A method for preparing a chloroform extract of *Gnaphalium affine* and its application in the preparation of drugs for treating breast cancer.

Abstract

This invention discloses a method for preparing a chloroform extract of *Ardisia crenata* and its application in the preparation of drugs for treating breast cancer, belonging to the field of biomedical technology. The method involves collecting fresh *Ardisia crenata*, washing and pulverizing it, extracting it with chloroform, collecting the chloroform extract, filtering and sterilizing it, and freeze-drying it to obtain a dry powder. This invention is the first to discover that the chloroform extract of *Ardisia crenata* has a good therapeutic effect on breast cancer and has promising application prospects in the biomedical field.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a method for preparing a chloroform extract of *Gnaphalium affine* and its application in the preparation of drugs for treating breast cancer. Background Technology

[0002] Breast cancer is a common cancer among women. Research data shows that in 2023, the number of newly diagnosed breast cancer patients reached 2.383 million, an increase of 1.77% year-on-year; while the number of newly diagnosed breast cancer patients in China reached 348,000, an increase of 1.2% year-on-year. Breast cancer manifests as symptoms such as breast lumps, nipple discharge, and enlarged axillary lymph nodes. In advanced stages, distant metastasis of cancer cells can lead to multi-organ complications, directly threatening the patient's life. Treating breast cancer remains a challenging clinical problem, and breast cancer has a significant impact on human health. The increase in the number of new breast cancer cases has driven the continuous growth of the breast cancer drug market.

[0003] Toxocarpus hainanensis Tsiang is a plant belonging to the genus Toxocarpus in the family Asclepiadaceae. A search revealed no literature currently reporting that Toxocarpus hainanensis or its extracts have the function of inhibiting breast cancer growth; nor have any reports been found domestically or internationally of Toxocarpus hainanensis being prepared into drugs for the treatment of breast cancer.

[0004] Currently, the treatment of breast cancer remains a pressing problem in clinical practice. Therefore, screening for drugs with low side effects and effective treatment of breast cancer is an urgent clinical need.

[0005] Therefore, providing a method for preparing a chloroform extract of *Gnaphalium affine* and its application in the preparation of drugs for treating breast cancer is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing a chloroform extract of *Gnaphalium affine* and its application in the preparation of drugs for treating breast cancer. The chloroform extract of *Gnaphalium affine* can significantly inhibit the growth of breast cancer and can be applied in the pharmaceutical field.

[0007] This invention discovers that a component for treating breast cancer can be obtained from the chloroform extract of the island plant *Gnaphalium affine*. Cell and mouse tumor-bearing experiments show that the chloroform extract can inhibit the growth of breast cancer.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A method for preparing a chloroform extract of *Gnaphalium affine*, comprising the following steps:

[0010] (1) Collect the vine of the bow, wash it repeatedly with tap water 2 to 3 times, then rinse it repeatedly with distilled water 1 to 2 times, dry the surface moisture and weigh it.

[0011] (2) Crush the dried aralia elata, add chloroform for extraction overnight and collect the chloroform extract; filter the above extract through a 0.22μm filter membrane for sterilization; the mass-volume ratio of the aralia elata to chloroform is 1:0.5-10;

[0012] (3) The filtered and sterilized extract was freeze-dried into powder to obtain the chloroform extract of *Gnaphalium affine*.

[0013] Furthermore, the application of the chloroform extract of *Gnaphalium affine* in the preparation of drugs for treating breast cancer.

[0014] Furthermore, the drug also contains pharmaceutically acceptable excipients or carriers.

[0015] Furthermore, the dosage form of the drug is tablets, capsules, pills, powders, granules, syrups, solutions, injections, sprays, aerosols, or patches.

[0016] Furthermore, the preparation method of the pills is as follows: First, dissolve the chloroform extract of *Gnaphalium affine* in sterile water, then mix it with methylcellulose. The weight ratio of the chloroform extract of *Gnaphalium affine* to methylcellulose is 1:3. Stir gently until the mixture is a uniform paste. Mix evenly to form pills and then air dry.

[0017] The chloroform extract of *Gnaphalium affine* described in this invention can be used alone or in combination with other drugs.

[0018] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a method for preparing chloroform extract of *Gnaphalium affine* and its application in the preparation of drugs for treating breast cancer, which has the following advantages:

[0019] Beneficial effects:

[0020] (1) This invention is the first to discover that the chloroform extract of *Gnaphalium affine* has a good therapeutic effect on breast cancer and has good application prospects in the field of biomedicine.

[0021] (2) The chloroform extract of the arrowroot of the present invention can be used to prepare a drug for treating breast cancer or as an active ingredient or drug precursor.

[0022] (3) The present invention contains a drug containing chloroform extract of *Gnaphalium affine*, which can be made into various product forms such as powder or injection. It can be used alone as a finished product or as an active ingredient in combination with adjuvants such as physiological saline or glucose solution. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 The attached figure shows the inhibition of breast cancer cell growth by the chloroform extract of *Gnaphalium affine* according to this invention;

[0025] Figure 2 The attached figure shows the inhibitory effect of the chloroform extract of *Gnaphalium affine* on the growth of BT-20 breast cancer cell line xenografts in nude mice.

[0026] Where: A: Mouse weight; B: Tumor weight; C: Tumor volume; D: Liver weight; E: Spleen weight; F: Physical morphology of tumor, liver, and spleen; G: Mouse morphology after drug administration;

[0027] Figure 3 The attached figures show the number of red blood cells (RBC), white blood cells (WBC), and platelets (PLT) in the blood and bone marrow tissues of each group of the present invention;

[0028] Where A: the number of red blood cells, white blood cells, and platelets in the blood; B: the number of red blood cells, white blood cells, and platelets in the bone marrow tissue. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0031] Example 1: Preparation of Chloroform Extract of *Gnaphalium affine*

[0032] A method for preparing a chloroform extract of *Gnaphalium affine*, comprising the following steps:

[0033] (1) Collect the vine of the bow, wash it twice with tap water, rinse it twice with distilled water, dry the surface moisture and weigh it.

[0034] (2) Crush the dried aralia elata and pass it through a 300-mesh sieve; add chloroform for extraction overnight and collect the chloroform extract; filter the extract through a 0.22μm filter membrane for sterilization; the mass-to-volume ratio of aralia elata to chloroform is 1:1;

[0035] (3) The filtered and sterilized extract was freeze-dried into powder to obtain the chloroform extract of *Gnaphalium affine*.

[0036] Example 2 Anti-breast cancer experiment

[0037] 1) Inhibitory effect of chloroform extract of *Gnaphalium affine* on the proliferation of breast cancer cells

[0038] Breast cancer cell lines SUM-159PT, BT-20, and MCF-7 were passaged in RPMI 1640 complete medium (containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin) at 37°C in a 5% CO2 incubator. Breast cancer cells in the logarithmic growth phase were then diluted with RPMI 1640 complete medium (containing 10% fetal bovine serum, 100 U / ml penicillin, and 100 μg / ml streptomycin) to a concentration of 4.0 × 10⁻⁶ cells / ml. 4 / ml cell suspension, seeded at 4.0×10 3 (0.1 ml) was incubated in 96-well plates at 37°C and 5% CO2 for 24 h. The culture medium was then replaced with fresh complete medium, and different final concentrations of DMSO (0.1%) solution of *Gnaphalium affine* chloroform extract prepared in Example 1 (20, 40, 80, 160, 320 μg / ml) were added for treatment. A control group was also included. After another 24 h of incubation, 20 μl of 5 mg / ml MTT solution was added, and incubation continued for 4 h. Then, 150 μl of DMSO was added per well, and the plates were shaken in the dark for 15 min. The OD value of each well was measured at 570 nm using a microplate reader. The inhibition rate of *Gnaphalium affine* chloroform extract against breast cancer cells was calculated. The results are shown in Table 1 and [Table data missing]. Figure 1 .

[0039] Table 1. Inhibition rate of chloroform extract of *Gynostemma pentaphyllum* on breast cancer cells

[0040]

[0041] Table 1 and Figure 1 The results showed that the proliferation ability of the three breast cancer cell lines decreased significantly with increasing concentration of *Gnaphalium affine* chloroform extract. The results also indicated that *Gnaphalium affine* chloroform extract could inhibit the proliferation of SUM-159PT, BT-20, and MCF-7 breast cancer cells to varying degrees in a dose-dependent manner, with IC50 values ​​of 77, 48, and 65 μg / ml, respectively.

[0042] 2) Chloroform extract of *Gnaphalium affine* inhibits the growth of BT-20 breast cancer cell line xenografts in nude mice.

[0043] Female nude mice of the BALB / C / nu strain, 4–6 weeks old, were housed and tested in an SPF-grade barrier system. An animal tumor model was established using xenograft cell culture; in this invention, BT-20 cells were used to establish the animal tumor model for in vivo drug efficacy observation. After large-scale culture and passage of BT-20 cells, and cell collection, the cell suspension was diluted to 1×10⁻⁶. 7 / ml, 0.2ml was injected subcutaneously into the right shoulder of nude mice. Successful inoculation occurred 7 days later, with a noticeable tumor mass at the inoculation site. Treatment began with chloroform extract of *Gnaphalium affine* at a dose of 200mg / kg; cisplatin was administered at a dose of 5mg / kg as a positive control; and physiological saline was administered at 0.3ml / mouse as a control group. Except for the cisplatin treatment group, which received the drug every other day, all other groups received oral administration daily for 27 days. During the treatment period, all nude mice maintained normal food and water intake, and their quality of life and changes in body weight and tumor growth volume were observed and recorded. The experiment ended 24 hours after the last administration by cervical dislocation. The body weight, tumor weight, liver weight, and spleen weight of the nude mice were measured, tumor size was measured, and the tumor inhibition rate was calculated. Results are shown in Table 2 and [Table data missing]. Figure 2 .

[0044] Table 2. Inhibitory effect of chloroform extract of *Gnaphalium affine* on the growth of breast cancer cell xenografts in nude mice.

[0045]

[0046] Compared with the saline control group: * P<0.05, ** P<0.01, *** P<0.001.

[0047] Table 2 and Figure 2 Experimental results showed that the chloroform extract of *Gnaphalium affine* significantly inhibited tumor growth, reduced tumor size in animals, and significantly improved the tumor inhibition rate. Observation of the liver and spleen of mice in the treatment group showed that the chloroform extract of *Gnaphalium affine* had no significant damage to the liver and spleen. Observation of weight changes after drug administration showed that the nude mice in the cisplatin-treated group were all thin and experienced significant weight loss, indicating certain toxic side effects, while the weight of mice treated with the chloroform extract of *Gnaphalium affine* remained stable, indicating lower toxic side effects. In summary, the chloroform extract of *Gnaphalium affine* at a dose of 200 mg / kg showed a certain inhibitory effect on mammary tumor xenografts in nude mice.

[0048] Blood and bone marrow tissue samples were collected from each group for analysis of red blood cell (RBC), white blood cell (WBC), and platelet (PLT) counts. Results are shown below. Figure 3 The results showed that *Gynostemma pentaphyllum* had an effect on red blood cells, white blood cells, and platelets in the blood. Figure 3 A) and red blood cells, white blood cells, and platelets in bone marrow tissue (A) Figure 3 B) No inhibitory effect.

[0049] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a chloroform extract of *Gnaphalium affine*, characterized in that, The specific steps are as follows: (1) Collect the vine of the bow, wash it repeatedly with tap water 2-3 times, then rinse it repeatedly with distilled water 1-2 times, dry the surface moisture and weigh it; (2) Crush the dried aralia elata, add chloroform for extraction overnight and collect the chloroform extract; filter the extract through a 0.22µm filter membrane for sterilization; the mass-to-volume ratio of the aralia elata to chloroform is 1:0.5-10; (3) The filtered and sterilized extract was freeze-dried into powder to obtain the chloroform extract of *Gnaphalium affine*.

2. The use of the chloroform extract of *Gnaphalium affine* as described in claim 1 in the preparation of a drug for treating breast cancer.

3. The application according to claim 2, characterized in that, The drug also contains pharmaceutically acceptable excipients.

4. The application according to claim 3, characterized in that, The dosage form of the drug is tablets, capsules, pills, powders, granules, solutions, injections, sprays, or patches.

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