Combination therapy of mixed herbal extract and docetaxel for the prevention or treatment of cancer

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating triple-negative breast cancer, comprising a mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen; and an anticancer agent, and a method for preventing or treating cancer using the pharmaceutical composition.

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for preventing or treating cancer, which comprises a mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen; and an anticancer agent, and a method for preventing or treating cancer using the pharmaceutical composition. [Background technology]

[0002] Anticancer drugs are a general term for chemotherapy agents used to treat malignant tumors, excluding surgery and radiation therapy, and most of them exhibit anticancer activity by inhibiting nucleic acid synthesis. Examples include compounds such as paclitaxel and docetaxel, which have been approved by the U.S. Food and Drug Administration and are used clinically. They exert their effects by excessively stabilizing cellular microtubules and inhibiting normal cell division.

[0003] Docetaxel (Taxol), a semisynthetic taxoid produced from the needles of the European yew (Taxus baccata), and its derivative paclitaxel are anticancer drugs used to treat solid tumors and highly metastatic malignancies, including breast cancer, ovarian cancer, non-small cell lung cancer, and colon cancer, and are among the most important new active chemotherapy agents on the market.

[0004] However, despite being an effective anticancer drug, docetaxel has limitations such as drug resistance, cancer recurrence, and side effects. For example, its use at high doses is known to induce another level of toxic reactions, and low or intermediate doses of docetaxel are known to show no significant antitumor activity in patients. Therefore, the use of docetaxel as a monotherapy for cancer is currently precluded. Therefore, there is a need for the development of combination therapeutic agents that exhibit synergistic anticancer effects when administered in combination with docetaxel.

[0005] Meanwhile, Astragalus membranaceus is a perennial plant of the dicotyledonous Rosales family, Fabaceae, and is found in Korea, Japan, Manchuria, northeastern China, eastern Siberia, and other areas. It is commonly cultivated as a medicinal herb, and in traditional Chinese medicine, it is harvested in the fall, the heads and lateral roots are removed, and the plant is dried in the sun. It is known for its medicinal properties when used whole, without peeling. It has effects such as strengthening the body, stopping sweating, diuretic, and reducing swelling, and is prescribed for physical weakness, fatigue, and cold sweats.

[0006] Dong qui (Angelica gigas) is the dried root of the perennial herb of the Apiaceae family, known for its sweet and spicy taste and warming properties. One of the benefits of Dong qui is its blood-replenishing effect, which promotes blood flow in the coronary arteries and stimulates red blood cell production. It has also been reported that Dong qui extract containing decursin and / or decursinol angelate can be used as an anticancer agent (Korean Patent Registered No. 10-1245328).

[0007] Trichosanthes kirilowii Maximowicz (Trichosanthes kirilowii Maximowicz) is the peeled root of the perennial trident or bitter gourd, which belongs to the Cucurbitaceae family. It has no odor, a bitter and sour taste, and is cold in nature. It is used to treat thirst, swelling, and pus when fluids are damaged by heat. It mainly reduces heat in the lungs and stomach, produces fluids, relieves thirst, and nourishes the body.

[0008] In this regard, the present inventors have confirmed the anti-cancer effects of a mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen (Korean Patent Publication No. 2014-0145087). However, the synergistic effects of a combined treatment of a mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen with existing anti-cancer drugs have not been clarified, and there has been no research on this topic. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Korean Patent 10-1245328 [Patent Document 2] Korean Patent Publication No. 2014-0145087 Summary of the Invention [Problem to be solved by the invention]

[0010] The present inventors have made extensive efforts to develop a novel anticancer agent that exhibits a synergistic effect when used in combination with existing anticancer agents. As a result, they have confirmed that a significantly more effective anticancer agent can be provided by combining herbal extracts of Astragalus Root, Angelica Root, and Angelica Root Pollen with existing anticancer agents, thereby completing the present invention. [Means for solving the problem]

[0011] One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer, which contains a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen, and which is administered in combination with an anticancer agent, and the anticancer agent is docetaxel or paclitaxel.

[0012] In one specific example of the pharmaceutical composition for preventing or treating cancer of the present invention, the pharmaceutical composition may reduce the expression level or activity of EGFR (Epidermal growth factor receptor).

[0013] In any one specific example of the pharmaceutical composition for preventing or treating cancer of the present invention, the mixed extract may be obtained by extracting a mixture of Astragalus Root, Dong Qi, and Angelica Root Pollen with one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof.

[0014] As a specific example of any one of the above-mentioned specific examples, the alcohol having 1 to 4 carbon atoms may be 20 to 40% (v / v) ethanol.

[0015] In any one specific example of the pharmaceutical composition for preventing or treating cancer of the present invention, the mixed extract of Astragalus Root, Dong Qi and Angelica Root Pollen may be mixed in a weight ratio of Astragalus Root: Dong Qi: Angelica Root Pollen = 0.5 to 5:1:1.

[0016] In any one specific example of the pharmaceutical composition for preventing or treating cancer of the present invention, the cancer may include breast cancer, lung cancer, stomach cancer, or prostate cancer.

[0017] In any one of the above embodiments, the breast cancer may include triple-negative breast cancer.

[0018] In any one of the above embodiments, the lung cancer may include non-small cell lung cancer.

[0019] In any one embodiment of the pharmaceutical composition for preventing or treating cancer of the present invention, the composition may further comprise a pharmaceutically acceptable carrier, excipient, or diluent.

[0020] In any one specific example of the pharmaceutical composition for preventing or treating cancer of the present invention, the composition may be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration.

[0021] Another object of the present invention is to provide a method for preventing or treating cancer, which comprises the step of administering the composition to an individual.

[0022] Another object of the present invention is to provide a composition containing a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen and an anticancer agent for the prevention or treatment of cancer.

[0023] Another object of the present invention is to provide a use of the mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen as an anti-cancer adjuvant.

[0024] Another object of the present invention is to provide a use of a composition containing a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen in combination with an anticancer agent.

[0025] Another object of the present invention is to provide a combination therapeutic agent comprising a composition containing a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen and an anti-cancer agent.

[0026] Another object of the present invention is to provide a method for preparing a composition containing a mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen and an anticancer agent. [Effects of the Invention]

[0027] The mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen of the present invention can be administered in combination with existing anticancer drugs to exhibit a synergistic anticancer effect, and can be used as an excellent anticancer therapeutic agent. [Brief explanation of the drawings]

[0028] [Figure 1] 1 is a graph showing the effect of the mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen of the present invention or docetaxel administered alone on the viability of BT-20 and MDA-MB-231 breast cancer cell lines. [Figure 2] 1 is a graph showing the effect of combined administration of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention and docetaxel on the viability of BT-20 breast cancer cell line. [Figure 3] 1 is a graph showing the effect of combined administration of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention and docetaxel on the viability of MDA-MB-231 breast cancer cell line. [Figure 4] 1 is a graph showing the effect of administration of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, alone or in combination with docetaxel, on the induction of apoptosis in BT-20 breast cancer cell line. [Figure 5] 1 is a graph showing the effect of administration of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, alone or in combination with docetaxel, on the inhibition of EGFR phosphorylation in BT-20 and MDA-MB-231 breast cancer cell lines. [Figure 6] 1 is a graph showing the effect of administration of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, alone or in combination with docetaxel, on the induction of apoptosis in BT-20 breast cancer cell line. [Figure 7] 1 is a graph showing the effect of the inventive mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen on tumor volume in a tumor xenograft mouse model when administered alone or in combination with docetaxel. Tumor size was measured daily, and the blue arrow indicates DTX administered intravenously at a dose of 15.28 mg / kg once a week, and the orange arrow indicates SH003 administered orally at a dose of 557.57 mg / kg three times a week. [Figure 8] FIG. 1 shows the effect of the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen of the present invention, administered alone or in combination with docetaxel, on tumor weight and size in a tumor xenograft mouse model. [Figure 9] FIG. 1 shows the effect of administration of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, alone or in combination with docetaxel, on body weight in a tumor xenograft mouse model. [Figure 10] 1 is a graph showing the effect of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, when administered alone or in combination with docetaxel, on the viability of H460 lung cancer cell line. [Figure 11] 1 is a graph showing the effect of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, when administered alone or in combination with docetaxel, on the viability of A549 lung cancer cell line. [Figure 12] 1 is a graph showing the effect of administration of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, alone or in combination with docetaxel, on the induction of apoptosis in H460 and A549 lung cancer cell lines. [Figure 13] 1 is a graph showing the effect of administration of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, alone or in combination with docetaxel, on the inhibition of EGFR phosphorylation in H460 and A549 lung cancer cell lines. [Figure 14]1 is a graph showing the effects of the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, administered alone or in combination with docetaxel, on tumor size (A), tumor volume (B), tumor weight (C), and body weight (D) in a tumor xenograft mouse model. Tumor size was measured daily, and the blue arrow indicates the administration of docetaxel, and the orange arrow indicates the administration of SH003. [Figure 15] 1 shows the results of measuring the Combination Index (CI) when the mixed extract of Astragalus Root, Dong Qi, and Angelica Root pollen of the present invention (SH003) was administered alone or in combination with docetaxel in the prostate cancer cell line DU145. [Figure 16] The results show that apoptosis-related proteins were identified by Western blot analysis to compare the efficacy of the present invention's mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen (SH003) administered alone or in combination with docetaxel on apoptosis in the prostate cancer cell line DU145. [Figure 17] After seven days of subcutaneous xenografting, the combined administration of the present invention's mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen (SH003) with docetaxel demonstrated relatively more effective tumor growth inhibition than the single-administration group (a), with no change in mouse weight confirmed as non-toxic (b), and H&E staining confirmed a reduction in cancer cell proliferation (c). Furthermore, the expression of Ki67, an indicator of cell proliferation, was reduced, the expression of cleaved caspase 3, an indicator of apoptosis, was increased, and EGFR phosphorylation and STAT3 expression were also suppressed. Consequently, when compared with in vitro results, it was confirmed that the combined extract inhibited EGFR-STAT3 signaling and induced apoptosis (d-e). [Figure 18] 1 shows the results of examining cell viability when the mixed extract of Astragalus membranaceus, Angelica acutiloba, and Angelica acutiloba pollen of the present invention (SH003) was administered alone or in combination with docetaxel to the gastric cancer cell line AGS. [Figure 19]These are the results of confirming whether apoptosis, one of the mechanisms for suppressing cancer cells, is induced when the mixed extract (SH003) of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention is administered alone or in combination with docetaxel. DETAILED DESCRIPTION OF THE INVENTION

[0029] This will be explained in more detail as follows. Meanwhile, each description and embodiment disclosed in the present invention also applies to each other description and embodiment. That is, any combination of various elements disclosed in the present application falls within the scope of the present invention. Furthermore, the following specific description is not considered to limit the category of the present application.

[0030] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising a mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen, wherein the pharmaceutical composition is administered in combination with an anticancer agent, and the anticancer agent is docetaxel or paclitaxel.

[0031] The mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen of the present invention can be administered in combination with existing anticancer drugs and can be usefully used as a cancer therapeutic agent by exhibiting a synergistic effect in anticancer treatment.

[0032] In the present invention, the term "Astragalus membranaceus" refers to the plant with the scientific name "Astragalus membranaceus"; "Angelica gigas" refers to the plant with the scientific name "Angelica gigas"; and "Trichosanthes kirilowii Maximowicz" refers to the plant with the scientific name "Trichosanthes kirilowii Maximowicz". In the present invention, the ethanol extracts of Astragalus Root, Angelica Root and Angelica Root Pollen are used synonymously with "SH003" and can be used in combination.

[0033] In the present invention, the Astragalus Root, Angelica Root or Angelica Root pollen may be purchased commercially or collected or cultivated from nature, but is not limited thereto. The Astragalus Root, Angelica Root or Angelica Root pollen extract may be extracted from natural, hybrid or variant plants, or may be extracted from plant tissue culture.

[0034] The term "extract" as used herein includes the extract itself and all forms of extract that can be prepared using the extract, such as the extract obtained by extracting a mixture of Astragalus Root, Angelica Root, and Angelica Root Pollen, diluted or concentrated solutions of the extract, a dried product obtained by drying the extract, a crude or purified product of the extract, a fraction of the extract, or a mixture thereof.

[0035] In one specific example of the pharmaceutical composition for preventing or treating cancer of the present invention, the mixed extract may be extracted from a mixture of Astragalus Root, Angelica Root, and Angelica Root Pollen with one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof, specifically, ethanol extract, but is not limited thereto.

[0036] As a specific example of any one of the above-mentioned specific examples, the alcohol having 1 to 4 carbon atoms may be 20 to 40% (v / v) ethanol, specifically 30% (v / v) ethanol, but is not limited thereto.

[0037] In the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen of the present invention, the method for extracting the mixture is not particularly limited and may be any method commonly used in the art. Non-limiting examples of the extraction method include hot water extraction, ultrasonic extraction, filtration, reflux extraction, etc., which may be used alone or in combination of two or more methods.

[0038] In addition, the extract may be prepared in the form of a dry powder after extraction and used, but is not limited thereto.

[0039] In any one specific example of the pharmaceutical composition for preventing or treating cancer of the present invention, the mixed extract of Astragalus Root, Angelica Root and Angelica Root pollen may be mixed in a weight ratio of Astragalus Root:Angelica Root:Angelica Root = 0.5 to 5:1:1, specifically, a weight ratio of Astragalus Root:Angelica Root:Angelica Root = 1 to 3:1:1, and more specifically, a weight ratio of Astragalus Root:Angelica Root:Angelica Root = 1:1:1, but is not limited thereto.

[0040] The term "anticancer agent" as used herein is a general term for known drugs used in existing cancer treatments that act on various metabolic pathways of cancer cells and exhibit cytotoxicity or growth-suppressing effects on cancer cells, and includes all metabolic antagonists, plant alkaloids, topoisomerase inhibitors, alkylating agents, anticancer antibiotics, hormones, and other drugs that have been developed to date.

[0041] Anticancer drugs contained in the pharmaceutical composition of the present invention and exhibiting a synergistic effect with the mixed extract of Astragalus Root, Dong Qi, and Angelica Root Pollen may include cisplatins such as cisplatin, carboplatin, and heptaplatin, gemcitabine, 5-fluorouracil, and taxols such as docetaxel and its derivative paclitaxel, specifically docetaxel or paclitaxel, more specifically docetaxel, but are not limited thereto. Such anticancer drugs may be prepared by known methods or commercially available products.

[0042] Docetaxel (Taxol), a semisynthetic taxoid produced from the needles of the European yew (Taxus baccata), and its derivative paclitaxel are anticancer drugs used to treat solid tumors and highly metastatic malignancies, and are among the most important new active chemotherapeutic agents on the market.

[0043] Although docetaxel is an effective anticancer drug, it has limitations such as drug resistance, cancer recurrence, and side effects. For example, its use at high doses is known to induce another level of toxic reactions, and low or intermediate doses of docetaxel are known to show no significant antitumor activity in patients. Therefore, the use of docetaxel as a monotherapy for cancer is currently precluded. Therefore, there is a need for the development of combination therapeutic agents that exhibit synergistic anticancer effects when administered in combination with docetaxel.

[0044] In this application, the term "cancer" refers to an abnormally proliferated mass due to autonomous overgrowth of bodily tissue, also called a tumor, and specifically, the cancer may include breast cancer, lung cancer, stomach cancer, or prostate cancer.

[0045] The term "breast cancer" as used herein refers to a carcinoma that subsequently forms abnormal tissue in the breast or spreads to other organs, and may specifically be triple-negative breast cancer.

[0046] The term "triple-negative breast cancer" as used herein refers to breast cancer that accounts for approximately 15% of all breast cancers and is generally known to have a faster recurrence rate and a relatively low survival rate than other types of breast cancer. Triple-negative breast cancer lacks expression of three hormone receptors: estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2). This type of cancer does not respond well to antihormonal agents or targeted therapeutic agents and has a poor prognosis.

[0047] The term "lung cancer" as used herein refers to cancer that occurs in the lung itself or that metastasizes to the lung from another organ, and lung cancer in the present invention may be non-small cell lung cancer.

[0048] The term "non-small cell lung cancer" in the present invention may be used interchangeably with non-small cell lung cancer. Non-small cell lung cancer is a type of epithelial carcinoma, and refers to all epithelial lung cancers, not just small cell lung cancer, accounting for approximately 85% to 90% of all lung cancers. Symptoms of non-small cell lung cancer include persistent cough, chest pain, weight loss, nail abnormalities, joint pain, and shortness of breath. However, non-small cell lung cancer generally progresses slowly, with few symptoms appearing in the early stages, making early detection and treatment difficult. It is likely to be discovered only after it has metastasized throughout the body, such as to the bones, liver, small intestine, and brain.

[0049] The term "gastric cancer" as used herein broadly refers to cancers that occur in the stomach. The majority of gastric cancers are gastric adenocarcinomas, but other rare cancers may also occur, such as lymphomas that develop in the lymphatic tissue of the stomach, gastrointestinal tumors that develop in the interstitial cells of the stomach, sarcomas that originate from non-epithelial tissue, and hormone-secreting neuroendocrine tumors.

[0050] The term "prostatic carcinoma" as used herein refers to cancer that develops in the prostate gland, most of which are adenocarcinomas that develop from prostate cells. Tumors are classified into different types depending on the degree of differentiation of the tumor tissue and the characteristics of the cells. The most widely used classification system was proposed by pathologist Donald Gleason, and is divided into grades from grade 1, representing the best differentiation, to grade 5, representing the lowest. It has been reported that the better the differentiation, the better the prognosis.

[0051] In the present invention, the cancer is not particularly limited as long as the symptoms can be alleviated, reduced, improved or treated by the pharmaceutical composition for preventing or treating cancer comprising the mixed extract of Astragalus Root, Angelica Root and Angelica Root Pollen provided by the present invention and an anticancer agent.

[0052] The term "prevention" in the present invention refers to any action of suppressing or delaying the onset of cancer by administering a pharmaceutical composition for preventing or treating cancer, which contains the mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen, and an anticancer agent.

[0053] The term "treatment" as used herein means any action in which the symptoms of individuals suspected of or affected by cancer are improved or beneficially altered by administering said pharmaceutical composition.

[0054] In this application, the term "co-administration" means simultaneous or sequential treatment.

[0055] In a specific example of the present invention, it was confirmed that when a mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen was administered in combination with docetaxel, a synergistic anti-cancer effect was observed, and in particular, the therapeutic effect was significantly increased when administered in combination.

[0056] In one specific example of the pharmaceutical composition for preventing or treating cancer of the present invention, the pharmaceutical composition may reduce the expression level or activity of EGFR (Epidermal growth factor receptor), but is not limited thereto.

[0057] The epidermal growth factor receptor (EGFR) is involved in regulating cellular functions important for the proliferation and survival of cancer cells and has therefore been identified as a target for the treatment of many cancers, particularly solid tumors. Increased expression of EGFR has been observed in bladder cancer, breast cancer, glioblastoma, head and neck cancer, lung cancer, and gastric cancer.

[0058] In the present invention, the pharmaceutical composition may exhibit a synergistic effect in anti-cancer treatment by decreasing the expression level or activity of EGFR (Epidermal growth factor receptor).

[0059] In any one embodiment of the pharmaceutical composition for preventing or treating cancer of the present invention, the composition may further comprise a pharmaceutically acceptable carrier, excipient, or diluent, but is not limited thereto.

[0060] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier.

[0061] The term "pharmaceutically acceptable" as used herein means that the composition exhibits the property of being non-toxic to cells or humans exposed to the composition.

[0062] Specifically, the pharmaceutical compositions can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as topical preparations, suppositories, and sterile injection solutions according to conventional methods. In the present invention, carriers, excipients, and diluents contained in the pharmaceutical compositions include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and are formulated by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid dosage forms include suspensions, oral solutions, emulsions, syrups, etc., which may contain various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Parenteral dosage forms include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0063] In any one specific example of the pharmaceutical composition for preventing or treating cancer of the present invention, the composition may be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or intrarectal administration, but is not limited thereto.

[0064] Another aspect of the present invention provides a method for preventing or treating cancer, comprising the step of administering the composition to an individual.

[0065] At this time, the definitions of cancer, prevention and treatment are as explained above.

[0066] The term "administration" as used herein means introducing a given substance into an individual by an appropriate method.

[0067] The term "individual" as used herein refers to any animal, including humans, that develops or may develop cancer, such as mice, rats, livestock, etc. Specific examples include, but are not limited to, mammals, including humans.

[0068] The method for preventing or treating cancer of the present invention specifically includes administering to an individual a pharmaceutical composition for preventing or treating cancer, which comprises a mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen; and an anticancer agent, and administering the pharmaceutical composition in a pharmaceutically effective amount.

[0069] As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment, and which does not cause adverse side effects. The effective dose level can be easily determined by one skilled in the art depending on factors including the patient's sex, age, weight, health condition, type and severity of the disease, drug activity, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, concomitant or concomitant drugs, and other factors well known in the medical field.

[0070] Specifically, the composition of the present invention can be administered at a daily dose of 0.0001 to 100 mg / kg body weight, more specifically, 0.001 to 100 mg / kg body weight, based on the solid content. The recommended dose can be administered once a day or in divided doses.

[0071] In the method for preventing or treating cancer of the present invention, the administration route and mode of administration of the composition are not particularly limited and may be any administration route and mode as long as the composition containing the composition can be delivered to the target site. Specifically, the composition may be administered via various routes, such as oral or parenteral, and non-limiting examples of the administration route include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, or inhalation administration.

[0072] Example Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding the present invention. However, the examples according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those skilled in the art.

[0073] Example 1. Preparation of fractions from mixed herbal ethanol extract (SH003) and preparation of docetaxel Astragalus membranaceus (Am), Angelica gigas (Ag), and Trichosanthes kirilowii Maximowicz (Tk) were mixed in a 1:1:1 weight ratio (w / w), placed in an extractor, and extracted with 30% (v / v) ethanol at 100°C for 3 hours. The extract was filtered, and the filtrate was concentrated under reduced pressure and dried to obtain a mixed extract of Astragalus membranaceus, Angelica gigas, and Trichosanthes kirilowii Maximowicz. The dried extract was dissolved in 30% ethanol and stored at -80°C until use.

[0074] Docetaxel (DTX, Sigma-Aldrich, St. Louis, MO, USA) was purchased, dissolved in DMSO, and stored at -20°C.

[0075] Experimental Example 1. Analysis of synergistic effects of combined administration of the mixed extract of the present invention and docetaxel in triple-negative breast cancer Experimental Example 1-1. Analysis of the synergistic effect of combined administration of the mixed extract of the present invention and docetaxel on the viability of triple-negative breast cancer cells To confirm the synergistic effect of the mixed extract prepared in Example 1 and docetaxel on the viability of triple-negative breast cancer cells, MTT assay was performed using a conventional method. BT-20 (TNBC, triple-negative breast cancer, non-invasive) and MDA-MB-231 (TNBC, highly metastatic) triple-negative breast cancer cell lines were used.

[0076] More specifically, MDA-MB-231 triple-negative breast cancer cell lines cultured in 96-well plates were treated with various concentrations of the mixed extract of Astragalus membranaceus, Angelica acutiloba, and Angelica dahurica pollen (SH003, 100, 300, and 500 μg / mL) prepared in Example 1 or docetaxel (DTX, 1, 10, 100, and 1,000 nM) and then cultured for 24 hours at 37°C and 5% CO2. The cells were then treated with MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and further cultured for 4 hours. The supernatant was then removed and 100 μL of DMSO (dimethyl sulfoxide) was added. Finally, absorbance was measured at a wavelength of 590 nm, and the absorbance values ​​were compared with those of a control group untreated with any substance.

[0077] The results are shown in Figures 1 to 3.

[0078] As shown in Figure 1(a) and Figure 1(b), it was confirmed that SH003 did not affect the viability of BT-20 and MDA-MB-231 cells. As shown in Figure 1(c) and Figure 1(d), it was confirmed that DTX reduced the viability of both BT-20 and MDA-MB-231 cells in a dose-dependent manner.

[0079] On the other hand, as shown in Figures 2 and 3, it was confirmed that the combined administration of DTX and SH003 significantly suppressed the viability of triple-negative breast cancer cells.

[0080] To determine whether SH003 had a synergistic effect with DTX, the combination index (CI) values ​​of the drug pairs were calculated using CompuSyn software. CI<1, CI=1, and CI>1 indicate synergistic, additive, and antagonistic effects, respectively. Fa indicates the inhibition rate. The results are shown in Tables 1 and 2 below.

[0081] [Table 1]

[0082] [Table 2]

[0083] As shown in Tables 1 and 2, the highest synergistic effect in BT-20 and MDA-MB-231 cells was observed with the combination of 100 μg / mL SH003 and 10 nM DTX (CI: 0.24) and the combination of 100 μg / mL SH003 and 100 nM DTX (CI: 0.1), respectively. These results suggest that SH003 and DTX block excessive toxic accumulation at relatively low concentrations of DTX, resulting in a synergistic effect.

[0084] Experimental Example 1-2. Confirmation of the apoptosis induction effect in triple-negative breast cancer cells by combined administration of the mixed extract of the present invention and docetaxel To investigate apoptosis, BT-20 and MDA-MB-231 cells were treated with SH003 and / or DTX for 24 hours, and then analyzed for apoptosis by flow cytometry.

[0085] Specifically, Annexin V / 7-AAD double staining was performed. More specifically, cells were stained with Annexin V and then with 7-AAD in the dark at room temperature for 15 minutes. The stained cells were then detected using a FACSCalibur (BD Biosciences, San Jose, CA, USA), and apoptotic cells were analyzed using CellQuest Pro version 5.2 (BD Biosciences, San Jose, CA, USA) software. The results are shown in Figures 4 and 5.

[0086] As shown in Figures 4 and 5, when SH003 and DTX were administered in combination, it was confirmed that the level of apoptotic cells in BT-20 cells was increased compared to the administration of SH003 or DTX alone, or similar to the administration of DTX alone.

[0087] These results suggest that the combined administration of the mixed extract of the present invention and docetaxel increases apoptosis and enhances the sensitivity of triple-negative breast cancer cells to SH003 or DTX.

[0088] Experimental Example 1-3. Confirmation of the apoptosis induction effect through the inhibition of the EGFR signaling pathway in triple-negative breast cancer cells by combined administration of the mixed extract of the present invention and docetaxel To confirm whether the combined administration of the mixed extract of the present invention and docetaxel inhibits the EGFR signaling pathway, Western blot analysis was performed, and the results are shown in Figure 6.

[0089] As shown in Figure 6(a) and Figure 6(b), it was confirmed that combined administration of SH003 and DTX suppressed EGFR phosphorylation in both BT-20 and MDA-MB-231 cells.

[0090] Experimental Example 1-4: Confirmation of the inhibitory effect of combined administration of the mixed extract of the present invention and docetaxel on triple-negative breast cancer tumor growth and EGFR phosphorylation in an animal model The combined administration effect of the mixed extract of the present invention and docetaxel was confirmed in an animal model. All animal studies were approved by the Kyung Hee University Animal Hospital Animal Care and Use Committee (KHU-IACUC). Five-week-old female BALB / c nude mice were purchased from Nara Biotech (Seoul, Republic of Korea). Mice were housed in appropriate isolation cages with free access to food and water, at room temperature (22-25°C) under a 12-hour / 12-hour light / dark cycle, and in a pathogen-free environment. To establish a tumor xenograft mouse model, a BT-20 cell suspension (1x10 cells) was dissolved in 100 μL of PBS. 7 Mice were subcutaneously inoculated into the right flank of the mice. Mice were divided into four groups: control (n = 3), SH003 (n = 4), DTX (n = 4), and SH003 + DTX (n = 5). Tumors with a volume of 100 mm 3 When the tumor size reached 100 mm, the drugs were injected. DTX was administered intravenously via the tail vein at 15.277 mg / kg once a week, while the control group or the SH003-only group received DMSO. SH003 was administered orally at 557.569 mg / kg three times a week, while the control group or the DTX-only group received saline. Body weight was measured three times a week, and tumor volume was analyzed daily for 17 days.

[0091] The mice were then euthanized and the tumors were isolated. The tumor tissues were fixed and then incubated with hematoxylin and eosin (H&E) solution on slides for H&E staining. H&E images were obtained under a microscope (Carl Zeiss, Germany) at 40x magnification. The results are shown in Figures 7 to 9.

[0092] As shown in FIG. 7, it was confirmed that the combined administration of SH003 and DTX most effectively inhibited tumor growth compared to the groups in which either drug was administered alone.

[0093] Furthermore, as shown in FIG. 8, it was confirmed that the combined administration of SH003 and DTX significantly reduced the tumor weight and size compared to the groups administered either alone.

[0094] On the other hand, as shown in FIG. 9, it was confirmed that the single administration and combined administration of SH003 and DTX did not affect body weight.

[0095] In summary, the present study demonstrated that the mixed extract of Astragalus membranaceus, Angelica acutiloba, and Angelica acutiloba pollen induces apoptosis in triple-negative breast cancer cells in vitro and in vivo without side effects, and enhances the anticancer effects of docetaxel by suppressing the EGFR signaling pathway.

[0096] Experimental Example 2. Analysis of synergistic effects of combined administration of the mixed extract of the present invention and docetaxel in non-small cell lung cancer Experimental Example 2-1. Analysis of the synergistic effect of combined administration of the mixed extract of the present invention and docetaxel on the viability of non-small cell lung cancer cells To confirm the synergistic effect of the mixed extract prepared in Example 1 and docetaxel on the viability of non-small cell lung cancer cells, cell viability was measured using WST colorimetric assay. Non-small cell lung cancer cell lines H460 (no. 30177, Korean Cell Line Bank, Seoul, Korea) and A549 (no. 10185, Korean Cell Line Bank, Seoul, Korea) were used.

[0097] Specifically, a total of 10 5 Cells were seeded into 6-well plates before treatment. After 24 hours, the cells were treated with SH003 (100, 300, and 500 μg / mL), DTX (1, ​​10, 100, and 1000 nM), or their combination for 24, 48, and 72 hours. Control medium contained 0.15% ethanol and 0.1% DMSO, which have no cytotoxic effect. After treatment, the cells were incubated with WST for 1 hour, and then the absorbance was measured at 450 nm using an ELISA reader (Molecular Devices, Palo Alto, CA, USA). The results are shown in Figures 10 and 11.

[0098] As shown in Figure 10A, it was confirmed that single administration of SH003 or DTX both dose-dependently inhibited the growth of H460 cells. As shown in Figure 10B, it was confirmed that combined administration of DTX and SH003 more effectively inhibited the growth of H460 cells than single administration.

[0099] As shown in Figure 11A, it was confirmed that the administration of either SH003 or DTX alone inhibited the growth of A549 cells in a dose-dependent manner. As shown in Figure 11B, it was confirmed that the combined administration of DTX and SH003 inhibited the growth of A549 cells more effectively than the administration of either alone.

[0100] To determine whether SH003 had a synergistic effect with DTX, the combination index (CI) values ​​of the drug pairs were calculated using CompuSyn software. CI<1, CI=1, and CI>1 indicate synergistic, additive, and antagonistic effects, respectively. Fa indicates the inhibition rate. The results are shown in Table 3 below.

[0101] [Table 3]

[0102] As shown in Table 3 above, it was confirmed that 300 μg / mL of SH003 and 1 nM DTX showed the lowest CI (CI=0.69) in H460 cells.

[0103] Experimental Example 2-2. Confirmation of the apoptosis-inducing effect in non-small cell lung cancer cells by combined administration of the mixed extract of the present invention and docetaxel To investigate the apoptosis-increasing effect of the combined administration of the mixed extract of the present invention and docetaxel, Annexin V / 7-AAD double staining was used for analysis.

[0104] Specifically, H460 and A549 cells were stained with Annexin V and then with 7-AAD in the dark at room temperature for 15 minutes. The stained cells were detected using a FACSCalibur (BD Biosciences, San Jose, CA, USA), and apoptotic cells were analyzed using CellQuest Pro version 5.2 (BD Biosciences, San Jose, CA, USA) software. The results are shown in Figure 3.

[0105] As shown in Figure 12, 24-hour DTX treatment had no significant effect on H460 and A549 cells compared to the control group, whereas SH003 and the combination treatment induced apoptosis. Specifically, the combination treatment significantly increased apoptosis in H460 (34.07%) and A549 cells (23.74%).

[0106] These results suggest that the combined administration of the mixed extract of the present invention and docetaxel increases apoptosis and enhances the sensitivity of non-small cell lung cancer cells to SH003 or DTX.

[0107] Experimental Example 2-3. Confirmation of the apoptosis induction effect through the inhibition of EGFR signaling pathway in non-small cell lung cancer cells by combined administration of the mixed extract of the present invention and docetaxel To confirm whether the combined administration of the mixed extract of the present invention and docetaxel inhibits the EGFR signaling pathway, Western blot analysis was performed, and the results are shown in Figure 4.

[0108] As shown in Figure 13A, the combined administration of SH003 and DTX synergistically suppressed p-EGFR (Y1068) expression and EGFR phosphorylation in H460 cells. Furthermore, as shown in Figure 13B, EGF treatment increased p-EGFR levels, whereas the combined administration of SH003 and DTX significantly reduced p-EGFR levels.

[0109] Experimental Example 2-4: Confirmation of the tumor growth and EGFR phosphorylation inhibitory effect of combined administration of the mixed extract of the present invention and docetaxel in an animal model The efficacy of the combined administration of the mixed extract of the present invention and docetaxel was confirmed using an animal model. All animal experiments, including maintenance and euthanasia, were approved by the Animal Care and Use Committee of Kyung Hee University Animal Hospital (KHU-IACUC-KHSASP-21-211). Five-week-old male Balb / c nude mice were purchased from NARA Biotech (Seoul, Korea) and adapted to a 12-hour light-dark cycle at 40-60% humidity and 22-23°C. The mice were divided into four groups: control (n=3), SH003-treated (n=4), DTX-treated (n=4), and combination-treated (n=5). To establish tumor xenograft mouse models, H460 cell suspensions (1x10 cells) were cultured in 100 μL of PBS. 7 Mice were subcutaneously inoculated with SH003 (557.569 mg / kg) into the right flank. Subsequently, 200 μL of SH003 (557.569 mg / kg) was administered orally three times a week, and 50 μL of DTX (15.277 mg / kg) was administered intravenously via the tail vein once a week for three weeks. Saline and DMSO were used as vehicles. Body weights were measured three times a week, and tumor volume was analyzed daily. On day 21, mice were sacrificed using carbon dioxide followed by cervical dislocation. After euthanasia, tumor tissue was isolated. The tumor tissue was fixed and mounted on a glass slide for hematoxylin and eosin (H&E) staining. The tissue was then incubated with H&E solution. H&E images were obtained using a microscope (Carl Zeiss, Germany) at 40x magnification. The results are shown in Figure 5.

[0110] As shown in Figure 14A to C, it was confirmed that the combined administration of SH003 and DTX significantly reduced the tumor volume and weight compared to the groups in which either was administered alone.

[0111] On the other hand, as shown in Figure 14D, it was confirmed that the single administration of SH003 and DTX, as well as the combined administration, did not affect body weight, suggesting that the combined administration of the mixed extract of the present invention and docetaxel did not cause toxicity in mice and synergistically suppressed tumor growth.

[0112] In summary, the present study demonstrated that the mixed extract of Astragalus membranaceus, Angelica acutiloba, and Angelica acutiloba pollen induces apoptosis in non-small cell lung cancer cells in vitro and in vivo without side effects, and enhances the anticancer effects of docetaxel by suppressing the EGFR signaling pathway.

[0113] Experimental Example 3: Analysis of synergistic effects of combined administration of the mixed extract of the present invention and docetaxel on prostate cancer and gastric cancer Using the same experimental methods as in Experimental Examples 1 and 2, the synergistic effect of combined administration of the mixed extract of the present invention and docetaxel on gastric cancer and prostate cancer was confirmed.

[0114] Experimental Example 3-1. Analysis of synergistic effects of combined administration of the mixed extract of the present invention and docetaxel in prostate cancer First, to verify the efficacy of the combined effect of the present mixed extract SH003 and Docetaxel on the prostate cancer cell line DU145, an MTT assay was performed for both single and combined treatments. The Combination Index (CI) was calculated based on the results of the combined treatment.

[0115] As a result, as can be seen in Figure 15, it was confirmed that 1 nM Docetaxel and 300 μg / mL SH003 showed an excellent synergistic effect. Here, a CI of less than 1 can be interpreted as a synergistic effect, 1 as a cooperative effect, and greater than 1 as an antagonistic effect.

[0116] Based on these results, we selected a concentration in subsequent experiments that resulted in close to 50% cell viability.

[0117] To compare the apoptosis efficacy of the combination of SH003 and Docetaxel in the prostate cancer cell line DU145, apoptosis-related proteins were examined using Western blot analysis. Apoptosis was most clearly observed with the combination of concentrations obtained through the MTT assay. A relatively higher number of cells exhibiting apoptosis was also confirmed through flow cytometry (Figure 16).

[0118] Finally, (a) after 7 days of subcutaneous xenografting, the combination therapy group showed relatively more effective tumor growth inhibition than the other groups. (b) There was no change in mouse weight, confirming the lack of toxicity. (c) H&E staining confirmed a decrease in cancer cell proliferation. (d-g) Ki67 expression, an indicator of cell proliferation, also decreased. (d-g) Cleaved caspase 3 expression, an indicator of apoptosis, increased, and EGFR phosphorylation and STAT3 expression were suppressed. Consequently, when compared with in vitro results, it was confirmed that the combination therapy inhibited EGFR-STAT3 signaling and induced apoptosis (Figure 17).

[0119] Experimental Example 3-2. Analysis of synergistic effect of combined administration of the mixed extract of the present invention and docetaxel in gastric cancer Using gastric cancer cell lines, it was confirmed whether the combined administration of the mixed extract of the present invention (SH003) also has a synergistic effect on gastric cancer.

[0120] First, the cell survival rate after combined treatment was confirmed. For reference, the specific experimental method was the same as that in Experimental Examples 1 and 2.

[0121] As a result, as can be seen in Figure 18, when SH003 and Docetaxel were co-treated in the gastric cancer cell line AGS, it was confirmed that when either drug was administered alone, it reduced AGS cell viability in a concentration-dependent manner. In particular, when the effect of the combination on cell viability was confirmed, it was confirmed that the combined treatment reduced cell viability significantly more than when either drug was administered alone.

[0122] Furthermore, the combination index (CI) was calculated to confirm whether the reduction in cell viability due to the combination treatment was synergistic compared to the individual treatments. The CI value was less than 1, clearly confirming the synergistic effect. This indicates that the combined treatment of the mixed extract of the present invention and docetaxel has a synergistic effect.

[0123] Based on these results, additional experiments were carried out at combined treatment concentrations with lower CI values.

[0124] Next, we investigated the apoptosis induction effect of the combined treatment of the mixed extract of the present invention with docetaxel, particularly to determine whether the combined treatment induces apoptosis, which is one of the mechanisms of cancer cell suppression.

[0125] As a result, as can be seen in Figure 19, the apoptosis induction effect was confirmed through Annexin V and 7-AAD staining, and it was confirmed that apoptosis increased with the combination treatment compared to the single treatment group. In addition, we confirmed whether the expression of PARP, cleaved caspase-3, and cleaved caspase-7, which are protein markers related to the apoptosis mechanism, was regulated, and it was confirmed that all apoptosis-related proteins increased with the combination treatment.

[0126] Additionally, to confirm whether the induction of apoptosis by the combination therapy was the primary mechanism of gastric cancer cell suppression, we examined changes in cell viability by treating them with a caspase inhibitor, an apoptosis-related protein. As a result, we confirmed that the expression of PARP and cleaved-caspase 7, which had increased with the combination therapy, was reduced when treated with the caspase inhibitor, thereby suppressing the inhibitory effect on cell viability induced by the combination therapy.

[0127] In conclusion, it was confirmed that the apoptosis-inducing effect of the combined treatment of the mixed extract of the present invention and docetaxel is the main inhibitory mechanism for the inhibition of AGS gastric cancer cell lines.

[0128] In summary, it was confirmed that the combined administration of the mixed extract of the present invention and an anticancer drug has a significantly superior cancer treatment effect compared to the administration of either drug alone, and that the mixed extract of the present invention significantly improves the anticancer effect of anticancer drugs, especially docetaxel, without any side effects.

[0129] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including within the meaning and scope of the claims below, and any modifications or alterations derived from the equivalent concepts thereof, rather than the above detailed description.

Claims

1. A pharmaceutical composition for preventing or treating cancer, comprising a mixed extract of Astragalus Root, Angelica Root, and Angelica Root Pollen, the pharmaceutical composition is administered in combination with an anticancer agent; A pharmaceutical composition for preventing or treating cancer, wherein the anticancer drug is docetaxel or paclitaxel.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition reduces the expression level or activity of EGFR (Epidermal growth factor receptor).

3. 2. The pharmaceutical composition according to claim 1, wherein the mixed extract is obtained by extracting a mixture of Astragalus Root, Angelica Root, and Angelica Root Pollen with one or more solvents selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixtures thereof.

4. 3. The pharmaceutical composition according to claim 2, wherein the C1-C4 alcohol is 20-40% (v / v) ethanol.

5. 2. The pharmaceutical composition according to claim 1, wherein the mixed extract of Astragalus Root, Dong Qi and Angelica Root Pollen is mixed in a weight ratio of Astragalus Root: Dong Qi: Angelica Root Pollen = 0.5-5:1:

1.

6. The pharmaceutical composition of claim 1 , wherein the cancer is breast cancer, lung cancer, gastric cancer, or prostate cancer.

7. The pharmaceutical composition of claim 6, wherein the breast cancer is triple-negative breast cancer.

8. The pharmaceutical composition of claim 6 , wherein the lung cancer is non-small cell lung cancer.

9. 10. The pharmaceutical composition of claim 1, wherein the composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

10. 10. The pharmaceutical composition of claim 1, wherein the composition is administered by intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, pulmonary, or rectal routes.

11. A method for preventing or treating cancer, comprising administering the composition according to any one of claims 1 to 10 to an individual.

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