CN110946948A

CN110946948A - Application of Huafengdan in preparation of anti-breast cancer drugs

Info

Publication number: CN110946948A
Application number: CN201911190541.4A
Authority: CN
Inventors: 宋晶睿; 李艳梅; 宋佳蕾; 饶青
Original assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Current assignee: Key Laboratory of Natural Product Chemistry of Guizhou Academy of Sciences
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2020-04-03

Abstract

The invention discloses an application of a wind-evil dispelling pill in preparing an anti-breast cancer medicament. The inventor finds that the known wind-evil dispelling pill has a good inhibition effect on the proliferation of breast cancer cells, can be used as an effective component for preparing an anti-breast cancer medicament, and provides a basis for developing a new application of the wind-evil dispelling pill. Moreover, the Huafengdan is a marketed Miao medicine, can be directly applied to human bodies without clinical safety evaluation, and can rapidly improve the market share of the product.

Description

Application of Huafengdan in preparation of anti-breast cancer drugs

Technical Field

The invention belongs to the technical field of biology and pharmacy, and particularly relates to an application of a wind-evil dispelling pellet in preparation of an anti-breast cancer medicine.

Background

The Miao medicine Feng Dan is produced and manufactured by Guizhou Wansheng pharmaceutical industry Limited liability company, and the pill has the following existing effects: extinguish wind, relieve spasm, eliminate phlegm and induce resuscitation. Can be used for treating wind phlegm obstruction, apoplexy, hemiplegia, epilepsy, facial paralysis, and facial distortion. The main components comprise more than 20 traditional Chinese medicines such as gastrodia elata, scorpion, stiff silkworm, giant typhonium rhizome, rhizoma atractylodis, rhizoma arisaematis, musk and the like. The cross section of a vermilion watered pill is brownish yellow; has strong fragrance and pungent taste.

Breast cancer is the malignant tumor with the highest morbidity and mortality of women all over the world, and seriously threatens the health of women. The existing comprehensive treatment comprises a series of treatment modes such as operation, chemotherapy, radiotherapy and the like which are generally accepted, however, complications brought by the treatment modes bring certain influence on patients, and the effect of further improving the curative effect of breast cancer is limited. Compared with the traditional treatment, the traditional Chinese medicine has the characteristics of multiple target points, small toxic and side effects, good effect and the like in treating the breast cancer.

Disclosure of Invention

The invention aims to provide a new application of the Huafengdan.

Aiming at the purposes, the invention provides an application of a wind-dispelling pill in preparing a medicine for preventing and treating breast cancer.

The application of the wind-evil dispelling pill in preparing the medicine for preventing and treating the breast cancer can directly prepare the wind-evil dispelling pill into a preparation for resisting the breast cancer or prepare a compound preparation by compounding the wind-evil dispelling pill with other medicines for resisting the breast cancer, or prepare the effective components of the wind-evil dispelling pill and a pharmaceutically acceptable carrier into a preparation for resisting the breast cancer. The preparation is any one of tablets, injections, capsules, oral liquid and dropping pills.

The cancer cell of the breast cancer is any one of human breast cancer cell MDA-231, human breast cancer cell BT474, rat breast cancer cell FE1.2 and rat breast cancer cell FE 12.5.

The invention discovers that the Huafengdan has obvious anti-breast cancer effect through in vitro (applying human breast cancer cells and rat breast cancer cells) and in vivo (breast cancer animal models) detection, and embodies the important potential of the Huafengdan as an anti-breast cancer medicament.

Compared with the existing usage of the wind-evil dispelling pill, the inventor finds that the wind-evil dispelling pill has good in-vivo and in-vitro inhibition effects on breast cancer, and finds new usage of the medicine. And the Huafengdan is a marketed Miao medicine, can be directly applied to a human body without clinical safety assessment, has good application prospect, and can quickly improve the market share of the product.

Drawings

FIG. 1 shows the results of the inhibition test of the growth of human breast cancer cells MDA-231 and BT474 by Huafengdan.

FIG. 2 shows the results of the inhibition test of the growth of rat breast cancer cells FE1.2 and FE12.5 by Huafengdan.

FIG. 3 is the detection of human breast cancer MDA-231 apoptosis by flow cytometry.

FIG. 4 is the detection of human breast cancer cell MDA-231 cell cycle by flow cytometry at 24h dosing.

FIG. 5 is a flow cytometer with 48h of medicine added to detect human breast cancer cell MDA-231 cell cycle.

FIG. 6 is the detection of the treatment of breast cancer in mice with Huafengdan.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Example 1

Application of Huafengdan in preparation of medicine for preventing and treating breast cancer

Detecting the activity of the Huafengdan for inhibiting the growth of the breast cancer cells

1. Experimental Material

Preparing a sample solution: the Liao's Huafengdan is prepared into sample solutions with the concentrations of 50 mug/mL, 25 mug/mL, 12.5 mug/mL, 6.25 mug/mL and 3.125 mug/mL by DMSO respectively.

Preparation of 5% MTT solution: 0.5g of MTT powder was weighed, 100mL of sterilized PBS was added, dissolved at 60 ℃, filtered and sterilized with a 0.22 μm filter membrane, and stored at 4 ℃.

Cell line: human breast cancer cells MDA-231 and BT474, rat breast cancer cell FE1.2 and FE12.5, both of which are chemical key laboratory preservations of natural products of Chinese academy of sciences of Guizhou province. Human breast cancer cells MDA-231 and BT474, rat breast cancer cells FE1.2 and FE12.5, 5% CO at 37 deg.C₂The culture was performed in a culture box using DMEM medium containing 5% FBS.

DMEM medium and pancreatin were purchased from Hycone, Fetal Bovine Serum (FBS) from Shanghai StartBiotech, Inc., and Tetramethylazo Living salt (MTT) and dimethyl sulfoxide (DMSO) from Beijing Solibao Biotech, Inc.

2. Inhibition of breast cancer cell proliferation in vitro

(1) MTT method for determining inhibition proliferation effect of different concentrations of Huafengdan on breast cancer cells

Respectively taking human breast cancer cells MDA-231 and BT474 and rat breast cancer cells FE1.2 and FE12.5 cells of logarithmic growth phase at a ratio of 2.5 × 10⁴Each cell was inoculated at a density of 190. mu.L of DMEM medium containing 5% FBS in a 96-well culture plate, and dosed 6 hours after the adherent cells were attached, 10. mu.L of each sample solution containing different concentrations of Plumbum preparatium (3.125. mu.g/mL, 6.25. mu.g/mL, 12.5. mu.g/mL, 25. mu.g/mL, 50. mu.g/mL) was added to each well, 5 duplicate wells were placed at each concentration, 5 cells were each in parallel, and 5% CO was added at 37 ℃ to each well₂Culturing for 72h in incubator, adding 20 μ L of 5% MTT solution (protected from light), and placing in CO₂The culture was continued for 4h in the incubator, centrifuged (1500rpm, 15min), the supernatant was discarded, 150. mu.L of DMSO was added to each well, the mixture was shaken on a shaker (10min, 180rpm, 25 ℃), the OD was measured at 490nm with a microplate reader, and the inhibition rate was calculated.

The detection result shows that: the Huafengdan has obvious inhibition effect on the growth of 4 breast cancer cells (see figure 1 and figure 2), has obvious concentration dependence on the growth inhibition of the breast cancer cells, and has IC (integrated Circuit) of human breast cancer cells MDA-231 and BT474 and rat breast cancer cells FE1.2 and FE12.5 cells₅₀The values were 28.6. mu.g/mL, 68.99. mu.g/mL, 8.73. mu.g/mL, and 20.99. mu.g/mL, respectively. From the data, the Huafengdan has potential value for developing anti-breast cancer drugs. In addition, the research shows that the effective components of the wind-dispelling pill are prepared into tablets, injections, capsules, oral liquid or dropping pills according to the experimental dataIt also has the same growth inhibitory effect.

(2) Flow cytometry for determining human breast cancer cell MDA-231 apoptosis change

Inoculating human breast cancer cell MDA-231 into a circular culture dish with the diameter of 60mm, placing the circular culture dish in an incubator at the temperature of 37 ℃ for 6h to allow the cell to adhere to the wall, adding sample solutions containing different concentrations of the propyza sativa L, wherein the final concentrations are respectively 5 mu g/mL, 10 mu g/mL and 20 mu g/mL, adding the same amount of DMSO into a control group, and adding 5% CO at the temperature of 37 ℃ to obtain a mixture₂Culturing in an incubator, collecting cells with drug addition of 24h and 48h, washing with PBS 3 times, adding 500 μ L of 1 × Binding Buffer to resuspend the cells, adding 5 μ L of annexin V-FITC and 10 μ L of propidium iodide, incubating for 15min at room temperature in a dark place, detecting and calculating the apoptosis rate by using a flow cytometer, and obtaining the result shown in figure 3. The results show that: the Huafengdan has obvious effect on the apoptosis of human breast cancer cells MDA-231.

(3) Flow cytometry for measuring human breast cancer cell MDA-231 cell cycle change

Inoculating human breast cancer cell MDA-231 into a circular culture dish with the diameter of 60mm, placing the circular culture dish in an incubator at the temperature of 37 ℃ for 6h to allow the cell to adhere to the wall, adding sample solutions containing different concentrations of the propyza sativa L, wherein the final concentrations are respectively 5 mu g/mL, 10 mu g/mL and 20 mu g/mL, adding the same amount of DMSO into a control group, and adding 5% CO at the temperature of 37 ℃ to obtain a mixture₂Culturing in incubator, collecting cells dosed for 24h and 48h, fixing with 500 μ L of 75% ethanol aqueous solution, staining with 50 μ g/mL propidium iodide solution (containing 100 μ g/mLRNase, prepared from PBS buffer) for 30min, and performing cell cycle analysis on flow cytometer, the results are shown in FIG. 4 and FIG. 5.

The streaming results of fig. 4 and 5 show that: the Huafengdan has no obvious influence on the MDA-231 of the human breast cancer cells when being added with the medicine for 24 hours, but has obvious influence on the MDA-231 cycle of the human breast cancer cells when being added with the medicine for 48 hours, and blocks the cell cycle in the G2/M phase, and the higher the medicine concentration is, the more obvious the block is.

3. Inhibition of breast cancer cell proliferation in vivo

Constructing an animal model: a xenograft-type experimental animal model, i.e., a human breast cancer cell line transplanted in an immunodeficient mouse, was used. The cell line used in this experiment was human breast cancer cell MDA-231, and the mouse used was SCID mouse purchased from Donghong in ChongqingThe animal technology company Limited, raised in SPF-level laboratory animal houses. Human breast cancer cell MDA-231 is cultured to the logarithmic phase of growth, 2.5 × 10⁶Each MDA-231 cell was injected subcutaneously into both axilla of 4-week-old SCID mice.

The administration mode comprises the following steps: the experiment adopts the mode of intragastric administration, and the mice with the breast cancer models are randomly divided into: the high-concentration administration group (Hfd-h) of the Huafengdan, the low-concentration administration group (Hfd-l) of the Huafengdan and the normal saline control group (NaCl) are 10 mice in each group. According to the Liao's Huafengdan instruction book and the conversion ratio of human body and mouse animal experiment, the high concentration of the experiment is 103.8mg/mL, and the low concentration is 55.36 mg/mL; the concentration of the physiological saline C is 9 mg/mL; the medicine is taken every two days after the model is made, 0.1mL of the medicine is taken every time, and the total amount of the medicine is 10 times. The length, width and body weight of the transplanted tumor nodules were measured after each dose. After the experiment, the mice were sacrificed by cervical dislocation, and the transplanted tumors were removed and weighed. The effect of treatment was analyzed and all animal studies followed institutional guidelines. The results of the experiment are shown in FIG. 6.

Comparing the solid tumor weight of the mice in the control group with that of the group to which the Huafengdan is administered, the result shows that the solid tumor weight of the mice in the control group is obviously greater than that of the group to which the Huafengdan is administered at high concentration, and the result shows that the feeding of the group to which the Huafengdan is administered at high concentration has obvious treatment effect on the breast cancer of the mice.

From the results, the Huafengdan has potential value in the aspect of preparing the medicine for preventing and treating the breast cancer or the breast cancer cell proliferation inhibitor.

Claims

1. Application of HUAFENGDAN in preparing medicine for treating breast cancer is provided.

2. The use of the pellet for treating breast cancer according to claim 1, wherein the pellet comprises: the gout-resolving pill is directly prepared into a preparation for resisting breast cancer or is compounded with other medicines for resisting breast cancer to prepare a compound preparation, or the effective components of the gout-resolving pill and a pharmaceutically acceptable carrier are prepared into the preparation for resisting breast cancer.

3. The use of the pellet for treating breast cancer according to claim 1, wherein the pellet comprises: the preparation is any one of tablets, injections, capsules, oral liquid and dropping pills.

4. The use of the pellet for treating breast cancer according to claim 1, wherein the pellet comprises: the cancer cell in the breast cancer is any one of human breast cancer cell MDA-231, human breast cancer cell BT474, rat breast cancer cell FE1.2 and rat breast cancer cell FE 12.5.