TW202220669A - USE OF CITRUS DEPRESSA HAYATA EXTRACTION TO INHIBIT ER-α PROTEIN EXPRESSION OF ESTROGEN RECEPTOR POSITIVE BREAST CANCER CELLS - Google Patents

Abstract

A method of using Citrus depressa Hayata extract to produce an effective ingredient for inhibiting breast cancer cells includes steps as follows: Citrus depressa Hayata is placed in an aqueous solvent to obtain the Citrus depressa Hayata extract, wherein the aqueous solvent includes 1:1 parts by weight of pure water and 95% by weight of alcohol. The Citrus depressa Hayata extract is mixed with additives to form the effective ingredients containing 100[mu]g/mL to 400 [mu]g/mL of Citrus depressa Hayata extract and having 160 [mu]M to 320 [mu]M laimonoids with the following structural formula selected from a group consisting of and the combination thereof, wherein the effective ingredient is used to inhibit the proliferation of estrogen receptor positive breast cancer cells.

Description

Method for preparing the active ingredient for inhibiting breast cancer cells using Taiwan citron extract

The present disclosure relates to a method for producing and applying an active ingredient for inhibiting breast cancer cells, particularly a method for producing and applying an active ingredient containing citrus extraction for inhibiting breast cancer cells.

Breast cancer is a malignant tumor that grows from the epithelial cells or lobules of the breast. It is one of the most common malignant tumors in women, accounting for 25% of all cancer cases and the leading cause of cancer death in women worldwide. With the changes in people's lifestyles and eating habits, the incidence of breast cancer is the number one cancer in women at home and abroad, and the incidence of breast cancer continues to increase globally.

Clinically, breast cancer can be divided into four categories according to the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor type II (Her2): (1) Female Hormone receptor (including estrogen receptor ER or progesterone receptor PR) positive (HR+), and human epidermal growth factor receptor type 2 negative (HER2-) luminal A breast cancer patients (2) Female hormone receptor-positive (HR+), and human epidermal growth factor receptor type II positive (HER2+) luminal B breast cancer patients; (3) Female hormone receptor negative, and type II human epidermal growth factor receptor-positive (HER2+) breast cancer patients; and (4) triple-negative (ER-, PR-, and HER-2) breast cancer patients who do not express all three receptors. Among them, estrogen receptor positive (ER+) and human epidermal growth factor receptor type II negative (Her2-) breast cancer patients are the most, accounting for about 70% of the overall breast cancer patients.

The main priority treatment for this type of breast cancer is hormonal therapy. The first drug used is selective estrogen-receptor modulators (SERMs), also known as anti-estrogens, which compete with estrogen for hormone receptors to block estrogen from promoting cancer. The effect of cell growth directly blocks the binding of estrogen to estrogen receptors, so that cancer cells die due to the lack of estrogen. The second type of drug is aromatase inhibitors (AIs), which block the activity of aromatase and prevent it from converting androgens into estrogen in postmenopausal women, thereby reducing the effect of estrogen on cancer cells . The third drug is luteinizing hormone-releasing hormone, which acts on the pituitary cells so that they no longer secrete luteinizing hormone-stimulating hormone, and the ovaries will no longer secrete estrogen if they lack hormones.

However, the above drugs will directly or indirectly block estrogen, which is responsible for regulating the function of the female reproductive system. Although women are used before or after menopause, these drugs are prone to side effects such as endometrial bleeding and abnormal thickening. The risk of developing endometrial cancer also increases the incidence of thromboembolism, which can easily lead to stroke and deep vein thrombosis in women over 50 years old.

Therefore, there is a need to provide an advanced active ingredient or method for inhibiting the growth of breast cancer cells to solve the side effects of current drugs.

及其組合所組成的族群，用於抑制雌激素受體陽性乳癌細胞的增殖。 The embodiment of the present specification discloses a method for preparing an active ingredient for inhibiting breast cancer cells with Taiwan citron extract, comprising: placing Taiwan citron in an aqueous solvent to obtain Taiwan citron extract. Wherein the aqueous solvent comprises 1:1 by weight of pure water and 95% by weight of alcohol; the Taiwan lemongrass extract is mixed with at least one additive, so as to form a composition comprising between 100 micrograms/milliliter (μg/mL) to 400 micrograms/ml Active ingredients of Taiwan citron extract. Wherein, the Taiwan lemongrass extract has a limonin compound between 160 micromoles/liter (µM) and 320 micromoles/liter, and the limonin compound is selected from the following structural formula:

A population composed of and combinations thereof are used to inhibit the proliferation of estrogen receptor-positive breast cancer cells.

In one embodiment of the present specification, the estrogen receptor positive breast cancer cells comprise MCF7 breast cancer cells.

In an embodiment of the present specification, the additive is selected from the group consisting of a solvent, an excipient, a water for injection, and a combination of the above.

In an embodiment of the present specification, the limonoid compound can be used to inhibit the proliferation of triple negative breast cancer cells.

In one embodiment of the present specification, the triple-negative breast cancer cells include MDA-MB-231 breast cancer cells.

In one embodiment of the present specification, the limonin compound can be used for inhibiting the proliferation of estrogen receptor negative and type 2 human epidermal growth factor receptor positive breast cancer cells.

In one embodiment of the present specification, the estrogen receptor negative and human epidermal growth factor receptor type 2 positive breast cancer cells comprise a plurality of HER2-enriched MDA-MBs -435S breast cancer cells.

According to the above-mentioned embodiment, the embodiment of the present specification is to provide a method for preparing an active ingredient for inhibiting breast cancer cells from Taiwan citronella extract. In an aqueous solvent containing 1:1 by weight of pure water and 95% by weight of alcohol, to obtain Taiwan Lemonade extract, it has a range of 160 micromoles/liter (µM) to 320 micromoles/liter Among the active ingredients of limonoid compounds. Wherein, the limonoid compound is selected from the group consisting of Limomin, Nomilin and the combination of the two. It can indeed inhibit the proliferation of estrogen receptor positive breast cancer cells, estrogen receptor negative and type 2 human epidermal growth factor receptor positive breast cancer cells and triple negative breast cancer cells as confirmed by Western blotting analysis. It is particularly effective in inhibiting the proliferation of estrogen receptor-positive breast cancer cells.

This specification is to provide a method for preparing an active ingredient for inhibiting breast cancer cells from Taiwan citron extract, so as to inhibit the proliferation of breast cancer cells. In order to make the above-mentioned embodiments and other objects, features and advantages of the present specification more clearly understood, a plurality of preferred embodiments are exemplified below and described in detail with the accompanying drawings.

However, it must be noted that these specific implementation cases and methods are not intended to limit the present invention. The present invention may still be practiced with other features, elements, methods and parameters. The preferred embodiments are provided only to illustrate the technical features of the present invention, and not to limit the scope of the present invention. Those with ordinary knowledge in the technical field will be able to make equivalent modifications and changes based on the description of the following specification without departing from the spirit and scope of the present invention. In different embodiments and drawings, the same elements will be represented by the same element symbols.

Please refer to FIG. 1 . FIG. 1 is a flow chart illustrating the steps of preparing an active ingredient for inhibiting breast cancer cells from Taiwan lemongrass according to an embodiment of the present specification. The method for making an active ingredient for inhibiting breast cancer cells includes the following steps: firstly, placing Taiwan citronella in an aqueous solvent to obtain Taiwan citronella extract (as shown in step S1).

In the examples of this specification, the used Taiwan citron is also known as flat lemon or flat orange, Hakka is called mountain orange, Hokkien is called sour orange, mountain orange, English name is Shiikuwasha or Sikuwasa, distributed in Taiwan, Ryukyu and Guam; is a plant belonging to the plant kingdom, Angiosperm, Dicotyledon, Rutales, Rutaceae, Citrus (Citrus) scientifically named Citrus depressa Hayata .

Take 200 grams of dried Taiwan lemons and crush them, add 1 liter by weight (95%) alcohol, 1 liter pure water (100% H ₂ O) and 1 liter by weight 1:1 pure water and 95 weight percent alcohol in an aqueous solvent for 3 hours to obtain three different Taiwan lemongrass extracts CD-1, CD-2 and CD-3.

Please refer to Figure 2. Figure 2 shows the MTT cell viability of human normal skin fibroblasts Detroit 551 with different concentrations of Taiwan lemongrass extracts CD-1, CD-2 and CD-3 respectively. Test results after analysis. Among them, the horizontal axis is the content of CD-1, CD-2 and CD-3 in Taiwan Lemon Extract; the vertical axis is the cell survival rate (that is, the addition of Taiwan Lemon Extract CD-1, CD-2 and CD-3 The ratio of the number of cells in the experimental group to the number of cells in the control group without the addition of Taiwan lemon extract CD-1, CD-2 and CD-3). From the experimental results, it was found that CD-1, CD-2 and CD-3 of Taiwan lemon extracts did not have a significant effect on human normal skin fibroblasts, even though Taiwan lemon extracts CD-1, CD-2 and CD The added concentration of -3 is higher than 400 μg/ml, but it still cannot effectively induce the death of human normal skin fibroblasts.

Please refer to Fig. 3A to Fig. 3C, Fig. 3A to Fig. 3C show the use of the above mentioned Taiwan lemon extract CD-1, CD-2 and CD-3 with different concentrations, respectively, on MCF7 breast cancer cells, MDA-MB435S breast cancer cells and MDA-MB231 breast cancer cells were detected after MTT cell viability assay. Among them, MCF7 breast cancer cells are estrogen receptor positive breast cancer cells; MDA-MB231 breast cancer cells are triple negative breast cancer cells; SKBR3 breast cancer cells and MDA-MB435S breast cancer cells are estrogen receptor negative (Estrogen Receptor Negative, ER-). and type II human epidermal growth factor receptor-positive (HER2+) breast cancer cells. MDA-MB435S breast cancer cells are a type II human epidermal growth factor receptor overexpressing (HER2-overexpressing) breast cancer cells.

The experimental results found that the Taiwan lemon extract CD-1 extracted with pure water did not significantly inhibit the proliferation of breast cancer cells. The Taiwan lemon extract CD-2 extracted with 95% by weight (95%) alcohol water has the ability to inhibit the proliferation of breast cancer cells, but under high concentration (400 μg/ml), there is still no way to remove MCF7 The survival rate of breast cancer cells, MDA-MB435S breast cancer cells and MDA-MB231 breast cancer cells dropped to below 60%. Using the Taiwan lemon extract CD-3 extracted with 1:1 weight of alcohol-containing aqueous solvent, the ability to inhibit the proliferation of breast cancer cells is the strongest. , MDA-MB435S breast cancer cells and MDA-MB231 breast cancer cells were reduced to less than 40% survival.

It can be seen that CD-3 from Taiwan lemongrass has the best ability to inhibit the proliferation of breast cancer cells. However, the ability of Taiwan lemon extract CD-3 to inhibit the proliferation of different breast cancer cells is still different. For example, in this example, the 50% inhibitory concentration of Taiwan lemon extract CD-3 against MCF7 breast cancer cells (even if the survival rate of breast cancer cells was reduced to 50%) was 200 μg/ml; while for MDA-MB435S breast cancer cells and The 50% inhibitory concentration of MDA-MB231 breast cancer cells is about 300 μg/ml. It was shown that CD-3 extracted from Taiwan citron has the strongest anti-proliferation ability on estrogen receptor-positive MCF7 breast cancer cells.

The components of the same amount of Taiwan citron extract CD-1, CD-2 and CD-3 were analyzed by High Performance Liquid Chromatography (HPLC). The results show that the major difference between Taiwan citron extract CD-1 and Taiwan citron extract CD-2 and CD-3 is that both Taiwan citron extract CD-2 and CD-3 contain the limonoid compound Limomin. and Nomilin, the structures of the two are shown as follows:

Among them, Taiwan lemon extract CD-3 has the highest content of limonin compounds. In this example, the content of the limonoid compound of the Taiwan lemon extract CD-3 is substantially between 160 micromoles/liter (µM) and 320 micromoles/liter. It can be inferred that the limonoid compound is the main key component of the Taiwan lemon extract CD-2 and CD-3 inhibiting the proliferation of breast cancer cells.

Then, the Taiwan lemon extract CD-3 is respectively mixed with an appropriate amount of solvent or excipient, such as 95% alcohol, water for injection (Water for Injection), or a combination of the above, to form an effective drug containing a drug for inhibiting the proliferation of breast cancer cells. ingredients (as shown in step S2).

Please refer to Figure 4A to Figure 4D, Figure 4A to Figure 4D respectively show the control group without the addition of Taiwan Lemongrass extract and the concentration of 100 μg/ml, 200 μg/ml and 400 μg/ml, respectively. The three active ingredients of the Taiwan lemon extract CD-3 were reacted with MCF7 breast cancer cells for 72 hours, and the DNA content of the cells was detected by flow cytometry. Among them, the vertical axis is the number of cells, and the horizontal axis is the fluorescence wavelength.

As shown in Figure 4A, in the control group without the addition of the Taiwan lemongrass extract CD-3, the ratio of the number of cells in the sub-G1 phase with apoptosis in MCF7 breast cancer cells was about 1.6%. The addition of the active ingredient containing Taiwan lemon extract CD-3 significantly increased the number of sub-G1 cells in MCF7 breast cancer cells with apoptosis. And with the increase of the concentration of Taiwan lemon extract CD-3, the concentration increased significantly (as shown in Figure 4B to Figure 4D). It is indicated that the active ingredient containing Taiwan Lemon Extract CD-3 can directly lead to the death of MCF7 breast cancer cells.

Furthermore, the western blotting method was used to test how the active ingredient containing Taiwan lemongrass extract CD-3 affects the expression of proteins related to the survival of MCF7 breast cancer cells, and the changes were observed. Please refer to Figure 5. Figure 5 shows the control group without the addition of the Taiwan lemongrass extract and the CD containing the Taiwan lemongrass extract at concentrations of 100 μg/ml, 200 μg/ml and 400 μg/ml, respectively. After the three active ingredients of -3 reacted with MCF7 breast cancer cells for 48 hours, the transfection images obtained by analyzing the expression levels of proteins regulating the growth and proliferation of breast cancer cells were analyzed by Western blotting method.

In this example, the proteins used to regulate the growth and proliferation of MCF7 breast cancer cells include proteins used to regulate mitogen-activated protein kinase (MAPK _S ), such as p-AKT protein, p-ERK1/2 protein, p- p38 protein, p-JNK protein and p-STAT3 protein. Among them, mitogen-activated protein kinases ( _MAPKS ) can transmit extracellular signals to the cytoplasm and nucleus through mutual regulation to regulate the gene expression, division, proliferation, differentiation, apoptosis and other processes of breast cancer cells. p-AKT protein, p-ERK1/2 protein, p-p38 protein, p-JNK protein and p-STAT3 protein can promote cell cycle progression, cell transformation and avoid apoptosis, and are considered to be oncogenic proteins.

As shown in Fig. 5, after treatment with the active ingredient containing Taiwan lemon extract CD-3, p-AKT protein, p-ERK1/2 protein, p-p38 protein, p-JNK protein in MCF7 breast cancer cells And the expression level of p-STAT3 protein was significantly decreased, and the protein expression level decreased with the increase of CD-3 content in Taiwan Lemongrass extract. It can be seen that the effective ingredients containing Taiwan lemon extract CD-3 can indeed inhibit the gene expression, division, proliferation and differentiation of MCF7 breast cancer cells, thereby preventing the cell growth, proliferation, cell migration and angiogenesis of MCF7 breast cancer cells.

In addition, the active ingredient containing Taiwan lemon extract CD-3 can also inhibit the protein expression of estrogen receptor (Estrogen Receptor-α, ER-α) that promotes the proliferation and differentiation of MCF7 breast cancer cells. Please refer to Figure 6. Figure 6 shows the control group without the Taiwan lemon extract and CD containing the Taiwan lemon extract at concentrations of 100 μg/ml, 200 μg/ml and 400 μg/ml, respectively. After the three active ingredients of -3 reacted with MCF7 breast cancer cells for 48 hours, the transfection images obtained by analyzing the expression level of ER-α protein by western blotting method. The results in Fig. 6 show that the expression level of ER-α protein in MCF7 breast cancer cells was significantly decreased with the increase of the concentration of Taiwan Lemongrass extract CD-3.

Next, the limonin compounds Limonin and Nomilin in the active ingredients were tested to analyze the effect of the active ingredients on the expression of ER-α protein. Please refer to Figures 7A and 7B. Figures 7 and 7B show that the four active ingredients of the limonin compounds Limonin and Nomilin containing 160 micromol/liter and 320 micromol/liter respectively, and After 48 hours of reaction in MCF7 breast cancer cells, the transfection images obtained by analyzing the expression of ER-α protein by western blotting method. The results in Figure 7 show that the active ingredients of the limonin compounds Limonin and Nomilin containing 160 μmol/L and 320 μmol/L can effectively inhibit the expression of ER-α protein in MCF7 breast cancer cells. All four active ingredients have the function of preventing the proliferation and differentiation of MCF7 breast cancer cells.

Please refer to Fig. 8A to Fig. 8D. Fig. 8A to Fig. 8D show that the active ingredient of the limonoid compound Nomilin containing 160 μmol/L was reacted with MCF7 breast cancer cells for 24 hours, 48 hours and After 72 hours, the transfection images obtained by analyzing the expression levels of YB-1 protein, TWIST protein, Hif1-α protein and ILK protein by Western blotting method. The results from Figure 8A to Figure 8D show that the active ingredient of the limonoid compound Nomilin containing 160 μmol/L can effectively inhibit the signaling pathway of TWIST protein and YB-1 protein in MCF7 breast cancer cells, and inhibit the Integrin-linked kinase (ILK) activity to induce cell cycle arrest and apoptosis in MCF7 breast cancer cells, thereby inhibiting estrogen receptor-α overexpression.

Please refer to Figures 9A and 9B. Figures 9A and 9B show that the control group without the addition of the limonoid compound Nomilin and the control group containing 80 micromol/liter, 160 micromol/liter and The active ingredient of limonoid compound Nomilin at 320 micromol/L was reacted with MCF7 breast cancer cells for 48 hours, and the biomarker proteins of epithelial-mesenchymal transition (E-cadherin protein, N-cadherin protein) were analyzed by Western blotting method. , Vimentin protein, Snail protein and Slug protein). The results in Figures 9A and 9B show that with the increase of Nomilin concentration, the expression of E-cadherin protein increases, which means that the cell type tends to be epithelial-form, and the cells are closely arranged and not easy to move. At the same time, the expression of N-cadherin protein, Vimentin protein, Snail protein and Slug protein decreased, indicating that Nomilin can inhibit the occurrence of epithelial-mesenchymal transition, which is not conducive to the crawling and metastasis of MCF7 breast cancer cells.

According to the above-mentioned embodiment, the embodiment of the present specification is to provide a method for preparing an active ingredient for inhibiting breast cancer cells from Taiwan citronella extract. In an aqueous solvent containing 1:1 by weight of pure water and 95% by weight of alcohol, to obtain Taiwan Lemonade extract, it has a range of 160 micromoles/liter (µM) to 320 micromoles/liter Among the active ingredients of limonoid compounds. Wherein, the limonoid compound is selected from the group consisting of Limomin, Nomilin and the combination of the two. It can indeed inhibit the proliferation of estrogen receptor positive breast cancer cells, estrogen receptor negative and type 2 human epidermal growth factor receptor positive breast cancer cells and triple negative breast cancer cells as confirmed by Western blotting analysis. It is particularly effective in inhibiting the proliferation of estrogen receptor-positive breast cancer cells.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone with ordinary knowledge in the technical field can make some changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the scope of the appended patent application.

CD-1: Taiwan Lemon Extract CD-2: Taiwan Lemon Extract CD-3: Taiwan Lemon Extract MCF7: Breast cancer cells MDA-MB-231: Breast cancer cells MDA-MB-435S: Breast cancer cells S1: Put Taiwan lemon in an aqueous solvent to obtain Taiwan lemon extract S2: combine Taiwan lemongrass extract with an appropriate amount of solvent or excipient to form an active ingredient for inhibiting the proliferation of breast cancer cells

In order to have a better understanding of the above-mentioned and other aspects of this specification, the following specific examples are given and described in detail with the accompanying drawings as follows: Fig. 1 is a flow chart showing the steps of preparing an active ingredient for inhibiting breast cancer cells from Taiwan citron according to an embodiment of the present specification; Figure 2 shows the results of MTT cell viability analysis on human normal skin fibroblasts Detroit 551 with different concentrations of Taiwan lemongrass extracts CD-1, CD-2 and CD-3 respectively; Fig. 3A to Fig. 3C show the treatment of MCF7 breast cancer cells, MDA-MB435S breast cancer cells and MDA-MB231 breast cancer cells with different concentrations of the above mentioned Taiwan lemongrass extracts CD-1, CD-2 and CD-3, respectively. The results of the cells after the MTT cell viability analysis; Figures 4A to 4D respectively show the control group without the addition of Taiwan lemongrass extract and the CD-3 containing Taiwan lemongrass extract at concentrations of 100 μg/ml, 200 μg/ml and 400 μg/ml, respectively. The three active ingredients of MCF7 were reacted with MCF7 breast cancer cells for 72 hours, and the DNA content of the cells was detected by flow cytometry. Figure 5 shows the three active ingredients of Taiwan lemon extract CD-3 with concentrations of 100 μg/ml, 200 μg/ml and 400 μg/ml in the control group without the Taiwan lemon extract. After reacting with MCF7 breast cancer cells for 48 hours, the blotted images obtained by analyzing the expression of proteins regulating the growth and proliferation of breast cancer cells were analyzed by Western blotting method; Figure 6 shows the three active ingredients of Taiwan lemon extract CD-3 with concentrations of 100 μg/ml, 200 μg/ml and 400 μg/ml, respectively, in the control group without the Taiwan lemon extract. After reacting with MCF7 breast cancer cells for 48 hours, the transfection images obtained by analyzing the expression of ER-α protein by Western blotting method; Figures 7 and 7B show that four active ingredients containing limonin compounds Limonin and Nomilin containing 160 μmol/L and 320 μmol/L, respectively, were reacted with MCF7 breast cancer cells for 48 hours, and the Transferred images obtained by Western blotting analysis of ER-α protein expression; Figures 8A to 8D show that the active ingredient of the limonin compound Nomilin containing 160 μmol/L reacted with MCF7 breast cancer cells for 24 hours, 48 hours and 72 hours, and analyzed by Western blotting method Transfection images obtained from the expression levels of YB-1 protein, TWIST protein, Hif1-α protein and ILK protein; and Figures 9A and 9B show, respectively, a control group without the addition of the limonoid compound Nomilin and a control group containing limonin at concentrations of 80 micromol/liter, 160 micromol/liter and 320 micromol/liter, respectively After reacting with MCF7 breast cancer cells for 48 hours, the active ingredient of the compound Nomilin was used to analyze the expression levels of biomarker proteins of Epithelial-Mesenchymal Transition (EMT) by Western blotting method.

S1: Put Taiwan lemon in an aqueous solvent to obtain Taiwan lemon extract

S2: combine Taiwan lemongrass extract with an appropriate amount of solvent or excipient to form an active ingredient for inhibiting the proliferation of breast cancer cells

Claims (8)

A method for preparing an active ingredient for inhibiting breast cancer cells from Citrus depressa extract, comprising: placing Citrus depressa in an aqueous solvent to obtain the Citrus depressa extract, wherein the aqueous solvent comprises parts by weight 1:1 pure water and 95 weight percent alcohol; and mixing the Taiwan citron extract with at least one additive to form the active ingredient, such that the active ingredient comprises between 100 micrograms/milliliter (µg/mL) to 400 microgram/ml of the Taiwan citron extract, and having the limonoid compound between 160 micromolar/liter (µM) and 320 micromolar/liter; and the limonoid compound is selected from By the following structural formula:

A group formed by any combination thereof is used to inhibit the proliferation of a plurality of estrogen receptor positive (Estrogen Receptor Positive, ER+) breast cancer cells. The method of claim 1, wherein the plurality of estrogen receptor positive (ER+) breast cancer cells comprise MCF7 breast cancer cells. The method of claim 1, wherein the at least one additive is selected from the group consisting of a solvent, an excipient, a water for injection (Water for Injection), and a combination of the above. The method of claim 1, wherein the active ingredient is further used to inhibit the proliferation of a plurality of triple-negative breast cancer cells. The method of claim 4, wherein the plurality of triple negative breast cancer cells comprises a plurality of MDA-MB-231 breast cancer cells. The method of claim 1, wherein the active ingredient is further used for inhibiting the proliferation of a plurality of estrogen receptor negative (ER-) and type 2 human epidermal growth factor receptor positive breast cancer cells. The method of claim 6, wherein the plurality of estrogen receptor negative and type 2 human epidermal growth factor receptor positive breast cancer cells comprise a plurality of HER2-overexpressing MDA-MB-435S breast cancer cells. The method of claim 1, wherein the active ingredient comprises 160 micromoles/liter (µM) of Nomilin.

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