089] The basic principle of flow cytometry for detecting apoptosis is based on the phenomenon of phosphatidylserine (PS) in the cell membrane flipping from the inside to the outside during apoptosis. In healthy living cells, PS is usually located on the inside of the cell membrane, while in apoptotic cells, PS is exposed on the outside of the cell membrane, becoming one of the markers of apoptosis. Annexin V is a Ca²⁺-dependent phospholipid-binding protein that specifically binds to PS. After being labeled with fluorescein (such as FITC or PE) or biotin, Annexin V can be used as a probe to detect whether cells have undergone apoptosis using flow cytometry or fluorescence microscopy. In addition, propidium iodide (PI) is a cell membrane-penetrating dye that binds to DNA, turning the nuclei of apoptotic or necrotic cells red.

[0090] The following are the specific steps using a 6-well plate as an example: After drug treatment, first centrifuge and digest the cells, collecting them into EP tubes. Discard the supernatant after centrifugation. Add 100 μL of pre-diluted 1× binding buffer to each tube and gently pipette to mix. Then, add 5 μL of labeled Annexin V / Alexa Fluor 488 and 5 μL of PI solution to each tube sequentially. After incubating at room temperature in the dark for 10 minutes, add 400 μL of 1× binding buffer to terminate the staining reaction and prepare the sample for detection. Simultaneously, prepare two tubes containing only 5 μL of Annexin V / Alexa Fluor 488 or 5 μL of PI solution as single-stain control samples. Additionally, prepare a blank tube without any reagents to adjust the voltage of the flow cytometer. After staining, transfer the cell suspension to flow cytometry tubes with cell filters and immediately perform flow cytometry analysis.

[0091] 3. Experimental Results

[0092] 3.1 Screening for the inhibitory activity of bamboo shoot extract on A549 cell proliferation

[0093] In this study, we used A549 and BEAS-2B cells as models. Figure 1 A shows the effects of these drugs on A549 cells, with darker colors indicating lower activity. Most drugs showed varying degrees of inhibitory effects on cell proliferation at this concentration, with *Bambusa textilis* (white arrow) showing a particularly significant inhibitory effect on A549 cells. Meanwhile, Figure 1 B demonstrates the effects of these drugs on BEAS-2B cells. Compared with A549 cells, *Bambusa textilis* had almost no significant effect on BEAS-2B cells, showing good selectivity. Figure 1 C and Figure 1D shows the effect of bamboo shoots on the viability of A549 and BEAS-2B cells using the MTT assay.

[0094] 3.2 Comparison of the inhibitory activities of four fractions of total extract of bamboo shoots on A549 cell proliferation

[0095] First, we obtained four polar components of the total extract of *Bambusa textilis*: DPF, DEF, DNF, and DWF. The effects of these different polar components on the proliferation of A549 and BEAS-2B cells were detected using the MTT assay. Figure 2 AD analysis showed that the DEF, DNF, and DWF components had almost no inhibitory effect on the proliferation of A549 cells, while the petroleum ether component showed a strong inhibitory effect. To further screen for active components, we performed repeated proliferation experiments in the H1299 cell model. Figure 2 E demonstrated that the petroleum ether fraction (DPF) had a significant inhibitory effect on H1299 cells. Based on these screening results, we selected the petroleum ether fraction (DPF) as the focus of further research.

[0096] 3.3 DPF inhibits the proliferation of NSCLC cells.

[0097] In this study, Figure 3 A and 3B show the significant inhibitory effect of DPF on colony formation in A549 and H1299 cells. To further verify the inhibitory effect of DPF, we used the Hoechst / PI double staining method to detect cell death. Figure 3 The results of C showed that DPF treatment significantly increased the proportion of PI-positive cells in A549 cells, suggesting that DPF has a significant pro-cell death effect. Similar changes were observed in H1299 cells, further demonstrating the cytotoxic effect of DPF on these two NSCLC cell types. Furthermore, we evaluated the anti-proliferative effects of different concentrations (200, 100, and 50 μg / mL) of DPF on A549 and H1299 cells using real-time cell viability monitoring. Figure 3 F and 3G show the inhibitory effect of DPF on the viability of A549 and H1299 cells at different concentrations, indicating that DPF significantly reduces cell proliferation. These results combined demonstrate that DPF can effectively inhibit the proliferation of A549 and H1299 cells and exhibits good cytotoxicity.

[0098] 3.4 Inhibitory effect of DPF on NSCLC cell migration

[0099] Cancer cell migration is a key step in the tumor metastasis process. To investigate the effect of DPF on NSCLC cell migration, this study conducted cell migration experiments. Figure 4The results showed that, compared with the control group, the diffusion rate of A549 and H1299 cells in the DPF-treated group was significantly slowed down, and the mass migration of cells was significantly inhibited. This indicates that DPF can effectively inhibit the migration ability of NSCLC cells.

[0100] 3.5 DPF activates the MEK / ERK pathway

[0101] Previous studies suggested that DPF might function through the MAPK signaling pathway. Since ERK (extracellular signal-regulated kinase) is a key member of the MAPK signaling pathway, we used the ERK inhibitor SCH to further explore the regulatory mechanism of DPF. In this experiment, we treated A549 cells with DPF (200 μg / mL) and different concentrations of SCH (5, 10, and 20 μM). The experimental results are shown below. Figure 5 This indicates that SCH can significantly attenuate the inhibitory effect of DPF on NSCLC cell activity, further supporting the key role of ERK in DPF-regulated NSCLC cell activity.

[0102] Since ERK inhibitors significantly reduce the effect of DPF, this indicates that activation of the ERK signaling pathway plays a crucial role in DPF's inhibition of NSCLC cell proliferation. Furthermore, ERK activation may further inhibit mTOR activity by phosphorylating AMPK or regulating upstream molecules of mTOR (such as TSC2), thereby relieving its negative regulatory effect on autophagy and promoting the initiation of autophagy and the formation of autophagosomes. Combined with the previously confirmed results that DPF can induce autophagy in NSCLC cells, we hypothesize that DPF may initially exert its effects by activating the MEK / ERK signaling pathway, subsequently regulating the AMPK / mTOR / ULK1 signaling pathway, and ultimately inducing autophagy.

[0103] To verify this hypothesis, we used Western blotting to detect the protein expression levels of MEK / ERK in DPF-treated NSCLC cells. Figure 6 As shown, DPF treatment significantly upregulated the protein expression of MEK and ERK in A549 and H1299 cells. This result initially supports the possibility that DPF activates the MEK / ERK signaling pathway, thereby initiating the downstream AMPK / mTOR / ULK1 pathway and inducing autophagy. However, future research needs to further verify whether ERK directly regulates the activity of AMPK or mTOR to clarify its upstream regulatory mechanism.

[0104] To further verify the hypothesis that DPF regulates autophagy through the MEK / ERK signaling pathway, we transiently transfected H1299 cells with GFP-LC3 and intervened with the MEK / ERK inhibitor SCH, then examined the effect of DPF on autophagy spot formation in the cells. The results are as follows: Figure 7 As shown, SCH significantly inhibited DPF-induced LC3 spot formation, indicating that the MEK / ERK signaling pathway plays a key role in DPF-induced autophagy.

[0105] 3.6 DPF induces apoptosis in NSCLC cells

[0106] As explained above, DPF can induce autophagy through the AMPK / mTOR / ULK1 signaling pathway. Furthermore, apoptosis also plays a crucial role in cancer cell death. Therefore, in this experiment, we used the Annexin V-FITC / PI apoptosis detection kit and flow cytometry to detect apoptosis in A549 and H1299 cells under DPF treatment. The results are shown below. Figure 8 .

[0107] To further verify the effect of DPF on NSCLC cell apoptosis, we treated A549 cells with a combination of the pan-cysteine ​​inhibitor Z-VAD-FMK (10 μM) and DPF (200 μg / mL). In the experiment, we first pretreated the cells with the inhibitor, and then assessed cell viability using the MTT assay 24 hours after treatment. The experimental results are as follows: Figure 9 As shown, this indicates that DPF can induce apoptosis in A549 cells.

[0108] Example 2: DPF inhibits tumor growth in tumor-bearing mice in vivo (involving breast cancer and pancreatic cancer).

[0109] Experimental Materials and Methods

[0110] 1. Experimental drugs, reagents and instruments

[0111] 1.1 Experimental Drugs

[0112] DPF (same as Part 1); Gefitinib (Gefi) was purchased from Taoshu.

[0113] 1.2 Experimental Reagents

[0114] Physiological saline solution.

[0115] 1.3 Experimental Apparatus

[0116] Clean workbench, Suzhou Antai Air Technology Co., Ltd., high-precision electronic scale, electronic vernier caliper, Tecos (Guangzhou) Technology Service Co., Ltd., Yantai Green Forest Tools, constant temperature and humidity incubator, Elburn China Co., Ltd., TS2R-inverted fluorescence microscope, Nikon Instruments (Shanghai) Co., Ltd.

[0117] 2 nude mice balb / c-nu

[0118] Male nude mice, purchased from Chengdu Yaokang Biotechnology Co., Ltd., were used in this experiment. The nude mouse phenotype is caused by a mutation in the Foxn1 gene. Because Foxn1 plays a crucial role in the development of the thymus and hair follicles, when the Foxn1 gene is mutated and in a homozygous state, the mice will exhibit the nude mouse phenotype: hairlessness and immunodeficiency.

[0119] 3 Experimental Methods

[0120] 3.1 Reagent Preparation

[0121] DPF was prepared as suspensions of 40 and 80 mg / kg using dd water:PEG:DMSO = 5:4:0.5; Gefi was prepared as a suspension of 20 mg / kg using dd water:PEG:DMSO = 5:4:0.5.

[0122] 3.2 Subcutaneous tumors in nude mice

[0123] Because of a genetic mutation in nude mice, they lack mature T cells and therefore do not exhibit a significant rejection response to transplanted tumors or other cells. Tumor cells are inoculated into the armpits of nude mice, where they absorb nutrients and proliferate rapidly to form measurable tumor tissue. The formation and growth of these tumors are then observed and monitored to assess tumor cell proliferation and the effectiveness and applicability of anti-tumor drug treatments.

[0124] The experiment typically uses nude mice aged 4-6 weeks. After one week of SPF culture to allow the mice to acclimatize, A549 cells are injected into the left axilla of each mouse at a rate of 1-3 million per mouse. During and after injection, the injection site is observed for any lumps or ulceration. Continuous observation for 7-10 days is maintained, and the flank of the mouse is examined for tumor formation. Drug treatment begins when the tumor reaches approximately 1 cm in length.

[0125] 3.3 Treatment of xenografts in nude mice

[0126] Nude mice successfully inoculated with A549 cells were divided into four groups: a negative control group, an experimental group (DPF 40 mg / kg, 80 mg / kg), and a positive control group (Gefi 20 mg / kg). Once the tumors had grown to a certain size, the prepared drug was administered intraperitoneally daily for two weeks. From the first administration, the mice were weighed every two days, and the length (a) and width (b) of the tumor were measured with calipers and recorded. Two weeks later, the mice were euthanized by cervical dislocation, and the tumors were removed, weighed, and their volume calculated. The removed tumors were then placed in EP tubes, 4% paraformaldehyde was added, and the tubes were frozen at -80°C for subsequent experiments. The tumor volume was calculated using the formula V = a × b. 2 ×1 / 2

[0127] 3.4 Hematoxylin-eosin staining method

[0128] Hematoxylin and eosin staining (H&E staining) is a widely used tissue section staining technique in pathology and histology. It can be used to observe the morphology and structure of tissues and the composition of cells. Hematoxylin is a basic dye that binds to DNA / RNA in cells, producing a blue-purple color. Eosin, an acidic dye, binds to the basic components of the cytoplasm, staining the cytoplasm, erythrocytes, and extracellular matrix pink. Therefore, it can reveal the general structural characteristics of various tissue or cellular pathologies.

[0129] 3.5 TUNEL fluorescent staining

[0130] TUNEL staining is a molecular biology technique used to detect apoptosis. The principle is that during apoptosis, the activation of endogenous nucleases leads to DNA fragmentation, resulting in numerous single-stranded or double-stranded breaks. TUNEL technology uses terminal deoxynucleotidyl transferase (TdT) to incorporate fluorescently labeled dUTP (deoxyuridine triphosphate) into the 3'-OH ends of the DNA breaks, allowing detection of apoptosis via fluorescence microscopy or flow cytometry. However, this staining method is rarely effective on normal cells without DNA breaks.

[0131] 3.6 Immunohistochemistry

[0132] Immunohistochemistry is a technique that utilizes the specific binding of antigens and antibodies to identify and locate target proteins in tissue sections or cell samples using enzyme-labeled or fluorescently labeled antibodies. It also uses signal amplification and visualization to detect the distribution and expression of target proteins in tissue sections or cell samples.

[0133] After fixing tumor tissue sections, several processing steps are required before acquiring images using a digital slide scanner. First, cell membranes are permeable by adding 1% Triton X-100, creating conditions for antibody entry into cells. Next, the sections are placed under high temperature and high pressure for antigen retrieval to restore antigenic epitopes. Then, the sections are incubated with a specific primary antibody, washed, and then incubated with a secondary antibody. After incubation, the sample is eluted again, and a chromogenic substrate is added to detect the expression signal of the target protein. Finally, the samples are mounted to prepare for subsequent scanning and analysis.

[0134] Experimental results

[0135] 1. DPF inhibits tumor growth in A549-bearing nude mice.

[0136] Based on previous experimental results, we found that DPF has a significant anti-proliferative effect on A549 and H1299 cells. Therefore, we further verified the inhibitory effect of DPF on NSCLC growth in an A549 tumor-bearing nude mouse model. Starting from day 10 of tumor formation, nude mice were intraperitoneally injected with DPF (40, 80 mg / kg) and Gefi (20 mg / kg) for 15 consecutive days. During this period, the weight of the nude mice was measured every two days, and the long and short diameters of the tumors were measured. The experimental results showed that compared with the vehicle group, the tumor volume and weight of the DPF and Gefi groups were significantly reduced ( Figure 10 AB). Of particular note is that no significant change in body weight was observed in the nude mice during the administration period. Figure 10 C), and there was no significant difference in the relative weights of the heart, liver, spleen, lungs, and kidneys in nude mice after drug treatment ( Figure 10 D).

[0137] These results indicate that DPF can effectively inhibit the proliferation of A549 cells in tumor-bearing nude mice. To further verify the inhibitory effect of DPF on tumor growth in A549-bearing nude mice, we performed H&E staining and TUNEL assays on tumor tissues. The experimental results are as follows: Figure 11 As shown, tissue sections treated with DPF and Gefi exhibited inhibited tumor cell proliferation and a significant increase in the number of apoptotic cells. These results indicate that DPF has a significant inhibitory effect on tumor growth in A549 tumor-bearing nude mice.

[0138] 2. Study on the mechanism by which DPF inhibits tumor growth in A549-bearing nude mice in non-small cell lung cancer (NSCLC)

[0139] To investigate the mechanism by which DPF inhibits tumor growth in A549 tumor-bearing mice, we performed immunohistochemical analysis on tumor tissues to detect the protein expression of LC3, ERK, mTOR, and Bax. Experimental results ( Figure 12The study showed that DPF upregulated the expression levels of these proteins in a concentration-dependent manner. The mechanism of action of DPF is consistent with the in vitro results, inhibiting tumor growth by promoting autophagy and inducing apoptosis.

[0140] Experimental conclusions

[0141] DPF significantly inhibited tumor growth in A549 tumor-bearing mice by inducing apoptosis and activating autophagy.

[0142] The crude extract (DE) of *Bambusa textilis* in this invention significantly inhibited the proliferation of A549 cells and exhibited low cytotoxicity against normal lung cells BEAS-2B, suggesting that DE has good anti-NSCLC potential. Further separation of DE yielded four fractions with different polarities: petroleum ether extract (DPF), ethyl acetate extract (DEF), n-butanol extract (DNF), and water extract (DWF). Among these, the petroleum ether extract (DPF) showed the strongest anti-proliferative activity against A549 and H1299 cells. Experimental results also showed that DPF significantly inhibited the migration, invasion, and colony formation of A549 and H1299 cells. Mass spectrometry analysis and transcriptome sequencing indicated that the anti-tumor effect of DPF may be related to the synergistic effect of autophagy and apoptosis pathways. Further validation experiments showed that DPF can promote NSCLC cell death by activating autophagy and apoptosis pathways. The anti-proliferative effect of DPF on A549 and H1299 cells was significantly weakened after the addition of the ULK1 inhibitor and the pan-caspase inhibitor Z-VAD-FMK.

[0143] Flow cytometry results showed that DPF treatment significantly increased the apoptosis rate of A549 and H1299 cells. Western blot analysis showed that DPF significantly upregulated the Bax / Bcl-2 ratio and promoted caspase-3 activation. DPF also induced fluorescent spot formation in U87 cells stably transfected with GFP-LC3. Western blot results showed that the LC3II / LC3I conversion rate was significantly increased in A549 and H1299 cells, indicating that DPF effectively induced autophagy in NSCLC cells. To further confirm the effect of DPF on autophagy, we labeled the autophagosomes of A549 and H1299 cells with the GFP-LC3 plasmid and found that the number of GFP-LC3 spots increased significantly with increasing DPF concentration. After the addition of 3-MA, CC, and ULK1 inhibitors, the formation of GFP-LC3 spots was significantly reduced, and A549 cell death was also inhibited, indicating that DPF promotes NSCLC cell death by activating autophagy.

[0144] Furthermore, Western blotting revealed that DPF can activate the MEK / ERK pathway, and confocal microscopy showed that the ERK inhibitor SCH significantly reduced the formation of GFP-LC3 fluorescent spots after DPF treatment, indicating its association with the AMPK / mTOR / ULK1 signaling pathway. In vivo experiments showed that compared with the vehicle group, the tumor volume and weight of mice in the DPF and Gefi groups were significantly reduced, indicating that DPF can effectively inhibit A549 tumor growth. During administration, mouse body weight did not change significantly, and TUNEL and H&E staining results showed that DPF significantly induced tumor cell apoptosis and inhibited proliferation. Immunohistochemical analysis showed that DPF significantly increased the expression of mTOR, ERK, LC3, and Bax proteins, further demonstrating that it inhibits tumor cell proliferation by activating autophagy and apoptosis pathways, effectively inhibiting tumor growth in A549 tumor-bearing mice.

[0145] Conclusion: This study confirms the potential of DPF in the treatment of NSCLC and demonstrates that it inhibits the proliferation of NSCLC cells by activating autophagy and inducing apoptotic cell death through the MEK / ERK and AMPK / mTOR / ULK1 pathways. This provides some evidence and options for the development of novel natural product-based anti-lung cancer drugs.

[0146] The above embodiments are merely specific implementations of the present invention, and the parameters described therein are quite specific and detailed. However, it should be noted that those skilled in the art can make several minor equivalent substitutions, modifications, and improvements based on the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. The application of a baiji bamboo shoot extract in the preparation of drugs for treating tumor diseases.

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

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

4. The application as described in claim 1, characterized in that, The tumor disease is pancreatic cancer.

5. The application as described in claims 1-4, characterized in that, The effective dosage of the *Bambusa textilis* extract is 40 mg / kg to 80 mg / kg.

6. The application as described in claim 1, characterized in that, The extract of *Bambusa textilis* is a petroleum ether extract of *Bambusa textilis*.

7. The application as described in claim 1, characterized in that, The preparation method of the Hundred-tail Bamboo Shoot Extract includes the following steps: (1) After crushing the bamboo shoots, weigh the coarse powder, add 10 times the volume of 75% ethanol solution, heat in a water bath and reflux for extraction, heat for 2 hours, repeat this process three times. After each extraction, collect the bamboo shoot extract. Combine the three extracts and then evaporate the bamboo shoot extract to obtain the bamboo shoot extract, which is stored at -20 ℃. (2) Take the total extract of bamboo shoots from step (1), suspend it in pure water, and then extract the aqueous suspension with petroleum ether, ethyl acetate and n-butanol in sequence. Repeat the extraction operation of each organic solvent 3 to 5 times until the extract solution is colorless and clear. Combine all petroleum ether extracts and vacuum dry them in a water bath at 60 ℃ to finally obtain the petroleum ether extract of bamboo shoots.

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