Application of tetrahydrohonokiol in preparation of medicine for preventing and / or treating tumors

Abstract

The invention provides application of tetrahydrohonokiol in preparation of a medicine for preventing and / or treating tumors. Tetrahydrohonokiol has relatively strong anti-cancer activity on cells of malignant tumors such as esophageal cancer, bladder cancer, brain glioma, liver cancer, colorectal cancer, lung cancer, pancreatic cancer and breast cancer. Therefore, the tetrahydrohonokiol can be used for preparing the anti-tumor medicine.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and relates to the application of honokiol derivatives in the preparation of drugs for preventing and / or treating tumors, in particular the application of tetrahydrohonokiol in the preparation of drugs for preventing and / or treating tumors. BACKGROUND

[0002] Esophageal cancer is a malignant tumor originating from the esophageal mucosa, and its clinical characteristics present significant challenges in diagnosis and treatment. The disease lacks typical clinical manifestations in the early stage, and patients often only show mild discomfort when eating. These atypical symptoms often do not attract enough attention. Due to the insidiousness and non-specificity of early symptoms, most patients have developed to the middle and advanced stages by the time of diagnosis, thus losing the best opportunity for radical surgery, directly affecting the overall treatment effect. Statistical data shows that the 5-year overall survival rate of esophageal cancer patients is only about 20%. In the current treatment strategy, the neoadjuvant chemoradiotherapy combined regimen has been established as the standard treatment mode for locally advanced resectable esophageal cancer. However, this treatment mode still shows obvious deficiencies in clinical application. Studies have shown that among patients receiving this treatment regimen, 40%-50% still have local regional recurrence or distant organ metastasis. It is worth noting that compared with simple surgery, neoadjuvant chemoradiotherapy has not significantly reduced the probability of distant metastasis, which suggests that this treatment strategy has limited effect on controlling systemic dissemination of the tumor. Based on the above clinical status, the field of esophageal cancer treatment urgently needs to explore more effective drug treatment regimens.

[0003] Bladder cancer occupies an important position in male urogenital malignant tumors, with high incidence and mortality. According to the depth of tumor invasion, bladder cancer can be divided into two types: muscle-invasive bladder cancer and non-muscle-invasive bladder cancer. This classification standard has important clinical guiding significance for developing treatment strategies. In terms of diagnostic techniques, cystoscopy combined with histopathological biopsy is still considered the "gold standard" for the diagnosis of bladder cancer. At the same time, urine exfoliative cytology and various imaging examination techniques as important auxiliary diagnostic methods provide strong support for clinical decision-making. It is worth noting that muscle-invasive bladder cancer and non-muscle-invasive bladder cancer show significant differences in treatment regimen selection and disease prognosis, which is mainly due to the essential difference in histopathological characteristics. Although a considerable proportion of patients have pathological results indicating non-muscle-invasive lesions at initial diagnosis and receive standardized transurethral resection of bladder tumors and postoperative intravesical instillation therapy, clinical follow-up data shows that disease recurrence and malignant progression still account for a high proportion. The above clinical status shows that the existing bladder cancer treatment system still has obvious limitations in controlling disease progression and improving long-term prognosis, and more precise and effective treatment strategies need to be developed.

[0004] Glioma is the most common primary malignant tumor of the central nervous system, which presents a wide age distribution, especially in the adult population, and has no significant gender difference. The disease is a major challenge in the field of neuro-oncology due to its high morbidity, recurrence rate and mortality. The current standard treatment for glioma mainly relies on maximum safe surgical resection combined with postoperative adjuvant radiotherapy and chemotherapy. However, clinical practice shows that for patients with high-grade glioma (WHO pathological grade Ⅲ-Ⅳ), even if they receive standardized multi-modal therapy, the median overall survival after surgery is still significantly limited, usually less than 12 months. In terms of chemotherapy drugs, Temozolomide (TMZ) as a standard first-line chemotherapy drug for high-grade glioma has not achieved the expected goal in terms of clinical efficacy. Clinical control study data shows that compared with simple surgical treatment, adjuvant chemotherapy with Temozolomide can only bring 3-5 months of survival benefit to patients with grade Ⅲ-Ⅳ glioma. This limited improvement in efficacy is far from meeting the clinical treatment needs. In view of the above clinical status, high-grade glioma still faces severe challenges in treatment strategies, and the existing chemotherapy drug system has obvious shortcomings in prolonging the survival of patients and improving the quality of life, and it is urgent to explore more effective treatment drugs and innovative treatment regimens.

[0005] Hepatocellular carcinoma (HCC) can be classified into two major types, primary and secondary, according to its origin. Primary HCC mainly originates from the malignant transformation of hepatocytes and bile duct epithelial cells, and has become one of the important malignant tumor types that seriously endanger global public health. From the perspective of clinical manifestations, HCC presents a typical stage-related symptom spectrum. Early-stage disease often lacks specific clinical signs, making early diagnosis difficult. As the disease progresses, patients in the middle and advanced stages may present a diverse group of clinical symptoms, mainly manifested as typical symptoms such as liver pain, abdominal fullness, loss of appetite, general weakness, and progressive weight loss, as well as signs of progressive liver enlargement or upper abdominal mass formation. In addition, some patients may also present with low fever, jaundice, diarrhea, and upper gastrointestinal bleeding. The diversity of these symptoms increases the complexity of clinical diagnosis. Currently, the treatment strategy for HCC in clinical practice mainly adopts a multidisciplinary comprehensive treatment model, which includes radical resection, transarterial chemoembolization, radiofrequency ablation, stereotactic radiotherapy, molecular targeted therapy, and traditional Chinese medicine adjuvant therapy. The choice of these treatment methods usually requires individualized assessment based on multiple factors such as tumor stage, liver function status, general condition, and comorbidities. However, despite the continuous development and improvement of existing treatment technologies, the overall prognosis of HCC patients remains unsatisfactory, with a relatively low 5-year survival rate. This situation highlights the limitations of the current treatment system, suggesting that we urgently need to develop more effective innovative treatment drugs and cutting-edge treatment strategies to significantly improve the long-term survival prognosis and quality of life of patients.

[0006] Colorectal cancer is a major public health problem worldwide, ranking third in the incidence of malignant tumors and second in mortality. It is an important component of digestive tract tumors, accounting for about 30% of all cases. Disease recurrence and distant metastasis are the core factors affecting the long-term survival of patients with colorectal cancer. Clinical statistics show that the 5-year overall survival rate of patients with recurrent disease is significantly lower. Even after standard radical surgery, 20%-30% of patients still face the risk of recurrence, highlighting the limitations of current treatment strategies. The prognosis of recurrent disease varies significantly depending on the treatment modality. For locally recurrent lesions, the median survival time is often limited to less than 10 months without effective intervention. The presence of distant metastases further complicates the disease, limiting the feasibility of sphincter-preserving surgery and significantly reducing the patient's expected survival time, creating a dual challenge of quality of life and length of life. This mode of disease progression has a profound impact on the physical and mental health of patients. Despite recent advances in early screening, accurate diagnosis, and individualized treatment of colorectal cancer, the existing treatment system still has significant shortcomings in dealing with recurrent and metastatic disease. Therefore, developing innovative drugs with higher efficacy and better safety characteristics and building a more comprehensive treatment strategy have become an urgent need and important direction for current oncology research.

[0007] Lung cancer, a malignant disease that occurs in the bronchial mucosa or glands of the lung, is a major disease burden that threatens human health worldwide. Epidemiological data show that the incidence and mortality rates of the disease are on the rise. In China, lung cancer ranks first among male malignant tumors and second among female groups, highlighting its important clinical significance. Based on the anatomical location of the lesion, lung cancer can be divided into peripheral and central types. The typical clinical manifestations of the disease include persistent cough with blood or hemoptysis symptoms, chest pain, chest tightness, dyspnea, and hoarseness. It is worth noting that this type of tumor has strong invasive and metastatic ability, and common metastatic target organs include the brain and skeletal system. In terms of treatment strategies, surgical radical resection is still the standard treatment for patients with peripheral lung cancer. However, for central lesions and advanced patients with distant metastasis, due to the loss of surgical treatment opportunities, clinical treatment relies on multidisciplinary treatment methods such as radiotherapy, systemic chemotherapy, and immune checkpoint inhibitors. Despite recent advances in lung cancer diagnosis and treatment, the prognosis of central lung cancer, especially metastatic cases, remains limited, and more effective treatment drugs and regimens are urgently needed to further improve patient survival benefits.

[0008] Pancreatic cancer is the most dangerous type of digestive tract malignancy in terms of biological behavior, and its malignancy and mortality rate ranks among the top in the spectrum of solid tumors. The clinical features of this disease are insidious onset and lack of specific early symptoms, resulting in a significantly low early detection rate. Epidemiological investigations have shown that only 15-20% of patients have indications for radical surgery at initial diagnosis, and the vast majority of cases are locally advanced or have distant metastasis at diagnosis, resulting in a five-year overall survival rate of less than 10%. Although some progress has been made in the field of multidisciplinary comprehensive treatment and precision medicine in recent years, the overall treatment effect of this disease is still not satisfactory, and it is urgent to achieve important breakthroughs in treatment strategies through in-depth basic medical research, translational medical exploration, and clinical trial innovation.

[0009] Breast cancer originates from malignant lesions of breast duct epithelial cells or lobular epithelial cells, and is one of the malignant tumor types with the highest incidence in the female population. The clinical symptoms at the early stage of this disease are atypical, often presenting as a painless mass in the breast, which is easily ignored by patients and leads to delayed diagnosis and treatment. With disease progression, patients may exhibit typical signs such as inward retraction of the nipple, "dimpled" or "orange peel-like" changes in the skin of the breast, and enlargement of the ipsilateral axillary lymph nodes. The current clinical treatment modalities mainly include surgery, systemic chemotherapy, radiotherapy, endocrine therapy, and molecular targeted therapy. Although some patients with certain subtypes can achieve good prognosis through standardized treatment, the long-term survival benefit of patients with triple-negative breast cancer (negative expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2) is still limited even after receiving standardized comprehensive treatment, which is mainly attributed to the lack of effective treatment targets and innovative drugs for this subtype.

[0010] Magnolia officinalis, the dried bark, root bark, and branch bark of Magnolia officinalis, belongs to the category of traditional Chinese medicinal materials. According to the Chinese Pharmacopoeia, it has a bitter and pungent taste, is warm in nature, and is associated with the spleen, stomach, lung, and large intestine meridians. It has the functions of drying dampness, reducing phlegm, and relieving fullness and abdominal distension. Modern pharmaceutical chemistry research has shown that Magnolia officinalis is rich in various bioactive compounds, including magnolol, honokiol, 4-O-methylhonokiol, and magnolol new phenol. CN103553888A isolated a new honokiol derivative from the mature fruits of Magnolia officinalis by active tracking separation method, and proved that the honokiol derivative has a significant effect of inhibiting tumor cell proliferation.

[0011] Tetrahydrohonokiol (2-(4-hydroxy-3-propylphenyl)-4-propylphenol; molecular formula: C 18 H 22 O2; molecular weight: 270.37; CAS number: 35406-31-6; formula I) is a derivative of honokiol, but its specific use is not clear at present.

[0012] Formula I. SUMMARY

[0013] In order to fill the blank of tetrahydro and magnolol in the field of preparing drugs for preventing and / or treating tumors, the application provides an application of tetrahydro and magnolol in preparing drugs for preventing and / or treating tumors.

[0014] The technical scheme adopted by the application is that tetrahydro and magnolol are applied in preparing drugs for preventing and / or treating tumors, and the tumors include esophageal cancer, bladder cancer, brain glioma, liver cancer, colorectal cancer, lung cancer, pancreatic cancer and breast cancer.

[0015] Preferably, the treatment and / or prevention includes reducing the proliferation ability, migration ability, apoptosis rate and / or division ability of tumor cells.

[0016] Preferably, the tumor cells include esophageal cancer KYSE70 cells, esophageal cancer TE1 cells, bladder cancer 5637 cells, bladder cancer T24 cells, brain glioma CT2A cells, brain glioma GL261 cells, brain glioma U251 cells, liver cancer HepG2 cells, liver cancer Huh7 cells, colorectal cancer RKO cells, colorectal cancer HCT8 cells, colorectal cancer HCT116 cells, colorectal cancer LOVO cells, colorectal cancer SW48 cells, colorectal cancer SW620 cells, colorectal cancer HT29 cells, lung cancer A549 cells, lung cancer NCIH196 cells, lung cancer H1299 cells, lung cancer H460 cells, lung cancer DMS114 cells, pancreatic cancer SW1990 cells, pancreatic cancer PANC-1 cells, breast cancer MDA-MB-231 cells and breast cancer Bcap37 cells.

[0017] The application provides a pharmaceutical composition for treating and / or preventing tumors, which comprises a therapeutically and / or preventively effective amount of tetrahydro and magnolol, and the tumors include esophageal cancer, bladder cancer, brain glioma, liver cancer, colorectal cancer, lung cancer, pancreatic cancer and breast cancer.

[0018] Preferably, the concentration of tetrahydro and magnolol in the pharmaceutical composition is not less than 10 μM.

[0019] Preferably, the treatment and / or prevention includes reducing the proliferation ability, migration ability, apoptosis rate and / or division ability of tumor cells.

[0020] Preferably, the tumor cells include esophageal cancer KYSE70 cells, esophageal cancer TE1 cells, bladder cancer 5637 cells, bladder cancer T24 cells, brain glioma CT2A cells, brain glioma GL261 cells, brain glioma U251 cells, liver cancer HepG2 cells, liver cancer Huh7 cells, colorectal cancer RKO cells, colorectal cancer HCT8 cells, colorectal cancer HCT116 cells, colorectal cancer LOVO cells, colorectal cancer SW48 cells, colorectal cancer SW620 cells, colorectal cancer HT29 cells, lung cancer A549 cells, lung cancer NCIH196 cells, lung cancer H1299 cells, lung cancer H460 cells, lung cancer DMS114 cells, pancreatic cancer SW1990 cells, pancreatic cancer PANC-1 cells, breast cancer MDA-MB-231 cells, breast cancer Bcap37 cells.

[0021] Further, the administration mode of the pharmaceutical composition includes oral administration, sublingual administration, topical administration, injection administration.

[0022] Further, the topical administration refers to a mode of administration that does not pass through the gastrointestinal absorption.

[0023] Further, the injection administration includes subcutaneous injection, intramuscular injection, intravenous injection, intradermal injection.

[0024] Further, the pharmaceutical composition further includes other pharmaceutically acceptable adjuvants, such adjuvants include adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, pH adjusting and / or buffering agents, solvents, surfactants or emulsifiers.

[0025] Further, the therapeutically and / or prophylactically effective amount refers to the modulated amount of tetrahydro and honokiol necessary to provide the desired therapeutic effect, whether used alone or in combination with another therapeutic agent. For example, an effective amount to prevent, alleviate, or ameliorate symptoms of a disease or extend the survival of the subject being treated.

[0026] In a specific embodiment, the therapeutically and / or prophylactically effective amount of the present application refers to the amount of tetrahydro and honokiol that is effective to provide the desired therapeutic effect, for example, an amount effective to prevent, alleviate, or ameliorate the symptoms of a disease or extend the survival of the subject being treated. As will be appreciated by those of ordinary skill in the art, the exact amount required will vary from subject to subject, depending on the age and general condition of the subject, the severity of the condition, and the particular compound and / or composition of matter administered, and the like, as will be appreciated by those of ordinary skill in the art. An appropriate therapeutically and / or prophylactically effective amount in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and / or by using routine experimentation.

[0027] In a more specific embodiment, the pharmaceutical composition is an anti- esophageal cancer drug containing 10-70 μΜ of tetrahydro and honokiol for inhibiting the proliferation ability of esophageal cancer KYSE70 cells and / or TE1 cells.

[0028] In a more specific embodiment, the pharmaceutical composition is an anti- bladder cancer drug containing 10-70 μΜ of tetrahydro and honokiol for inhibiting the proliferation ability of bladder cancer 5637 cells and / or T24 cells.

[0029] In a more specific embodiment, the pharmaceutical composition is an anti- brain glioma drug containing 10-70 μΜ of tetrahydro and honokiol for inhibiting the proliferation ability of brain glioma CT2A and / or GL261 and / or U251 cells.

[0030] In a more specific embodiment, the pharmaceutical composition is an anti- liver cancer drug containing 10-70 μΜ of tetrahydro and honokiol for inhibiting the proliferation ability of liver cancer HepG2 cells and / or Huh7 cells.

[0031] In a more specific embodiment, the pharmaceutical composition is an anti- colorectal cancer drug containing 10-70 μΜ of tetrahydro and honokiol for inhibiting the proliferation ability of colorectal cancer RKO and / or HCT8 and / or HCT116 and / or LOVO and / or SW48 and / or SW620 and / or HT29 cells, and for increasing the apoptosis rate of colorectal cancer SW620 and / or HT29 cells, and for decreasing the colony formation ability of colorectal cancer SW620 and / or HT29 cells.

[0032] In a more specific embodiment, the pharmaceutical composition is an anti- lung cancer drug containing 10-70 μΜ of tetrahydro and honokiol for inhibiting the proliferation ability of lung cancer A549 and / or NCIH196 and / or H1299 and / or H460 and / or DMS114 cells.

[0033] In a more specific embodiment, the pharmaceutical composition is an anti- pancreatic cancer drug containing 10-70 μΜ of tetrahydro and honokiol for inhibiting the proliferation ability of pancreatic cancer SW1990 and / or PANC-1 cells, and for increasing the apoptosis rate of pancreatic cancer SW1990 and / or PANC-1 cells, and for decreasing the colony formation ability of pancreatic cancer SW1990 and / or PANC-1 cells.

[0034] In a more specific embodiment, the pharmaceutical composition is an anti-breast cancer drug containing 10-70 μM tetrahydro and honokiol for inhibiting the proliferation ability of breast cancer MDA-MB-231 and / or Bcap37 cells.

[0035] The present application provides a method for inhibiting the proliferation ability, migration ability, division ability of tumor cells and / or increasing the apoptosis rate of tumor cells for non-therapeutic purposes in vitro, the method comprising the following steps: using tetrahydro and honokiol or a composition thereof to treat tumor cells; wherein the tumor includes esophageal cancer, bladder cancer, brain glioma, liver cancer, colorectal cancer, lung cancer, pancreatic cancer, breast cancer.

[0036] Preferably, the tumor cells include esophageal cancer KYSE70 cells, esophageal cancer TE1 cells, bladder cancer 5637 cells, bladder cancer T24 cells, brain glioma CT2A cells, brain glioma GL261 cells, brain glioma U251 cells, liver cancer HepG2 cells, liver cancer Huh7 cells, colorectal cancer RKO cells, colorectal cancer HCT8 cells, colorectal cancer HCT116 cells, colorectal cancer LOVO cells, colorectal cancer SW48 cells, colorectal cancer SW620 cells, colorectal cancer HT29 cells, lung cancer A549 cells, lung cancer NCIH196 cells, lung cancer H1299 cells, lung cancer H460 cells, lung cancer DMS114 cells, pancreatic cancer SW1990 cells, pancreatic cancer PANC-1 cells, breast cancer MDA-MB-231 cells, breast cancer Bcap37 cells.

[0037] The present application provides an application of tetrahydro and honokiol in inhibiting the proliferation ability, migration ability, division ability of tumor cells and / or increasing the apoptosis rate of tumor cells in vitro, wherein the tumor includes esophageal cancer, bladder cancer, brain glioma, liver cancer, colorectal cancer, lung cancer, pancreatic cancer, breast cancer.

[0038] The present application has the following beneficial effects: tetrahydro and honokiol have strong anticancer activity on malignant tumor cells such as esophageal cancer, bladder cancer, brain glioma, liver cancer, colorectal cancer, lung cancer, pancreatic cancer and breast cancer. Therefore, tetrahydro and honokiol can be used for preparing an anti-tumor drug. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1The graph shows the effects of tetrahydromethylenetetramine and honokiol on the proliferation of malignant tumor cells, including esophageal cancer (KYSE70 and TE1), bladder cancer (5637 and T24), glioma (CT2A, GL261 and U251), colorectal cancer (RKO, HCT8, HCT116, LOVO, SW48, SW620 and HT29), lung cancer (A549, NCIH196, H1299, H460 and DMS114), pancreatic cancer (SW1990 and PANC-1), and breast cancer (MDA-MB-231 and Bcap37).

[0040] Figure 2 The results show the effects of different concentrations of tetrahydromethane and honokiol (THH) on apoptosis in colorectal cancer cells (SW620 and HT29) and their quantitative analysis.

[0041] Figure 3 The image shows the effects of different concentrations of tetrahydromethane and honokiol (THH) on apoptosis in pancreatic cancers (SW1990 and PANC-1) and their quantitative analysis.

[0042] Figure 4 The effects of different doses of tetrahydromethylenetetra ... Detailed Implementation

[0043] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0044] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0045] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0046] When the embodiments give numerical ranges, it is understood that, unless the present invention indicates otherwise, each numerical range's two endpoints and any number between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to specific methods, devices, materials, etc. described in the embodiments, any methods, devices, and materials similar or equivalent to those described in the embodiments of the present invention can be used in the practice of the present invention, according to the knowledge of those skilled in the art and the description of the present invention.

[0047] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all use conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields.

[0048] In the following examples of the present application, the sources of the reagents used are as follows:

[0049] Tetrahydro and Magnolol (Shanghai Yuan Ye Biotechnology Co., Ltd.); 10% fetal bovine serum (Vistec, SA); 1640, IMDM, DMEM medium (Shanghai Yuan Pei Biotechnology Co., Ltd.); 0.25% trypsin (Shanghai Yuan Pei Biotechnology Co., Ltd.); CCK8 kit (Dalian Milen Biotechnology Co., Ltd.); Crystal violet dye (Beyotime); Apoptosis detection kit (Shanghai Yisheng Biotechnology)

[0050] In the following examples of the present application, the cell strains used are as follows:

[0051] Esophageal cancer (KYSE70 and TE1), bladder cancer (5637 and T24), brain glioma (CT2A, GL261 and U251), colorectal cancer (RKO, HCT8, HCT116, LOVO, SW48, SW620 and HT29), lung cancer (A549, NCIH196, H1299, H460 and DMS114), pancreatic cancer (SW1990 and PANC-1) and breast cancer (MDA-MB-231 and Bcap37) were purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0052] Cell Counting Kit 8 (CCK8 method) was used to detect cell proliferation, including the following steps:

[0053] (1) Esophageal cancer (KYSE70 and TE1), bladder cancer (5637 and T24), brain glioma (CT2A, GL261 and U251), colorectal cancer (RKO, HCT8, HCT116, LOVO, SW48, SW620 and HT29), lung cancer (A549, NCIH196, H1299, H460 and DMS114), pancreatic cancer (SW1990 and PANC-1) and breast cancer (MDA-MB-231 and Bcap37) cells in good growth state and in logarithmic growth phase were digested with trypsin, terminated by adding complete culture medium containing 10% FBS, blown into cell suspension and transferred to EP tubes, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and complete culture medium was added to prepare cell suspension;

[0054] (2) Counted using a cell counting plate, seeded 5000 cells in each well of a 96-well plate with a volume of 100 μL per well, and added an equal amount of PBS solution in the four peripheral wells to prevent edge effects;

[0055] (3) Incubated overnight at 37°C in a 5% CO2 incubator with conventional culture medium; aspirated the liquid medium in each well, and added different concentrations of tetrahydro and magnolol prepared in gradient (10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM) under light-free conditions, and added to different cells in each well for continuous culture;

[0056] (4) After 24 h of culture, aspirated the culture medium in the plate under light-free conditions, prepared CCK8 solution required for the experiment, added 100 μL volume to each well (to prevent air bubbles during liquid addition to avoid inaccurate measurement by the enzyme marker), and incubated in an incubator for 1-2 h;

[0057] (5) Adjusted the wavelength of the full-automatic enzyme marker to 450 nm, and measured the OD value of each well;

[0058] (6) According to the OD value, the growth curve of each group of cells was drawn, and the cell proliferation was calculated and analyzed by Graphpad 10.1.2, and the results are shown in Figure 1 .

[0059] As shown in Figure 1As shown, the effect of different concentrations of tetrahydro and magnolol on the growth and proliferation of esophageal cancer (KYSE70 and TE1), bladder cancer (5637 and T24), brain glioma (CT2A, GL261 and U251), colorectal cancer (RKO, HCT8, HCT116, LOVO, SW48, SW620 and HT29), lung cancer (A549, NCIH196, H1299, H460 and DMS114), pancreatic cancer (SW1990 and PANC-1) and breast cancer (MDA-MB-231 and Bcap37) cells was detected by CCK8 method. The growth and proliferation of lung cancer, intestinal cancer and pancreatic cancer cells were inhibited by each experimental concentration of tetrahydro and magnolol, and the cell growth inhibition rate gradually increased with the increase of the dose, which had obvious dose dependence.

[0060] Apoptosis experiment, including the following steps:

[0061] (1) Select intestinal cancer cells (SW620 and HT29) and pancreatic cancer cells (SW1990 and PANC-1) in good growth state and in logarithmic growth phase, digest with trypsin, terminate with complete culture medium containing 10% FBS, centrifuge, resuspend, count;

[0062] (2) According to 3x10 5 / well, the cells were inoculated in 6-well plates, shaken gently, and then placed in an incubator overnight. When the cell density reached 70%-80%, the culture medium was discarded, the cells were washed with PBS, and different concentrations of tetrahydro and magnolol were added in the dark. The concentration of tetrahydro and magnolol is shown in Figure 2 and Figure 3 ;

[0063] (3) Collect the cell supernatant culture based on the labeled 15 ml centrifuge tube, wash with PBS, trypsin digestion, stop digestion in complete culture medium, gently blow the cells, try to reduce mechanical death, and transfer the cell-containing suspension to the 15 ml centrifuge tube containing the collected supernatant;

[0064] (4) Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells with 500 μL PBS and collect them in 1.5 mL EP tube, centrifuge at 1000 rpm for 5 min, discard the supernatant;

[0065] (5) Add 200 μL 1x binding buffer to each EP tube containing cell precipitate, mix well, then add 3 μL Annexin V-FITC and 3 μL PI dye solution respectively, mix well, then transfer to flow tube, incubate at room temperature for 15 min in the dark;

[0066] (6) The pipette gun is gently blown evenly, and the flow cytometer is used to analyze the cell staining and count the apoptosis of the cells. The results are shown in Figure 2 and Figure 3 .

[0067] As shown in Figure 2 and Figure 3 , the effects of different concentrations of tetrahydro and magnolol on the growth and proliferation of intestinal cancer and pancreatic cancer cells were detected by apoptosis experiment. According to the experimental data, different concentrations of tetrahydro and magnolol were used to treat different cancer cells. With the increase of drug concentration, the apoptosis rate of the cells treated with tetrahydro and magnolol was significantly increased in a dose-dependent manner compared with the control group.

[0068] The plate monoclonal formation experiment includes the following steps:

[0069] (1) Select the logarithmic growth phase, good growth state and moderate density of intestinal cancer cells SW620 and HT29, pancreatic cancer cells SW1990 and PANC-1, digest, centrifuge, resuspend, count;

[0070] (2) The cells are inoculated in 6 cm dishes at 2000 cells / dish, gently shaken and placed in an incubator overnight. After the cells adhere and grow normally, the culture medium is discarded, and the culture medium containing or not containing the drug is added to the culture dish according to the drug concentration gradient (this operation is in the dark). After 24 h, the fresh culture medium is replaced; the concentration of tetrahydro and magnolol is shown in Figure 4 ;

[0071] (3) Place the culture dish in the incubator for static culture for about two weeks, and replace the fresh culture medium every 2-3 days during this period;

[0072] (4) Observe the cells, stop the culture when the monoclone cell mass is visible to the naked eye, discard the culture medium, wash with PBS, and fix the cells with 3 mL of 4% paraformaldehyde fixing solution for 30 min;

[0073] (5) Remove the fixing solution, wash with PBS, and stain the cells with 0.1% crystal violet solution for 30 min;

[0074] (6) Recover the crystal violet solution, slowly rinse the culture dish with running water to wash away the excess dye, place the treated culture dish upside down on the absorbent paper to dry, take a photo, and the results are shown in Figure 4 .

[0075] As shown in Figure 4The effect of tetrahydro and magnolol on the proliferation of SW620, HT29, SW1990 and PANC-1 was detected by a plate cloning experiment. The results showed that the number of cell clones decreased with the increase of the concentration of tetrahydro and magnolol, indicating that tetrahydro and magnolol can inhibit the proliferation of SW620, HT29, SW1990 and PANC-1.

[0076] In summary, tetrahydro and magnolol have strong anticancer activity on esophageal cancer, bladder cancer, brain glioma, colorectal cancer, lung cancer, pancreatic cancer and breast cancer and other malignant tumor cells, thereby effectively playing an antitumor role; therefore, tetrahydro and magnolol can be used for preparing antitumor drugs.

[0077] The above examples only illustrate the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical idea disclosed by the present application should be covered by the claims of the present application.

Claims

1. Use of tetrahydro and honkyphenol for the preparation of a medicament for the prevention and / or treatment of neoplasms, characterized in that, The tumor includes esophageal cancer, bladder cancer, brain glioma, liver cancer, colorectal cancer, lung cancer, pancreatic cancer, breast cancer.

2. Use according to claim 1, wherein The treatment and / or prevention include reducing the proliferation ability, migration ability, and / or division ability of the tumor cells.

3. The use according to claim 1, wherein The tumor cells include esophageal cancer KYSE70 cells, esophageal cancer TE1 cells, bladder cancer 5637 cells, bladder cancer T24 cells, brain glioma CT2A cells, brain glioma GL261 cells, brain glioma U251 cells, liver cancer HepG2 cells, liver cancer Huh7 cells, colorectal cancer RKO cells, colorectal cancer HCT8 cells, colorectal cancer HCT116 cells, colorectal cancer LOVO cells, colorectal cancer SW48 cells, colorectal cancer SW620 cells, colorectal cancer HT29 cells, lung cancer A549 cells, lung cancer NCIH196 cells, lung cancer H1299 cells, lung cancer H460 cells, lung cancer DMS114 cells, pancreatic cancer SW1990 cells, pancreatic cancer PANC-1 cells, breast cancer MDA-MB-231 cells, breast cancer Bcap37 cells.

4. A pharmaceutical composition for treating and / or preventing a tumor, characterized by, The pharmaceutical composition includes a therapeutically and / or prophylactically effective amount of tetrahydro and honokiol, and the tumor includes esophageal cancer, bladder cancer, brain glioma, liver cancer, colorectal cancer, lung cancer, pancreatic cancer, breast cancer.

5. The pharmaceutical composition of claim 4, wherein The concentration of tetrahydro and honokiol in the pharmaceutical composition is not less than 10 μM.

6. The pharmaceutical composition of claim 4, wherein The treatment and / or prevention include reducing the proliferation ability, migration ability, and / or division ability of the tumor cells.

7. The pharmaceutical composition of claim 4, wherein The tumor cells include esophageal cancer KYSE70 cells, esophageal cancer TE1 cells, bladder cancer 5637 cells, bladder cancer T24 cells, brain glioma CT2A cells, brain glioma GL261 cells, brain glioma U251 cells, liver cancer HepG2 cells, liver cancer Huh7 cells, colorectal cancer RKO cells, colorectal cancer HCT8 cells, colorectal cancer HCT116 cells, colorectal cancer LOVO cells, colorectal cancer SW48 cells, colorectal cancer SW620 cells, colorectal cancer HT29 cells, lung cancer A549 cells, lung cancer NCIH196 cells, lung cancer H1299 cells, lung cancer H460 cells, lung cancer DMS114 cells, pancreatic cancer SW1990 cells, pancreatic cancer PANC-1 cells, breast cancer MDA-MB-231 cells, breast cancer Bcap37 cells.

8. A method for inhibiting the proliferation, migration, and / or division of tumor cells and / or increasing the apoptosis rate of tumor cells in vitro for non-therapeutic purposes, characterized in that, The method includes the following steps: using tetrahydro and honokiol or a composition thereof to treat tumor cells; wherein the tumor includes esophageal cancer, bladder cancer, brain glioma, liver cancer, colorectal cancer, lung cancer, pancreatic cancer, breast cancer.

9. The method of claim 8, wherein, The tumor cells include esophageal cancer KYSE70 cells, esophageal cancer TE1 cells, bladder cancer 5637 cells, bladder cancer T24 cells, brain glioma CT2A cells, brain glioma GL261 cells, brain glioma U251 cells, liver cancer HepG2 cells, liver cancer Huh7 cells, colorectal cancer RKO cells, colorectal cancer HCT8 cells, colorectal cancer HCT116 cells, colorectal cancer LOVO cells, colorectal cancer SW48 cells, colorectal cancer SW620 cells, colorectal cancer HT29 cells, lung cancer A549 cells, lung cancer NCIH196 cells, lung cancer H1299 cells, lung cancer H460 cells, lung cancer DMS114 cells, pancreatic cancer SW1990 cells, pancreatic cancer PANC-1 cells, breast cancer MDA-MB-231 cells, breast cancer Bcap37 cells.

10. Use of tetrahydro and honkyphenol for inhibiting the ability of tumor proliferation, migration, division and / or increasing the rate of apoptosis of tumor cells in vitro, characterized in that, The tumors include esophageal cancer, bladder cancer, brain glioma, liver cancer, colorectal cancer, lung cancer, pancreatic cancer, breast cancer.

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