CN108578407B

CN108578407B - Application of liensinine perchlorate in preparation of anti-colorectal cancer drugs

Info

Publication number: CN108578407B
Application number: CN201810527429.4A
Authority: CN
Inventors: 李斌; 何庆瑜; 李杨葭
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-05-29
Filing date: 2018-05-29
Publication date: 2020-04-14
Anticipated expiration: 2038-05-29
Also published as: CN108578407A

Abstract

The invention discloses application of liensinine perchlorate in preparation of an anti-colorectal cancer drug. The liensinine perchlorate is white needle crystal, is a quaternary ammonium salt derivative generated by combining liensinine which is a main effective component of lotus plumule with perchloric acid, and has a structural formula I shown in the specification. The embodiment of the invention proves that the liensinine perchlorate can inhibit the proliferation of human colorectal cancer cells and promote apoptosis, can inhibit the growth of human colorectal cancer cell transplantation tumor in a nude mouse body, and can not cause abnormal weight increase and decrease of the nude mouse. The liensinine perchlorate is cheap and can be widely applied to the preparation of anti-colorectal cancer drugs.

Description

Application of liensinine perchlorate in preparation of anti-colorectal cancer drugs

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of liensinine perchlorate in preparation of an anti-colorectal cancer medicine.

Background

Cancer is one of the most refractory diseases in the world. Colorectal cancer (CRC) is the second leading mortality in malignant tumors worldwide. At present, the treatment of intestinal cancer is limited to radiotherapy, chemotherapy and the like. Although the radiotherapy and chemotherapy can inhibit most of cancer cells, normal cells can be killed while the cancer cells are killed, gastrointestinal dysfunction, bone marrow inhibition and other toxic and side effects can be caused, and the life quality of patients is greatly reduced. Meanwhile, drug resistance is easily generated by malignant tumor cells after long-term chemotherapy. Most of the drugs for treating cancer are expensive, and the application of the drugs is limited. Therefore, the search for a safe anticancer drug with little toxic and side effect, good drug effect and low price is urgent.

The liensinine perchlorate is white needle crystal, and is quaternary ammonium salt derivative produced by combining liensinine as the main effective component of lotus plumule with perchloric acid. The relationship between liensinine perchlorate and colorectal cancer cell growth is not clear.

Disclosure of Invention

In order to overcome the defects and shortcomings in the prior art, the invention aims to provide the application of liensinine perchlorate in preparing anti-colorectal cancer drugs.

The liensinine perchlorate has a structural formula shown as I, and is characterized by being applied to preparation of anti-colorectal cancer drugs.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the liensinine perchlorate is a natural product with homology of medicine and food, has small toxic and side effect and low price, can be dissolved by water and taken orally, and can be used for adjuvant therapy of colorectal cancer.

(2) The lotus plumule alkali perchlorate is cheap and has good development and utilization prospect.

Drawings

FIG. 1 shows the results of experiments on the growth inhibition of DLD-1 by liensinine perchlorate in example 1.

FIG. 2 shows the results of the experiment on the growth inhibition of HT29 by liensinine perchlorate in example 1.

FIG. 3 is an apoptotic flow image of DLD-1 treated with liensinine perchlorate in example 2.

FIG. 4 is a flow chart of apoptosis quantification statistics of example 2 liensinine perchlorate on DLD-1 treatment.

FIG. 5 is an apoptotic flow image of HT29 treatment with liensinine perchlorate in example 2.

FIG. 6 is a flow chart of the apoptosis quantification statistic of example 2 liensinine perchlorate on HT29 treatment.

FIG. 7 is a set of graphs showing the results of experiments on subcutaneous neoplasia HT29 in nude mice in example 3.

FIG. 8 is a graph showing the growth of experimental tumors of HT29 subcutaneous tumor formation in nude mice of example 3.

FIG. 9 is a graph showing the body weight changes of mice in the experimental group and the control group in the experiment of subcutaneous tumorigenesis of HT29 in nude mice of example 3.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.

The following examples refer to HT29 (human colorectal adenocarcinoma epithelial cells, available from the cell bank of Chinese academy of sciences), DLD-1 (human colon carcinoma cells, available from the cell bank of Chinese academy of sciences).

Example 1

In vitro experiments: growth inhibition of liensinine perchlorate on colorectal cancer cells. Detection was by the WST-1 method. The method comprises the following specific steps:

(1) HT29 and DLD-1 were plated into 96-well plates, 3000 cells per well, respectively;

(2) dissolving liensinine perchlorate (purchased from Shanghai ceramic Biotechnology Co., Ltd.) in DMSO (dimethyl sulfoxide) to prepare stock solution with concentration of 10mM, and diluting the stock solution with RPMI1640 cell culture medium (purchased from Life Technologies, Gaithersburg, Md., USA) to prepare working solution with final concentration of 0 μ M, 5 μ M, 10 μ M, 20 μ M;

(3) after the cells in the step (1) adhere to the wall, adding the working solution prepared in the step (2) with different concentrations into corresponding holes according to the volume of 100 mu L per hole, and detecting the activity of the cells by using a WST-1 cell proliferation and cytotoxicity detection kit (purchased from Bilun sky biotechnology, Inc.) after the working solution is treated for 48 hours;

(4) 3 biological replicates were performed over the above 3 experimental steps.

Example 2

In vitro experiments: the apoptosis-promoting effect of liensinine perchlorate on colorectal cancer cells. The method comprises the following steps:

HT29 and DLD-1 were placed in 12-well plates, 2X 10 per well, respectively⁷After the cells adhered to the wall, the cells were treated with a working solution with a concentration gradient (0 μ M, 5 μ M, 10 μ M, 20 μ M), 1mL of the working solution was added to each well for 48 hours, the cells were treated with a cell cycle and apoptosis assay kit (available from Byuntian Biotechnology Co., Ltd.) and then subjected to apoptosis assay with a C6Flow Cytometer (available from BD Accuri Co., Ltd.), and the above experimental procedures were repeated 3 times (the sources of HT29 and DLD-1 used in this step and the preparation method of the working solution were the same as in example 1).

Example 3

In vivo experiments: the inhibition effect of liensinine perchlorate on the growth of human colorectal cancer cell transplantation tumor in nude mice. The method comprises the following steps:

(1) 12 female nude mice (balb/c-nu/nu) (purchased from southern medical university) of 6 weeks old were selected and randomly divided into 6 each of the control group and the experimental group to construct a tumor metastasis model.

(2) Counting and re-suspending the digested HT29 in PBS buffer solution, uniformly mixing the digested HT29 with matrigel according to the volume ratio of 1:1, anesthetizing the nude mice before the experiment, evaluating the anesthesia degree through painless and painful stimulation, and determining that the nude mice are in an anesthetic state; nude mice were injected subcutaneously with HT29 resuspended in PBS using a 25G needle microinjector, which was 2X 10 per nude mouse⁶HT 29.

(3) After day 0 of HT 291injection, mice were dosed intragastrically. The liensinine perchlorate is dissolved in pure water, and the drug concentration of each group of mice is 0mg/kg (control group) and 30mg/kg (experimental group), each mouse is perfused with 100 mu L of stomach, and the drug is administrated once every 1 day for 3 weeks, and the weight and the tumor volume of the mice are measured every 2 days.

Explanation regarding the drawings:

(1) liensinine perchlorate can inhibit proliferation and promote apoptosis of human colorectal cancer cells.

As shown in fig. 1, 2, the proliferative activity of colorectal cancer cells decreased with increasing concentrations of liensinine perchlorate, and this effect was more pronounced with time; as shown in fig. 4 and 6, apoptosis of colorectal cancer cells increased with the increase of the concentration of liensinine perchlorate.

(2) Liensinine perchlorate can inhibit the growth of human colorectal cancer cell transplantation tumor in nude mice.

FIG. 7 is a real figure showing the results of experiments on subcutaneous tumor formation in nude mice, FIG. 8 is a tumor growth curve, and FIGS. 7 and 8 show that subcutaneous tumor formation in nude mice is significantly inhibited by liensinine perchlorate. In fig. 9, it is shown that there is no significant difference in body weight between the two groups of mice, demonstrating the safety of use of liensinine perchlorate as an antitumor drug.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. Application of liensinine perchlorate in preparing medicine for treating colorectal cancer is provided.