Application of penthorum chinense pursh glycoside A in preparation of products for treating or preventing colorectal cancer

Abstract

The invention provides application of penthorum chinense pursh glycoside A in preparation of products for treating or preventing colorectal cancer, and relates to the technical field of biological medicine, penthorum chinense pursh glycoside A can effectively inhibit proliferation and migration of colorectal cancer cell lines (HCT116 cells and HT29 cells) and induce cell cycle arrest so as to inhibit colorectal cancer, and in addition, penthorum chinense pursh glycoside A can be used for preparing products for treating or preventing colorectal cancer. The penthorum chinense pursh glycoside A can effectively inhibit the growth of colorectal tumors, reduce the weight of the colorectal tumors and reduce the volume of the colorectal tumors, and is good in safety and high in clinical applicability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an application of a chases yellow grass glycoside A in preparation of a product for treating or preventing colorectal cancer. BACKGROUND

[0002] Colorectal cancer is a high-incidence digestive tract malignant tumor occurring in the colorectal region, which is formed by the genetic mutation and abnormal growth of normal cells in the colorectal region. Although the surgical, chemotherapy, immunotherapy and other schemes are constantly iterated, the prevention and treatment of colorectal cancer have achieved remarkable results, but still face difficulties such as multi-drug resistance and narrow treatment window. Furthermore, traditional treatment methods such as chemotherapy can control colorectal tumors, but it often causes adverse reactions such as bone marrow suppression and immune function impairment, which seriously affects the quality of life of patients after surgery, increases the risk of metastasis and recurrence of colorectal tumors, and ultimately affects the long-term survival of patients.

[0003] With the development of scientific research, by inhibiting cell proliferation, metastasis and angiogenesis, inducing cell apoptosis, targeting ferroptosis, improving intestinal microecology, reversing chemotherapy resistance, regulating immune response, and involving the regulation of multiple signaling pathways, it has become the focus of current cancer research. Among them, traditional Chinese medicine has the characteristics of multi-component, multi-target and multi-pathway, with low toxicity, which can enhance the immunity of patients and improve the quality of life of patients during treatment and after surgery. Its relatively low cost makes it have broader application potential in developing countries. In clinical application, traditional Chinese medicine can significantly improve the quality of life of patients, curb disease progression, and have a positive impact on the prognosis of patients.

[0004] Chases yellow grass is a traditional food and medicine dual-purpose plant derived from the Miao area of China. Studies have shown that it has strong antioxidant capacity, can reduce lung damage, prevent alcoholic liver disease, and shows significant liver protection characteristics, and also shows treatment potential in the field of hepatocellular carcinoma treatment. Glycoside A is the main bioactive flavonoid compound in chases yellow grass, which has beneficial effects on various diseases such as diabetic vascular calcification, diabetic renal fibrosis and Alzheimer's disease. In addition, chases yellow grass glycoside A also has a wide range of pharmacological effects, including liver protection, antibacterial, antiviral, anti-liver cancer and regulation of angiogenesis. These studies provide very valuable potential basis for the development of chases yellow grass glycoside A in the application of treating or preventing colorectal cancer products. SUMMARY

[0005] Based on this, the purpose of the present application is to provide an application of chases yellow grass glycoside A in preparation of a product for treating or preventing colorectal cancer.

[0006] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: In one aspect, the application provides a use of a chases grass glycoside A in the preparation of a product for treating or preventing colorectal cancer, wherein the chases grass glycoside A has a chemical structure as shown in formula (I):

[0007] (I).

[0008] In one aspect of the above technical solution, the colorectal cancer cells include one or more of HCT116 cells and HT29 cells.

[0009] In one aspect of the above technical solution, the product is a drug for inhibiting the growth, proliferation and migration of HCT116 cells and HT29 cells, and inducing cell cycle arrest of HCT116 cells and HT29 cells.

[0010] In one aspect of the above technical solution, the product is a drug for inhibiting the growth of colorectal tumors, reducing the volume of colorectal tumors, and reducing the weight of colorectal tumors.

[0011] In one aspect of the above technical solution, the content of the chases grass glycoside A in the drug is 0.01wt%-99.99wt%.

[0012] In one aspect of the above technical solution, the drug further includes other pharmacodynamic components for treating or assisting in treating colorectal cancer.

[0013] In one aspect of the above technical solution, the drug further includes any one of a pharmaceutically acceptable carrier or excipient, including an excipient, a solubilizer, a preservative, a stabilizer, a wetting agent, an emulsifier, a salt for adjusting osmotic pressure, and a buffer.

[0014] In one aspect of the above technical solution, the dosage form of the drug is any one of a pharmaceutically acceptable dosage form, including a powder injection, an injection solution, a tablet, a pill, a capsule, a spray, and a dispersion.

[0015] In one aspect of the above technical solution, the administration route of the drug is any one of a pharmaceutically acceptable route, including oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and transdermal, intranasal or oral inhalation.

[0016] In one aspect of the above technical solution, the chases grass glycoside A is extracted from chases grass.

[0017] Due to the above technical solution, the application has the following advantages and beneficial effects: The active compound in traditional Chinese medicinal materials, chases the yellow grass glycoside A, can effectively inhibit the growth, proliferation and migration of colorectal cancer cell lines, and induce the colorectal cancer cell lines to be blocked in the cell cycle. Experiments prove that the chase yellow grass glycoside A can effectively inhibit the proliferation and migration of colorectal cancer cell lines (HCT116 cells and HT29 cells), and induce cell cycle arrest, thereby realizing the inhibition of colorectal cancer, and showing a concentration-dependent effect of inhibiting colorectal cancer.

[0018] Secondly, the chase yellow grass glycoside A can effectively inhibit the growth of colorectal tumors, reduce the weight of colorectal tumors, and reduce the volume of colorectal tumors. Mouse model experiments prove that the chase yellow grass glycoside A can significantly inhibit the growth of colorectal tumors and reduce the tumor weight, and the effect is equivalent to that of the chemotherapeutic drug 5-fluorouracil. The chase yellow grass glycoside A is extracted from the traditional Chinese medicine chase yellow grass and belongs to natural flavonoids, which has the characteristics of multiple components and multiple targets. During the administration process, the weight of the mice does not change, and the safety is good. Moreover, the low dose (5 mg / kg) is effective, and the clinical applicability is strong.

[0019] In addition, the chase yellow grass glycoside A can be extracted from the plant chase yellow grass at a low cost compared to chemical synthesis, and the plant chase yellow grass can also be cultivated on a large scale. It can be made into injection, oral preparation and other preparations, covering various administration routes, reducing production cost and reducing the burden on patients. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The CCK-8 experimental detection results of the chase yellow grass glycoside A on colorectal cancer cell lines (HCT116 cells and HT29 cells) in Example 1 of the present application are shown in the schematic diagram; Figure 2 The cell scratch experiment results of the chase yellow grass glycoside A on colorectal cancer cell lines (HCT116 cells and HT29 cells) in Example 2 of the present application are shown in the schematic diagram; Figure 3 The colony formation experiment results of the chase yellow grass glycoside A on colorectal cancer cell lines (HCT116 cells and HT29 cells) in Example 3 of the present application are shown in the schematic diagram; Figure 4 The flow cytometry cycle results of the chase yellow grass glycoside A on HCT116 cells in Example 4 of the present application are shown in the schematic diagram; Figure 5 The flow cytometry cycle results of the chase yellow grass glycoside A on HT29 cells in Example 4 of the present application are shown in the schematic diagram; Figure 6 The change of the weight of the mice in different groups in Example 5 of the present application is shown in the schematic diagram; Figure 7 The change of the volume of the mice in different groups in Example 5 of the present application is shown in the schematic diagram; Figure 8 The colorectal tumor effect diagram of the mice in different groups in Example 5 of the present application is shown in the schematic diagram; Figure 9 A schematic diagram of tumor weight of mice in different groups in Example 5 of the present application; The following detailed description will further describe the present application with reference to the above-mentioned drawings. DETAILED DESCRIPTION

[0021] For the purpose of understanding the present application, a more complete description of the present application will be made with reference to the relevant drawings. Several embodiments of the present application are shown in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the specification of the present application herein is only for the purpose of describing specific embodiments and is not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the associated listed items.

[0023] The present application provides a use of Thonningianin A in the preparation of a product for treating or preventing colorectal cancer, wherein the Thonningianin A has a chemical structure as shown in formula (I):

[0024] (I).

[0025] Further, the English name of Thonningianin A is Thonningianin A; the molecular formula is C 42 H 34 O 21 .

[0026] In a preferred embodiment, the cells of colorectal cancer of the present application include one or more of HCT116 cells and HT29 cells.

[0027] In a preferred embodiment, the product is a drug for inhibiting the growth, proliferation and migration of HCT116 cells and HT29 cells, and inducing cell cycle arrest of HCT116 cells and HT29 cells.

[0028] In some preferred embodiments, the product is a drug for inhibiting the growth of colorectal tumor, reducing the volume of colorectal tumor, and reducing the weight of colorectal tumor.

[0029] In some preferred embodiments, the content of Thonningianin A in the drug is 0.01wt%-99.99wt%. Thonningianin A is extracted from Thonningia Sanderi.

[0030] In some preferred embodiments, the medicament further comprises other pharmaceutical ingredients for treating or adjuvant treating colorectal cancer.

[0031] In some preferred embodiments, the medicament further comprises any one of pharmaceutically acceptable carriers or excipients, including excipients, solubilizers, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers.

[0032] In some preferred embodiments, the medicament is in any one of pharmaceutically acceptable dosage forms, including powder injection, injection solution, tablet, pill, capsule, spray, dispersion.

[0033] In some preferred embodiments, the medicament is administered by any one of pharmaceutically acceptable routes, including oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and transdermal, intranasal or oral inhalation.

[0034] Further, the chases Huangcao glycoside A is extracted from chases Huangcao.

[0035] In addition, the colorectal cancer includes cancer of the colon, cancer of the rectum, and cancer of both the colon and the rectum.

[0036] The present application is further illustrated in the following specific examples: In the figures, **** indicates p<0.0001, the probability of difference caused by random error is less than 0.01%, the statistical evidence is extremely strong; *** indicates p<0.001, the probability of difference caused by random error is less than 0.1%, the statistical evidence is strong; * indicates p<0.05, the probability of difference caused by random error is less than 5%, the statistical evidence is moderate.

[0037] Example 1 CCK-8 cell viability assay Cell counting kit-8 (CCK-8) was used to quantitatively evaluate cell activity. The specific steps are as follows: colorectal cancer cell lines (HCT116 cells and HT29 cells) were cultured, and HCT116 cells and HT29 cells were respectively inoculated at 1×10 4HCT116 and HT29 cells were seeded at a density of [number] cells / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 hours. After adherence, the appropriate drugs (glucosinolate A: 1 μM, 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM) were added, and the cells were cultured for another 24 hours. After drug treatment, 10 μl of CCK-8 reagent was added to each well using a pipette. The plates were then incubated in a controlled environment for 1 hour to ensure adequate reaction and color development. After incubation, the optical density (OD) value at 450 nm was measured using a microplate reader, and the viability of HCT116 and HT29 cells was quantitatively analyzed by colorimetric intensity.

[0038] like Figure 1 Figure A (schematic diagram of CCK-8 assay results for HCT116 cells) and Figure B (schematic diagram of CCK-8 assay results for HT29 cells) show that the horizontal axis LogC represents the logarithm of the concentration of ginsenoside A. The results show that as the concentration of ginsenoside A increases, the inhibition rate of both HCT116 cells and HT29 cells shows a significant upward trend, indicating that the concentration of ginsenoside A has a significant inhibitory effect on the proliferation of colorectal cancer cell lines (HCT116 cells and HT29 cells), and this inhibitory effect is concentration-dependent.

[0039] Example 2 Wound healing experiment Logarithmic growth state colorectal cancer cell lines (HCT116 cells and HT29 cells) were respectively fed with 1.8 × 10⁻⁶ cells. 6 Cells were seeded per well in six-well plates and incubated at 37°C for 24 h in a 5% CO2 incubator. Once HCT116 and HT29 cells reached confluence (95% confluence), a straight line perpendicular to the back of the plate was drawn using a 200 μL sterile pipette tip. After washing with PBS to remove cell debris, cells were administered different concentrations of scutellarin A (0, 6.25 μM, 12.5 μM), with a control group (without scutellarin A). Scratch images were captured and recorded at 0 and 24 hours, and cell migration area was measured using ImageJ software.

[0040] like Figure 2As shown in FIG. 1A (a cell scratch effect diagram of the colorectal cancer cell line), FIG. 1B (a schematic diagram of the cell migration rate experiment results of the colorectal cancer cell line), the results show that, from the control group, compared with 0 h, after 24 h of culture, the scratch area of HCT116 cells and HT29 cells has a certain degree of healing, indicating that HCT116 cells and HT29 cells have migration ability. With the increase of the concentration of Chuanhuangcao Glycoside A, the ability of HCT116 cells and HT29 cells to fill the scratch is gradually weakened, which shows that Chuanhuangcao Glycoside A can effectively inhibit the migration of colorectal cancer cell lines (HCT116 cells and HT29 cells).

[0041] Example 3 Cloning experiment A 6-well plate was spread with 1000 colorectal cancer cell lines (HCT116 cells and HT29 cells) per well and cultured overnight, and then treated with culture medium containing different concentrations of Chuanhuangcao Glycoside A (0, 6.25 μM, 12.5 μM, 25 μM), wherein no Chuanhuangcao Glycoside A was added as a control group (Control). The culture medium was replaced every 3 days. After the HCT116 cells and HT29 cells were visible to the naked eye, they were washed with PBS twice, fixed in a 4 wt% paraformaldehyde solution for 30 minutes, and stained with crystal violet for 15 minutes. Then, they were washed with distilled water, dried, photographed in a place with sufficient light, and analyzed using ImageJ software.

[0042] As Figure 3 As shown in FIG. 2A (a cell cloning effect diagram of the colorectal cancer cell line), FIG. 2B (a schematic diagram of the monoclonal number experiment results of the colorectal cancer cell line), the results show that, with the increase of the concentration of Chuanhuangcao Glycoside A, the number and size of the clones formed by HCT116 cells and HT29 cells are significantly reduced. At a higher concentration of Chuanhuangcao Glycoside A (such as 12.5 μM and 25 μM), the cloning ability is almost completely inhibited, which further confirms the inhibitory effect of Chuanhuangcao Glycoside A on the proliferation and survival ability of colorectal cancer cell lines (HCT116 cells and HT29 cells).

[0043] Example 4 Flow cytometry cycle experiment Using flow cytometry of cell cycle, 3x10 5Density spread 6-well plates of two colorectal cancer cell lines (HCT116 cells and HT29 cells) and grow overnight. After the HCT116 cells and HT29 cells adhere to the wall, different concentrations of Chaseside A (0, 2.5 μM, 5 μM) are added, and no Chaseside A is added as the control group (Control). After 24 hours, the cells are collected and rinsed twice with pre-cooled PBS. The HCT116 cells and HT29 cells cycle is detected by using propidium iodide (PI) staining solution. After mixing the sample with the staining solution, incubate at 37°C for 30 minutes in the dark, and analyze the HCT116 cells and HT29 cells cycle by using a flow cytometer.

[0044] As shown in Figs. 13A (the effect diagram of the flow cytometry cycle experiment results of HCT116 cells), B (the schematic diagram of the flow cytometry cycle experiment results of HCT116 cells), Figure 4 As shown in Figs. 13A (the effect diagram of the flow cytometry cycle experiment results of HCT116 cells), B (the schematic diagram of the flow cytometry cycle experiment results of HCT116 cells), Figure 5 As shown in Figs. 13A (the effect diagram of the flow cytometry cycle experiment results of HCT116 cells), B (the schematic diagram of the flow cytometry cycle experiment results of HCT116 cells),

[0045] For HCT116 cells, compared with the control group, the proportion of HCT116 cells in the G1 phase significantly increased, and the proportions in the S phase and the G2 phase decreased after the treatment of 5.0 μM Chaseside A. The similar trend was also shown by the treatment of 2.5 μM Chaseside A, but the degree was relatively light.

[0046] For HT29 cells, compared with the control group, the proportion of HT29 cells in the G1 phase increased, and the proportions in the S phase and the G2 phase decreased after the treatment of 5.0 μM Chaseside A. The proportion of HT29 cells in the G1 phase slightly increased, the proportion in the S phase decreased, and the proportion in the G2 phase changed little after the treatment of 2.5 μM Chaseside A.

[0047] This shows that Chaseside A can inhibit the proliferation of HCT116 cells and HT29 cells by inducing the G1 phase arrest of HCT116 cells and HT29 cells. This indicates that there may be some differences in the cell cycle regulation of Chaseside A on different colorectal cancer cell lines (HCT116 cells and HT29 cells), but Chaseside A generally shows the inhibitory effect on the cell cycle of HCT116 cells and HT29 cells.

[0048] Example 5 Construction of a mouse subcutaneous colorectal tumor model A total of 24 female BALB / c nude mice, 6 weeks old, weighing 18 g-22 g, were raised in a barrier facility under standard conditions with a 12-hour light and dark cycle. HT29 cells (5 x 10 6subcutaneously injected into the left axillary region of each mouse. The following formula was used to measure the colorectal tumor volume every three days: [0.5 x (long diameter) x (short diameter) 2 ]. And the body weight of mice was measured every three days.

[0049] Animal experimental dosing regimen When the colorectal tumor volume reached about 50mm 3 (approximately one week), the mice were randomly divided into 4 groups of 6 animals each: control group (Control, 1% absolute ethanol), Tachalins A low dose group (TA-L, 5 mg / kg), Tachalins A high dose group (TA-H, 10 mg / kg), and 5-fluorouracil positive control group (5-Fu, 15 mg / kg). All dosing was administered by gavage at a volume of 100 μL per day. Daily dosing was continued for 19 days, after which the animals were anesthetized and subsequently sacrificed by cervical dislocation, the colorectal tumor was excised and weighed, and then half of the colorectal tumor was fixed in 4 wt% paraformaldehyde solution and the other half was stored frozen at -80°C.

[0050] As shown in Figure 6 , the results show that Tachalins A was able to inhibit colorectal tumor growth while not affecting the body weight of the mice. There was no significant difference in the body weight of the mice in each group throughout the dosing period, indicating that Tachalins A treatment did not cause significant toxicity.

[0051] As shown in Figure 7 A (graph of the results of the colorectal tumor volume experiment after 19 days of dosing), B (graph of the change in colorectal tumor volume during the 19 days of dosing), by measuring the colorectal tumor volume, it was found that the colorectal tumor growth in the Tachalins A low dose group and the Tachalins A high dose group was significantly inhibited compared to the control group. In particular, the colorectal tumor volume in the Tachalins A high dose group was significantly reduced, with a trend similar to that of the 5-fluorouracil positive control group. By the end of the 19 days of dosing, the colorectal tumor volume in the Tachalins A high dose group was about 50% lower than that of the control group.

[0052] At the end of the experiment, the colorectal tumors were excised and weighed. As shown in Figures 8-9 , the colorectal tumor weight in the Tachalins A low dose group and the Tachalins A high dose group was significantly lower than that of the control group. Among them, the colorectal tumor weight in the Tachalins A high dose group was most significantly reduced, similar to the 5-fluorouracil positive control group. This confirms that Tachalins A can effectively inhibit colorectal tumor growth without showing significant toxicity.

[0053] In summary, when the two colorectal cancer cell lines HCT116 and HT29 were treated with Chuanhuangcao glycoside A, the cell proliferation of the two colorectal cancer cell lines was inhibited in a concentration-dependent manner, and the cell proliferation inhibition rate gradually increased with the increase of the concentration of Chuanhuangcao glycoside A, indicating that Chuanhuangcao glycoside A had a direct inhibitory effect on the two tumor cells and could hinder their growth and proliferation process.

[0054] In addition, the proportions of HCT116 cells and HT29 cells in G1, S, and G2 phases changed with different concentrations of Chuanhuangcao glycoside A, indicating that Chuanhuangcao glycoside A could interfere with the normal process of the cell cycle of HCT116 cells and HT29 cells, make HCT116 cells and HT29 cells arrest at a certain specific cycle phase, and thus prevent the further division and proliferation of HCT116 cells and HT29 cells.

[0055] The drug administration experiment in nude mice showed that compared with the control group, the growth of colorectal tumor volume and weight was significantly inhibited after administration of Chuanhuangcao glycoside A, and the tumor inhibition effect showed a certain dose-dependent manner, further confirming that Chuanhuangcao glycoside A had an inhibitory effect on the growth of colorectal tumors and could slow down or even prevent the development of colorectal tumors in vivo.

[0056] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0057] The above-described embodiments only express several implementation manners of the present application, and the description is more specific and detailed, but it should not be understood as a limitation on the scope of the patent of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Use of aesculin A in the preparation of a product for the treatment or prevention of colorectal cancer, characterized in that, The chases the yellow grass glycoside A as formula (I) shows chemical structure: （I） 。 2. Use according to claim 1, characterized in that, The colorectal cancer cells include one or more of HCT116 cells and HT29 cells.

3. Use according to claim 2, characterized in that, The product is a drug for inhibiting the growth, proliferation and migration of HCT116 cells and HT29 cells, and inducing cell cycle arrest of HCT116 cells and HT29 cells.

4. Use according to claim 2, characterized in that, The product is a drug for inhibiting the growth of colorectal tumor, reducing the volume of colorectal tumor, and reducing the weight of colorectal tumor.

5. Use according to claim 3 or 4, characterized in that, The content of the chases the yellow grass glycoside A in the drug is 0.01wt%-99.99wt%.

6. Use according to claim 5, characterized in that, The drug further includes other pharmaceutical ingredients for treating or assisting in treating colorectal cancer.

7. Use according to claim 5, characterized in that, The drug further includes any one of pharmaceutically acceptable carriers or excipients, including excipients, solubilizers, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers.

8. Use according to claim 5, characterized in that, The dosage form of the drug is any one of pharmaceutically acceptable dosage forms, including powder injection, injection, tablet, pill, capsule, spray, dispersion.

9. Use according to claim 5, characterized in that, The administration route of the drug is any one of pharmaceutically acceptable routes, including oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and transdermal, intranasal or oral inhalation.

10. The use according to claim 1, characterized in that, The chases the yellow grass glycoside A is extracted from the chases the yellow grass.

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