Use of a chicory ethyl acetate extract

Abstract

The present application relates to the use of chicory ethyl acetate extract. In vitro experiments, using CellTiter-Glo luminescence method to evaluate the effect of chicory ethyl acetate extract on triple negative breast cancer cells, the results show that the extract significantly inhibits tumor cell proliferation. In vivo experiments: the anti-tumor effect of chicory ethyl acetate extract is verified by triple negative breast cancer animal model, the results show that: chicory ethyl acetate extract can effectively inhibit tumor growth. Further experiments show that the anti-tumor effect of chicory ethyl acetate extract is related to its regulation on PI3K / Akt / mTOR signaling pathway. The chicory ethyl acetate extract provided by the present application can be used for preparing drugs or pharmaceutical compositions for preventing and treating triple negative breast cancer.

Description

Technical Field

[0001] This invention belongs to the field of natural medicine and tumor prevention and treatment technology, specifically relating to the use of ethyl acetate extract of chicory. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), accounting for approximately 15% to 20% of all breast cancers. This type of breast cancer is characterized by its high invasiveness, rapid growth, high recurrence rate, and tendency to metastasize to distant sites, resulting in a poor prognosis for patients.

[0003] Because triple-negative breast cancer lacks clearly defined molecular targets, there are currently no specific targeted therapies available in clinical practice. Treatment primarily relies on chemotherapy drugs, such as anthracyclines, taxanes, and alkylating agents. However, long-term or high-dose chemotherapy can easily lead to significant toxic side effects and may induce drug resistance in tumor cells, thus limiting treatment efficacy. Therefore, developing novel treatment strategies and candidate drugs with clear anti-tumor effects and fewer toxic side effects has become a key focus in the field of triple-negative breast cancer research.

[0004] Chicory (Cichorium glandulosum Boiss. et Huet.) is an annual herb belonging to the genus Cicorium in the Asteraceae family. It is a commonly used medicinal and edible plant in Xinjiang. The chicory in this invention originates from the standardized chicory cultivation base in the agricultural park of Moyu County, Xinjiang. Chicory possesses various biological activities, including hepatoprotective, hypoglycemic, and anti-inflammatory effects, and is rich in active ingredients such as sesquiterpene lactones. Studies have found that the ethyl acetate extract of chicory can enrich various active substances, exhibiting good biological activity potential. Existing research suggests that sesquiterpenes in chicory have certain anti-tumor effects, but the use and mechanism of action of the ethyl acetate extract of chicory in the treatment of triple-negative breast cancer remain unclear. This invention is the first to systematically propose the use of ethyl acetate extract of chicory for the treatment of triple-negative breast cancer, and verifies its anti-tumor activity through in vitro and in vivo experiments. This extract significantly downregulates the phosphorylation levels of AKT and S6 in tumor cells by regulating the PI3K / AKT / mTOR signaling pathway, thereby effectively inhibiting tumor growth, which is significantly innovative. This invention is based on this and explores related applications and mechanisms of action.

[0005] Novelty Analysis Explanation: Currently, publicly available information on existing technologies related to chicory can be mainly divided into three categories:

[0006] The first category comprises methods for the extraction and separation of compounds / extracts from chicory or wild chicory. For example, CN107266400B discloses a method for extracting lactucinoids from chicory, yielding lactucin and lactucinogen, focusing on the extraction and purification process itself; CN101099566B discloses a method for preparing lactucin and its use as a PTP1B inhibitor in diabetes-related applications; CN102600223A and CN101869348A focus on the hepatoprotective and antioxidant uses of chicory extracts, respectively. The common thread among these patents is that they focus on compound separation, processes, or non-TNBC indications, and do not directly target the use of chicory ethyl acetate extract for triple-negative breast cancer.

[0007] The second category includes other pharmacological uses of chicory extracts or isolated compounds. For example, CN120241818A discloses the application of chicory extracts and their isolated compounds in anti-inflammatory applications, emphasizing the inhibitory effects of chicorylolide A, chicorylolide B, chicorylin, and chicorylin on RAW264.7 cells; CN112939912A relates to the extraction of lactucin from chicory for anti-inflammatory and hepatoprotective purposes. Although these technologies are related to the active parts or components of chicory, their indications and efficacy evaluation systems differ from those of TNBC.

[0008] The third category consists of publicly available literature on TNBC. Our research group previously published articles on the in vitro antitumor activity of chicory sesquiterpenes and its potential mechanisms, and on the induction of G2 / M cell cycle arrest and apoptosis in triple-negative breast cancer by lactucin via the Wnt / β-catenin signaling pathway. These articles explicitly mentioned that lactucin (monomer) has an inhibitory effect on MDA-MB-468 cells and suggested an AKT / mTOR-related mechanism. This literature pertains to the study of the role of monomeric compounds in an in vitro TNBC model, rather than the use of chicory ethyl acetate extract (CG-EA) as the whole extract in an integrated in vitro-in vivo evidence chain for anti-TNBC activity.

[0009] Therefore, compared with the prior art, the proposed solution has clear differences in research subjects, indications, and efficacy evidence system, and has a basis for claiming novelty. Summary of the Invention

[0010] The purpose of this invention is to provide the use of ethyl chicory extract. In in vitro experiments, the effect of ethyl chicory extract on triple-negative breast cancer cells was evaluated using CellTiter-Glo luminescence assay. The results showed that the extract significantly inhibited tumor cell proliferation. In vivo experiments: By constructing a triple-negative breast cancer xenograft model, the antitumor effect of ethyl chicory extract was verified. The results showed that ethyl chicory extract effectively inhibited tumor growth. Further experiments indicated that the antitumor effect of ethyl chicory extract is related to its regulation of the PI3K / Akt / mTOR signaling pathway. The ethyl chicory extract provided by this invention can be used to prepare drugs or pharmaceutical compositions for the prevention and treatment of triple-negative breast cancer.

[0011] The present invention relates to the use of an ethyl acetate extract of chicory in the preparation of a drug for the prevention of triple-negative breast cancer.

[0012] The use of the ethyl acetate extract of chicory in the preparation of a drug for treating triple-negative breast cancer.

[0013] The triple-negative breast cancer mentioned above is breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2).

[0014] Furthermore, the ethyl acetate extract of chicory was prepared into a drug for inhibiting the occurrence or development of triple-negative breast cancer.

[0015] Furthermore, the ethyl acetate extract of *Chicorydalis* of this invention regulates the proliferation, survival, and apoptosis of tumor cells by directly inhibiting the phosphorylation levels of key proteins (such as p-AKT and p-S6) in the PI3K / AKT / mTOR signaling pathway. Particularly in triple-negative breast cancer cells, this extract exhibits anti-tumor activity, and its mechanism of action is closely related to the abnormal activation of this signaling pathway. The uses of the ethyl acetate extract of *Chicorydalis* of this invention, including its preparation:

[0016] Take dried whole chicory (Cichorium glandulosum) herb, pulverize it, and add 8-12 times its weight of ethanol aqueous solution. Reflux extraction is performed 1-3 times at 60-70℃, 1-2 hours each time. Combine the extracts and concentrate under reduced pressure until no alcohol odor remains. Dissolve the obtained concentrate in an appropriate amount of distilled water, and extract multiple times with an equal volume of ethyl acetate. Combine the ethyl acetate layers, concentrate under reduced pressure, and dry to obtain the chicory ethyl acetate extract.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention uses ethyl acetate extract of chicory as the research object. In in vitro pharmacodynamic experiments, its inhibitory effect on triple-negative breast cancer cells was determined by cell viability assay, and the half-maximal inhibitory concentration (IC50) was obtained. 50 The results showed that the ethyl acetate extract of chicory exhibited good inhibitory activity against the triple-negative breast cancer cell line MDA-MB-468. In in vivo experiments, administration of the ethyl acetate extract of chicory to a mouse triple-negative breast cancer xenograft model inhibited tumor growth, demonstrating anti-tumor effects.

[0019] The results show that the ethyl acetate extract of chicory provided by this invention can be used to prepare drugs or pharmaceutical compositions for the prevention and treatment of triple-negative breast cancer. This technical solution is derived from a natural plant, the raw materials are readily available, the preparation process is simple, and the application safety is relatively high, providing a technical basis for the development of drugs against triple-negative breast cancer. Attached Figure Description

[0020] Figure 1 Example 1 of this invention illustrates the effect of ethyl acetate extract of chicory on the viability of MDA-MB-468 triple-negative breast cancer cells.

[0021] Figure 2 Example 3 of this invention demonstrates the effect of chicory ethyl acetate extract on the in vivo inhibition of triple-negative breast cancer tumor growth;

[0022] Figure 3 HE staining morphological observation of tumor tissue treated with ethyl chicory ethyl acetate extract in Example 3 of this invention; wherein: A - model group; B - cyclophosphamide (CTX) group; C - docetaxel group; D - low-dose CG-EA group; E - high-dose CG-EA group; F - low-dose CG-EA + CTX group; G - low-dose CG-EA + Docetaxel group; HE staining: ×40; scale bar = 50 μm;

[0023] Figure 4 The image shows the immunohistochemical staining results of Ki-67 tumor tissue treated with ethyl chicory ethyl acetate extract in Example 3 of this invention. A represents the model group; B represents the cyclophosphamide (CTX) group; C represents the docetaxel group; D represents the low-dose CG-EA group; E represents the high-dose CG-EA group; F represents the low-dose CG-EA + CTX group; and G represents the low-dose CG-EA + Docetaxel group. HE staining: ×40; scale bar = 50 μm.

[0024] Figure 5The results of TUNEL apoptosis index detection in tumor tissue treated with ethyl chicory extract in Example 3 of this invention are shown below; where A is the model group; B is the cyclophosphamide (CTX) group; C is the docetaxel group; D is the low-dose CG-EA group; E is the high-dose CG-EA group; F is the low-dose CG-EA + CTX group; G is the low-dose CG-EA + Docetaxel group; scale bar = 50 μm (×20).

[0025] Figure 6 This invention relates to the analysis of the PI3K / AKT / mTOR signaling pathway mechanism in tumor tissue treated with ethyl chicory extract in Example 3 of this invention; wherein G2 is the model group; G3 is the cyclophosphamide (CTX) group; G4 is the docetaxel group; G5 is the low-dose CG-EA group; G6 is the high-dose CG-EA group; G7 is the low-dose CG-EA + CTX group; and G8 is the low-dose CG-EA + Docetaxel group. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments.

[0027] Example 1

[0028] Inhibitory effect of ethyl acetate extract of chicory on the proliferation of triple-negative breast cancer cells (in vitro experiment):

[0029] The human triple-negative breast cancer cell line MDA-MB-468 was selected as the experimental subject and cultured in a medium containing 10% fetal bovine serum at 37°C and 5% CO2. After the cells grew to the logarithmic growth phase, they were seeded into 96-well plates and treated with ethyl chicory extract at logarithmic gradients of concentrations ranging from 30.48 to 200,000 ng / mL.

[0030] After 72 h of culture following drug administration, cell viability was assessed using CellTiter-Glo luminescence assay to evaluate the effect of ethyl chicory extract on the proliferation of triple-negative breast cancer cells. The results showed that under the experimental conditions, ethyl chicory extract inhibited the proliferation of MDA-MB-468 triple-negative breast cancer cells.

[0031] Example 2

[0032] Antitumor effect of ethyl acetate extract of chicory on triple-negative breast cancer-bearing mice:

[0033] A tumor-bearing animal model was constructed using triple-negative breast cancer cells. Human triple-negative breast cancer cells were inoculated into experimental animals. After the tumors grew to a predetermined volume, the experimental animals were randomly divided into a blank control group (Naive), a model control group (Vehicle), and a drug treatment group. The drug treatment group was treated with ethyl acetate extract of chicory, while the blank control group and the model control group were treated with the same volume of solvent under the same conditions. During the drug treatment period, the tumor volume of each group of experimental animals was measured regularly, and changes in the animals' activity, food and water intake, weight loss, appearance changes of hair and eyes, death, and other clinical symptoms were observed and recorded. After the experiment, the tumor tissue of each group of experimental animals was removed and weighed to evaluate the effect of ethyl acetate extract of chicory on tumor growth.

[0034] The experimental results showed that, compared with the model control group, the tumor volume growth in the group treated with ethyl chicory extract was inhibited, indicating that ethyl chicory extract has an inhibitory effect on the growth of triple-negative breast cancer tumors under in vivo conditions. The above results suggest that ethyl chicory extract can be used as a natural active extract for the preparation of drugs or drug compositions for the prevention and treatment of triple-negative breast cancer; see Table 1.

[0035] Table 1. Antitumor effects of the test drugs in each group ( )

[0036]

[0037] Example 3

[0038] Results of the detection of organ indices and tumor tissues in triple-negative breast cancer-bearing mice by ethyl chicory extract:

[0039] Based on Example 1, further tests were conducted on the tumor tissue and related indicators of the experimental animals after drug administration:

[0040] a. Effects on liver and spleen indices in experimental animals:

[0041] After the experiment, the experimental animals in each group were dissected, and the liver and spleen were weighed. The liver index and spleen index were calculated. The results showed that under the drug administration conditions, the liver index and spleen index of the group treated with ethyl chicory extract did not show any abnormal changes compared with the model control group (see Table 2).

[0042] Table 2 Spleen index and liver index of tumor-bearing mice in each group ( )

[0043]

[0044] b. HE staining observation of tumor tissue:

[0045] Tumor tissues from each group of experimental animals were stained with hematoxylin and eosin (HE) and their morphology was observed. The results showed that the tumor tissues in the model control group had relatively dense cell arrangement and relatively intact tissue structure; the tumor tissues in the group treated with ethyl acetate extract of chicory had relatively loose cell arrangement and local tissue structure changes.

[0046] c. Immunohistochemical detection of Ki-67 expression in tumor tissue:

[0047] Immunohistochemistry was used to detect the expression of Ki-67 in tumor tissues. The results showed that, compared with the model control group, the positive expression level of Ki-67 in tumor tissues treated with chicory ethyl acetate extract was decreased.

[0048] d. Detection of tumor tissue apoptosis index based on TUNEL staining:

[0049] The TUNEL assay was used to detect cell apoptosis in tumor tissues. The results showed that, compared with the model control group, the proportion of TUNEL-positive cells in the tumor tissues of the group treated with ethyl chicory extract increased.

[0050] e. Detection of proteins related to the PI3K / Akt / mTOR signaling pathway:

[0051] The expression of proteins related to the PI3K / Akt / mTOR signaling pathway in tumor tissues was detected by Western blotting. The results showed that compared with the model control group, the phosphorylation levels of Akt and S6 were reduced in the group treated with ethyl acetate extract of chicory, while the total protein expression level did not change significantly.

[0052] In summary, the above results showed that HE staining revealed significant changes in the morphology and structure of tumor cells, indicating that the ethyl acetate extract of chicory exerts its anti-tumor effect by inhibiting tumor cell proliferation or promoting apoptosis. Ki-67 immunohistochemical staining results showed that the drug effectively inhibited cell proliferation and reduced tumor cell growth, further confirming its inhibitory effect on tumors. TUNEL assay results showed that the ethyl acetate extract of chicory could inhibit tumor growth by promoting apoptosis and increasing the proportion of apoptotic cells. These biological changes are closely related to the inhibitory effect of the ethyl acetate extract of chicory on the PI3K / AKT / mTOR signaling pathway. Inhibition of the PI3K / AKT / mTOR pathway may lead to cell cycle arrest and activate apoptotic signals, thereby significantly affecting the proliferation and survival of tumor cells. This mechanism of action provides a clear molecular biological basis for the anti-tumor effect of the ethyl acetate extract of chicory and further supports its potential as a candidate drug for the treatment of triple-negative breast cancer.

Claims

1. The use of an ethyl acetate extract of chicory, characterized in that... The use of the ethyl acetate extract of chicory in the preparation of a drug for the prevention of triple-negative breast cancer.

2. The use of an ethyl acetate extract of chicory, characterized in that... The use of the ethyl acetate extract of chicory in the preparation of a drug for treating triple-negative breast cancer.

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