Pharmaceutical composition containing prunella vulgaris l. extract for preventing or treating cancer and method for preparing the same

Abstract

The present invention discloses a pharmaceutical composition for the prevention or treatment of cancer containing a Prunella vulgaris extract and a method for preparing the same. Since the present invention can prevent or treat cancer by including a Prunella vulgaris extract as an active ingredient, it can be utilized as a pharmaceutical composition or a health functional food. This project (result) is the result of research (2024-IT-RD-0069-01-101) conducted in 2024 with funding from the government (Ministry of Science and ICT) and support from the Korea R&D Special Zone Foundation. "Project Title: Research on the Commercialization of Neuro-stabilizing Materials for Insomnia Improvement"

Description

Technology Field

[0001] The present invention relates to a pharmaceutical composition for the prevention or treatment of cancer containing a Prunella vulgaris extract and a method for preparing the same. More specifically, the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer containing a Prunella vulgaris extract capable of reducing tumor size by inhibiting at least one of A549 and HCC1937 cancer cells, and a method for preparing the same. Background Technology

[0003] Cancer is the most representative disease threatening human life, and it is predicted that in 2022 alone, there will be 1.9 million new cases and nearly 600,000 cancer deaths in the United States. As of 2019, the number of new cancer cases in Korea was 254,718, and the number of cancer-related deaths was 81,203, accounting for 27% of total deaths; this figure ranks first among all deaths caused by disease. Although various strategies and treatments to overcome cancer have been developed over the past few decades, the number of new cases and deaths in Korea continues to steadily increase. In the United States, the cancer mortality rate has shown a gradual downward curve since the 1990s, but to this day, cancer accounts for 21% of deaths caused by disease, ranking second after heart disease.

[0004] Cancer generally involves a highly complex and multifaceted carcinogenesis process consisting of initiation, promotion, progression, and metastasis. Among these, metastasis is known to be a fatal cause, accounting for the highest proportion of mortality rates among human diseases. The process of metastasis involves metastatic malignant cancer cells infiltrating new tissues from the site of initial cancer development to form secondary tumors, and it consists of three major stages: adhesion, invasion, and angiogenesis.

[0005] Angiogenesis, the most important factor influencing tumor growth and metastasis, involves the degradation of the basement membrane and extracellular matrix, as well as the migration, proliferation, matrix formation, and maturation of endothelial cells, and is regulated by the balance between angiogenesis-promoting and angiogenesis-inhibiting factors. These factors act directly on endothelial cells or stimulate the secretion of enzymes that promote angiogenesis, while indirectly stimulating stromal cells to secrete enzymes that dissolve the extracellular matrix.

[0006] However, most angiogenesis inhibitors developed to date are in the form of compounds or proteins, which have problems such as difficulty in maintaining excellent activity for a long time, difficulty in mass production, and high manufacturing costs, as well as low pharmacological properties and the potential for easy denaturation.

[0007] Meanwhile, Prunella vulgaris L. is the inflorescence of Prunella vulgaris var. lilacina and P. vulgaris L., which are perennial herbaceous plants belonging to the Lamiaceae family. Prunella vulgaris has long been widely used as a folk medicine in the Eastern medical sphere of China, Japan, and Korea, as well as in Europe. In the East, it was used for liver cirrhosis and hypertension, while in Europe, it has been used since the 17th century for reducing throat inflammation, fever reduction, and promoting wound healing.

[0008] Recent studies on the physiological activities of *Hagocho* have reported anti-allergic, anti-inflammatory, antioxidant, antimicrobial, and antiviral effects, but research on the anticancer activity of *Prunella vulgaris* is limited.

[0009] This project (result) is the result of research (2024-IT-RD-0069-01-101) conducted in 2024 with funding from the government (Ministry of Science and ICT) and support from the Korea R&D Special Zone Foundation. "Project Title: Research on the Commercialization of Neuro-stabilizing Materials for Insomnia Improvement" Prior art literature

[0011] Korean Publication No. 10-2025-0014243, "Composition for the prevention or treatment of cancer containing Guaran extract as an active ingredient" The problem to be solved

[0012] Embodiments of the present invention aim to provide a pharmaceutical composition for the prevention or treatment of cancer containing a self-heal extract as an active ingredient, and a method for preparing the same.

[0013] An embodiment of the present invention aims to provide a health functional food for the prevention or treatment of cancer containing Prunella vulgaris extract as an active ingredient. means of solving the problem

[0015] A pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention comprises a self-heal extract as an active ingredient.

[0016] The above-mentioned Prunella vulgaris extract may be the flower of at least one of Prunella vulgaris var. lilacina and P. vulgaris L.

[0017] The above-mentioned Prunella vulgaris extract can induce at least one of inhibition of cancer cell growth and inhibition of cancer metastasis.

[0018] The above-mentioned Prunella vulgaris extract can induce inhibition of proliferation of at least one of the cancer cell lines A549 and HCC1937.

[0019] The above-mentioned Prunella vulgaris extract can regulate the production of nitric oxide (NO).

[0020] The above-mentioned Prunella vulgaris extract can be extracted with a solvent comprising at least one of water, C1 to C4 lower alcohols, an aqueous solution of lower alcohols, hexane, chloroform, and ethyl acetate.

[0021] The dosage concentration of the above pharmaceutical composition for the prevention or treatment of cancer may be 25 mg / L to 200 mg / L.

[0022] The above composition may further include at least one of an excipient, a filler, a carrier, a diluent, and a herbal medicine.

[0023] The above cancer may include at least one of lung cancer and breast cancer.

[0024] A health functional food composition for the prevention or treatment of cancer according to an embodiment of the present invention comprises Prunella vulgaris extract as an active ingredient.

[0025] A method for preparing a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention comprises the steps of: preparing a Prunella vulgaris extract by extracting Prunella vulgaris with a solvent comprising at least one of water, a C1 to C4 lower alcohol, an aqueous solution of a lower alcohol, hexane, chloroform, and ethyl acetate; obtaining a filtrate by filtering the Prunella vulgaris extract; preparing a Prunella vulgaris concentrate by concentrating the filtrate; and preparing a Prunella vulgaris extract by drying the Prunella vulgaris concentrate. Effects of the invention

[0027] According to an embodiment of the present invention, the Prunella vulgaris extract suggests the potential to inhibit the growth and proliferation of tumor cells while simultaneously influencing cancer metastasis processes such as tumor angiogenesis and invasion, and can be utilized as a novel natural product-based anticancer material that inhibits the progression of cancer. Brief explanation of the drawing

[0029] Figure 1 is a graph showing the drug administration schedule in a zebrafish xenograft model. Figure 2 is an image illustrating a xenograft model. Figure 3 is a graph showing the relative rate of change in tumor size (%) (4 dpi / 1 dpi) of A549 xenograft larvae treated with paclitaxel (PTX) and a candidate drug (NC-1) at specified concentrations. Figure 4 is a fluorescence microscope image of a zebrafish model administered with A549, Figure 5 is a fluorescence microscope image of a zebrafish model administered with A549 and control paclitaxel, Figure 6 is a fluorescence microscope image of a zebrafish model administered with 25 mg / L of A549 and NC-1 extract, Figure 7 is a fluorescence microscope image of a zebrafish model administered with 50 mg / L of A549 and NC-1 extract, and Figure 8 is a fluorescence microscope image of a zebrafish model administered with 100 mg / L of A549 and NC-1 extract. Figure 9 is a graph showing the relative rate of change in tumor size (%) (4 dpi / 1 dpi) of HCC1937 xenograft larvae treated with paclitaxel (PTX) and a candidate drug (NC-1) at specified concentrations. Figure 10 is a fluorescence microscope image of a zebrafish model administered HCC1937, Figure 11 is a fluorescence microscope image of a zebrafish model administered HCC1937 and a control Paclitaxel, Figure 12 is a fluorescence microscope image of a zebrafish model administered 25 mg / L of HCC1937 and NC-1 extract, Figure 13 is a fluorescence microscope image of a zebrafish model administered 50 mg / L of HCC1937 and NC-1 extract, and Figure 14 is a fluorescence microscope image of a zebrafish model administered 100 mg / L of HCC1937 and NC-1 extract. Figure 15 is a graph showing the cell viability of RAW 264.7 macrophages. Figure 16 is a graph showing NO production in RAW 264.7 macrophages. Specific details for implementing the invention

[0030] Embodiments of the present invention will be described in detail below with reference to the attached drawings and the contents described therein, but the present invention is not limited or restricted by the embodiments.

[0031] The terms used herein are for describing the embodiments and are not intended to limit the invention. In this specification, the singular form includes the plural form unless specifically stated otherwise in the text. As used herein, "comprises" and / or "comprising" do not exclude the presence or addition of one or more other components or steps mentioned in the description.

[0032] As used herein, terms such as “examples,” “examples,” “aspects,” “examples,” etc., are not to be interpreted as implying that any described aspect or design is superior or more advantageous than other aspects or designs.

[0033] Furthermore, the term 'or' refers to an inclusive or rather an exclusive or. That is, unless otherwise noted or is clear from the context, the expression 'x uses a or b' refers to any one of the natural inclusive permutations.

[0034] Additionally, singular expressions (“a” or “an”) used in this specification and claims should generally be interpreted to mean “one or more” unless otherwise stated or it is clear from the context that they relate to the singular form.

[0035] The terms used in the following description have been selected as common and universal in the relevant technical field, but other terms may exist depending on technological development and / or changes, conventions, preferences of the skilled technician, etc. Therefore, the terms used in the following description should not be understood as limiting the technical concept, but as illustrative terms to explain the embodiments.

[0036] In addition, in specific cases, there are terms arbitrarily selected by the applicant, and in such cases, their detailed meanings will be described in the relevant explanatory section. Therefore, the terms used in the description below must be understood not merely as their names, but based on their meanings and the content throughout the specification.

[0037] Unless otherwise defined, all terms used in this specification (including technical and scientific terms) may be used in a meaning that is commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0038] Meanwhile, in describing the present invention, if it is determined that a detailed description of related known functions or configurations could unnecessarily obscure the essence of the invention, such detailed description will be omitted. Furthermore, the terminology used in this specification is used to appropriately express embodiments of the present invention, and such terminology may vary depending on the intent of the user or operator, or the conventions of the field to which the invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification.

[0039] In this specification, "active ingredient" means an ingredient that exhibits the intended activity alone or can exhibit activity together with a carrier that is inactive itself.

[0041] A pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention comprises a self-heal extract as an active ingredient.

[0042] Prunella vulgaris extract can be of great help in the treatment of cancer because it possesses activity that inhibits proliferation and reduces tumor size in breast and lung cancer cell lines.

[0043] Accordingly, a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention may include a self-heal extract as an active ingredient and exhibit an effect of inhibiting the growth of cancer cells or inhibiting cancer metastasis.

[0044] Specifically, Hagocho belongs to the above-ground parts (roots, stems, and flower stalks) of Prunella vulgaris, and among the extracts from each part, the activity of physiologically active substances such as phenolic compounds and flavonoid compounds may be highest in the flower stalk extract.

[0045] Therefore, the Prunella vulgaris extract may contain the flower heads of at least one of Prunella vulgaris var. lilacina and P. vulgaris L.

[0046] Prunella vulgaris extract can induce at least one of inhibition of cancer cell growth and inhibition of cancer metastasis.

[0047] Cancer may include at least one of lung cancer and breast cancer.

[0048] The active ingredient of the Prunella vulgaris extract may include Dansensu.

[0049] Dansensu has antioxidant and inflammation-regulating effects.

[0050] Prunella vulgaris extract may contain various bioactive components in addition to Dansensu.

[0051] Rosmarinic acid plays a role in removing free radicals generated in the body, thereby preventing human aging and various diseases, and can have antiviral and anticancer activities.

[0052] Ursolic acid exhibits tumor-inhibiting effects in various cancer cell lines, which can block the expression of oncogenes by inhibiting the activity of the NF-κB pathway.

[0053] Chlorogenic acid has been extensively studied for its physiological activity, and it has anticancer activity and immune-enhancing effects.

[0054] Caffeic acid is a polyphenol compound that acts as a carcinogen inhibitor and can inhibit the induction of oxidative stress and the production of aflatoxins.

[0055] Prunella vulgaris extract can induce inhibition of proliferation in at least one of the cancer cell lines A549 and HCC1937.

[0056] The A549 cell line is a cell line used to search for substances with lung cancer-inhibiting ability.

[0057] The HCC1937 cell line is a cell line used to search for substances with breast cancer suppression ability.

[0058] Specifically, major bioactive components in Prunella vulgaris extract (rosmarinic acid, ursolic acid, dansensu, etc.) can increase NO production by stimulating the TLR (Toll-like-receptor) signaling pathway of macrophages and promoting INOS expression.

[0059] NO directly attacks cancer cells and induces apoptosis, and NO can play a role in inhibiting cancer cells by binding with RNS (Reactive Nitrogen Species) to induce DNA damage in cancer cells.

[0060] Therefore, the pharmaceutical composition for the prevention or treatment of cancer according to the embodiment of the present invention can be of great help in the treatment of cancer diseases because it has activity exhibiting inhibition of proliferation and reduction of tumor size against breast cancer or lung cancer cell lines.

[0061] A pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention can control the amount of nitric oxide (NO) produced.

[0062] NO production is primarily regulated by increased expression of the INOS (Iducible Nitric Oxide Synthase) gene, and major bioactive components in Prunella vulgaris extract (rosmarinic acid, ursolic acid, dansensu, etc.) can increase NO production by stimulating the TLR (Toll-like-receptor) signaling pathway in macrophages and promoting INOS expression.

[0063] NO directly attacks cancer cells and induces apoptosis, and NO can play a role in inhibiting cancer cells by binding with RNS (Reactive Nitrogen Species) to induce DNA damage in cancer cells.

[0064] A pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention can be extracted with a solvent comprising at least one of water, C1 to C4 lower alcohols, an aqueous solution of lower alcohols, hexane, chloroform, and ethyl acetate.

[0065] Preferably, the pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention may be extracted using an edible solvent, and more preferably, may be extracted using ethanol.

[0066] For example, in the case of solvents such as methanol, there is a problem in that methanol extracts cannot be used in pharmaceutical compositions because their high toxicity makes them inedible. Furthermore, methanol extraction requires additional processes (fractionation, spray drying, nitrogen concentration, etc.) to completely remove residual solvent, making the process complex. Additionally, continuous quality control regarding toxic substances to ensure the absence of residual solvent is required, making it ineffective in terms of cost.

[0067] The active ingredient may be administered in a pharmaceutically effective amount. A "pharmaceutically effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitantly used drugs, and other factors well known in the medical field.

[0068] A pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses.

[0069] It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, taking into account all of the aforementioned factors, and this can be easily determined by a person skilled in the art.

[0070] For example, in a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention, the effective amount of the active ingredient may vary depending on the patient's age, gender, and weight. However, since it may increase or decrease depending on the route of administration, severity of the disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0071] The pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention can control the size of a tumor depending on the administration concentration. For example, as the concentration of the extract increases, the size of the tumor can be inhibited, but if the concentration of the extract increases too much, the efficacy of inhibiting the size of the tumor may actually decrease. In addition, excessive NO production at high concentrations of the extract may lead to immune hypersensitivity reactions and abnormal symptoms.

[0072] Therefore, the administration concentration of a pharmaceutical composition for the prevention or treatment of cancer must be appropriately controlled.

[0073] The administration concentration of the pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention may be 25 mg / L to 200 mg / L, and if the administration concentration is less than 25 mg / L, the anticancer effect of suppressing tumor size may not be observed, and if it exceeds 200 mg / L, there is a problem that the effect of suppressing tumor size is actually reduced.

[0074] A pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention may further include at least one of an excipient, a volume expander, a carrier, a diluent, and a herbal medicine.

[0075] In addition, it can be used by being formulated into pharmaceutically acceptable formulations such as oral formulations like powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external formulations, suppositories, sterile injectable solutions, or injectables, according to conventional methods.

[0076] Carriers, excipients, and diluents that may be included in a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0077] Pharmaceutically acceptable carriers are not limited to these, but include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0078] When formulating a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0079] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the above composition.

[0080] In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0081] Liquid formulations for oral administration include suspensions, liquid formulations, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included.

[0082] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Additionally, non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0083] Witepsol, Macrogol, Tween 61, Cacao, Laurin, Glycerozelatin, etc. can be used as bases for suppositories.

[0084] A pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention may be administered to an individual by various routes. Any mode of administration may be anticipated, for example, by oral, intravenous, intramuscular, subcutaneous, or intraperitoneal injection.

[0085] In addition, the pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention may be used as an anticancer agent or an anticancer adjuvant.

[0086] An anticancer adjuvant refers to a preparation that can improve, enhance, or increase the anticancer effect of an anticancer drug.

[0087] For example, a preparation that does not exhibit anticancer activity on its own but can improve, enhance, or increase the anticancer effect of an anticancer drug when used together with an anticancer drug may be used as an anticancer adjuvant.

[0088] In addition, when a preparation exhibiting concentration-dependent anticancer activity is used in combination with an anticancer agent at a level where it does not exhibit anticancer activity on its own, it can be used as an anticancer adjuvant to improve, enhance, or increase the anticancer effect of the said anticancer agent. In this case, the said anticancer adjuvant can be used as an anticancer agent or an anticancer adjuvant depending on the treatment concentration, and can be used as an anticancer adjuvant within the treatment concentration range where it does not exhibit anticancer activity on its own.

[0089] An anticancer adjuvant may be administered in combination with an anticancer agent or an anticancer adjuvant exhibiting anticancer activity to enhance the anticancer effect of the anticancer agent. An anticancer adjuvant may be administered in combination with a known anticancer agent. When an anticancer agent is administered with a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention, an equivalent level of anticancer therapeutic effect can be achieved even when the anticancer agent is administered at a lower dose than the conventional dose of the anticancer agent, thereby enabling safer anticancer treatment.

[0090] Not limited to, any known anticancer agents, for example, DNA alkylating agents, mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; Anticancer antibiotics include, but are not limited to, dactinomycin (actinomycin D), doxorubicin (adriamycin), daunorubicin, idarubicin, mitoxantrone, plicamycin, mitomycin C, and bleomycin; and plant alkaloids include vincristine, vinblastine, paclitaxel, docetaxel, etoposide, teniposide, topotecan, and iridotecan.

[0091] The route of administration for anticancer adjuvants may be any general route as long as it can reach the target tissue. Depending on the purpose, anticancer adjuvants may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonaryly, or rectally, but are not limited thereto. Additionally, anticancer adjuvants may be administered by any device capable of delivering the active substance to the target cells.

[0093] A health functional food composition for the prevention or treatment of cancer according to an embodiment of the present invention comprises Prunella vulgaris extract as an active ingredient.

[0094] A health functional food composition for the prevention or treatment of cancer according to an embodiment of the present invention may include the same components as a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention, and a description of the same components is omitted.

[0095] Health functional foods can be used in various ways, such as in foods and beverages, that are effective in preventing or improving cancer. For example, the health functional food composition for the prevention or treatment of cancer according to the embodiment of the present invention may include a food science-acceptable food additive and may be used in various foods, beverages, spices, gums, teas, vitamin complexes, health supplements, etc., and may be used in the form of powder, granules, tablets, capsules, suspensions, or beverages.

[0096] The active ingredient can generally be added in an amount of 0.01 to 15% by weight of the total food weight, and the health drink composition can be added in a ratio of 0.02 to 10g, preferably 0.3 to 1g, based on 100ml.

[0097] If the content of Prunella vulgaris extract included in the health drink composition is relatively high, the bitter taste may increase due to changes in flavor, and the appearance may change and turn brown during long-term storage due to excessive melanoidin reactions. In addition, phenolic or polysaccharide components in the health drink may aggregate over time to form precipitates, and abnormal symptoms may appear due to immune hypersensitivity reactions caused by excessive NO generated from high concentrations of extract; therefore, an appropriate content is important.

[0098] In addition to containing the above-mentioned compound as an essential ingredient in the prescribed proportions, the health functional food may contain food-grade acceptable food additives, such as natural carbohydrates and various flavoring agents, as additional ingredients.

[0099] Examples of natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, as well as sugar alcohols such as xylitol, sorbitol, and erythritol.

[0100] As flavoring agents, natural flavoring agents such as stevia, taumatin, rebaudioside A, or glycyrrhizin, and synthetic flavoring agents such as saccharin and aspartame may be used. The ratio of natural carbohydrates is generally about 1 to 20g, preferably about 5 to 12g, per 100ml of the health functional food of the present invention.

[0101] In addition, health functional foods may contain various nutritional supplements, vitamins, minerals, flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0102] In addition, health functional foods may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, vegetable beverages, etc. These ingredients may be used independently or in combination. The proportion of these additives is generally selected in the range of 0.01 to about 20 parts by weight per 100 parts by weight of the active ingredient of the present invention.

[0104] A method for preparing a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention comprises the steps of: preparing a Prunella vulgaris extract by extracting Prunella vulgaris with a solvent comprising at least one of water, a C1 to C4 lower alcohol, an aqueous solution of a lower alcohol, hexane, chloroform, and ethyl acetate (S110); obtaining a filtrate by filtering the Prunella vulgaris extract (S120); preparing a Prunella vulgaris concentrate by concentrating the filtrate (S130); and preparing a Prunella vulgaris extract by drying the Prunella vulgaris concentrate (S140).

[0105] First, a method for preparing a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention proceeds with the step (S110) of preparing a Prunella vulgaris extract by extracting Prunella vulgaris with a solvent comprising at least one of water, a C1 to C4 lower alcohol, an aqueous solution of a lower alcohol, hexane, chloroform, and ethyl acetate.

[0106] About 1 to 100 times (v / w), preferably about 5 to 20 times (v / w) the weight of the dried Hagocho sample, water, C1 to C4 lower alcohol or a mixed solvent thereof, preferably water, ethanol or a mixed solvent thereof, more preferably a mixed solvent of water and ethanol, and more preferably 60 to 95% ethanol are added, and the sample can be extracted by at least one of cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, and heat extraction at 30°C to 110°C, preferably 50°C to 100°C, for 1 to 5 hours, preferably 1 to 3 hours.

[0107] Afterwards, the method for preparing a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention proceeds to the step (S120) of filtering a Prunella vulgaris extract to obtain a filtrate.

[0108] Prunella vulgaris extract can be filtered to remove residue and obtain only the filtrate. The filtration process can remove impurities by filtering the extract through cotton wool or filter paper with a diameter of 0.06 mm or less.

[0109] Afterwards, the method for preparing a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention proceeds to the step (S130) of concentrating the filtrate to prepare a concentrated extract of Prunella vulgaris.

[0110] The concentration process can concentrate the filtrate from which impurities have been removed under reduced pressure at a temperature range of 50°C to 60°C.

[0111] Finally, the method for preparing a pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention proceeds with the step (S140) of preparing a Prunella vulgaris extract by drying a Prunella vulgaris concentrate.

[0112] The drying process may include a spray drying process or a freeze drying process.

[0113] The freeze-drying process can rapidly freeze the Prunella vulgaris concentrate using an ultra-low temperature freezer at a temperature of minus 50°C to minus 70°C.

[0114] In addition, freeze-dried Prunella vulgaris concentrate can be rapidly frozen and then dried using a vacuum freeze-dryer for 24 to 48 hours to obtain a final dried product.

[0116] In the following, examples are presented to aid in understanding the present invention; however, the following examples are merely illustrative of the present invention and the scope of the present invention is not limited to the following examples.

[0117] The pharmaceutical composition for the prevention or treatment of cancer according to an embodiment of the present invention was subjected to both in-vitro and in vivo experiments.

[0119] [Experimental Example 1] Method for evaluating the in vivo anticancer efficacy of NC-1 extract

[0120] Figure 1 is a graph showing the drug administration schedule in a zebrafish xenograft model.

[0121] Referring to Figure 1, two types of cancer cells (A549, HCC1937) were labeled with CM-Dil and injected into the perivitelline space (PVS) and yolk of 2 dpf zebrafish.

[0122] Paclitaxel, used as a positive control, and Prunella vulgaris extract, the test drug, were diluted to their final concentrations in E3 medium immediately before use, and each was exposed to concentrations ranging from 3 dpf to 6 dpf for 3 days, with the drugs being replaced daily.

[0124] [Experimental Example 2] Evaluation of in vivo anticancer efficacy of NC-1 extract

[0125] Figure 2 is an image illustrating a xenograft model.

[0126] A549, HCC1937 were cultured in RPMI 1640 medium (WEL GENE, Cat.LM011-03, Korea). Cells were maintained at 37°C and 5% CO2 humidity, and mycoplasma detection was performed regularly using the MycoAlert™ Mycoplasma Detection Kit (Lonza, Basel, Switzerland) in accordance with the manufacturer's specifications.

[0127] Cells were harvested with 0.25% tripsin-EDTA (Gibco, USA) to label them with CellTracker™ CM-DiI (Invitrogen, Thermo Fisher Scientific, USA). 1–2 x 10⁷ cells were labeled with 4 μM CM-DiI, washed with DPBS (WEL GENE, Cat. LB001-01, Korea), and cell viability was confirmed by trypan blue staining (viability usable for the experiment was 95% or higher). The labeled cells were resuspended in 40% Geltrex™ (ThermoFisher, USA) serum-free medium to achieve a final density of 2.7 x 10⁵ cells / μl, and then stored on ice.

[0128] 2dpf zebrafish fry were anesthetized with 0.4 mg / mL tricaine and mounted on a 2% low-melting agarose gel, and about 300 to 450 cells (500 to 700 cells for SW620) were injected into the yolk using borosilicate capillaries and a microinjector μPump (World Precision Instruments, USA). The larvae injected with cells were transferred to E3 (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 and 0.33 mM MgSO4, pH adjusted to 7.0-7.2) / 0.1 mM PTU medium and incubated at 34℃.

[0129] Zebrafish fry 1 day (1 dpi) and 4 days (4 dpi) after cell injection were anesthetized with 1X Tricaine and imaged using a fluorescence microscope.

[0130] The tumor size of the PVS site where cell injection was performed was quantified using Image J software, and the relative change in tumor size (%) (4dpi / 1dpi) was measured.

[0132] [Example 1: NC-1]: Prunella vulgaris extract

[0133] Prunella vulgaris flower stalks were purchased at Gyeongdong Market in Seoul. Ethanol was added at a ratio of 20 to 30 times the dry weight of the prunella vulgaris, and the mixture was extracted at 70°C for 3 hours. After extraction, the mixture was filtered to remove insoluble substances and an ethanol extract was prepared.

[0134] The prepared ethanol extract was concentrated in a thin film concentrator at a temperature of 40°C to 60°C until it reached 15 Brix or higher.

[0135] Maltodextrin of 30% to 40% by weight was added to a concentrated Prunella vulgaris extract and dried at an inlet temperature of approximately 180°C to 200°C to produce a final Prunella vulgaris flower stem extract powder (Extract 1) with a yield of approximately 15%.

[0137] [Control group] : Control anticancer agent used for anticancer activity

[0138] To compare and measure the anticancer activity of the ethanol extract of Prunella vulgaris, paclitaxel, a drug currently used as an anticancer agent, was purchased from Supelco and used as a positive control.

[0140] [Experimental Example 3] Results of In vivo Anticancer Efficacy Evaluation of NC-1 Extract

[0142] Figure 3 is a graph showing the relative rate of change in tumor size (%) (4 dpi / 1 dpi) of A549 xenograft larvae treated with paclitaxel (PTX) and a candidate drug (NC-1) at specified concentrations.

[0143] Figure 4 is a fluorescence microscope image of a zebrafish model administered with A549, Figure 5 is a fluorescence microscope image of a zebrafish model administered with A549 and a control paclitaxel, Figure 6 is a fluorescence microscope image of a zebrafish model administered with 25 mg / L of A549 and NC-1 extract, Figure 7 is a fluorescence microscope image of a zebrafish model administered with 50 mg / L of A549 and NC-1 extract, and Figure 8 is a fluorescence microscope image of a zebrafish model administered with 100 mg / L of A549 and NC-1 extract.

[0144] Referring to Figures 2 to 8, the breast cancer efficacy of the control group Paclitaxel and 25 mg / L Prunella vulgaris extract was demonstrated, which inhibited tumor size similarly to Paclitaxel.

[0145] In other words, to evaluate the anticancer efficacy of NC-1, cancer A549 and HCC1937 cells were injected into zebrafish (xenograft), and it was confirmed that it inhibited the proliferation of A549 and HCC1937 cells, and the relative change in tumor size (%) was statistically significantly reduced. This indicates that the NC-1 extract is an active ingredient with an inhibitory effect on cancer cell growth.

[0146] Therefore, it can be seen that NC-1 exhibits anticancer efficacy.

[0148] Figure 9 is a graph showing the relative rate of change in tumor size (%) (4 dpi / 1 dpi) of HCC1937 xenograft larvae treated with paclitaxel (PTX) and a candidate drug (NC-1) at specified concentrations.

[0149] Figure 10 is a fluorescence microscope image of a zebrafish model administered HCC1937, Figure 11 is a fluorescence microscope image of a zebrafish model administered HCC1937 and a control Paclitaxel, Figure 12 is a fluorescence microscope image of a zebrafish model administered 25 mg / L of HCC1937 and NC-1 extract, Figure 13 is a fluorescence microscope image of a zebrafish model administered 50 mg / L of HCC1937 and NC-1 extract, and Figure 14 is a fluorescence microscope image of a zebrafish model administered 100 mg / L of HCC1937 and NC-1 extract.

[0150] Referring to Figures 9 to 14, the lung cancer efficacy effect was demonstrated in that the control group Paclitaxel and 25 mg / L Prunella vulgaris extract inhibited tumor size similarly to Paclitaxel.

[0152] Accordingly, the pharmaceutical composition for the prevention or treatment of cancer according to the embodiment of the present invention confirmed that the extract of *Prunella vulgaris* induced inhibition of proliferation of A549 and HCC1937 cancer cell lines in in vivo experiments and exhibited an effect of statistically significantly reducing tumor size. These results suggest that the extract of *Prunella vulgaris* may inhibit the growth and proliferation of tumor cells while simultaneously influencing cancer metastasis processes such as tumor angiogenesis and invasion, and can be utilized as a novel natural product-based anticancer material that inhibits the progression of cancer.

[0154] [Experimental Example 4] In-vitro Immunostimulating Effect of Honey Water Extract (NC-01)

[0155] RAW 264.7 macrophages are monocytic leukemia cell lines derived from BALB / c mice and are one of the most widely used cells (ATCC) in immune-related research. Lipopolysaccharide (LPS) is known to stimulate immune cells such as macrophages and dendritic cells to induce inflammatory responses, and it is mainly used in immune-related research because it can be confirmed that treatment with LPS in RAW 264.7 macrophage experiments increases inflammatory cytokines (TNF-α, IL-6, IL-1β, etc.) and nitric oxide (NO).

[0156] Nitric oxide (NO), produced by inducible nitric oxide synthase (iNOS) in immune cells such as macrophages, contributes to eliminating pathogens and inhibiting tumor cell growth, and functions as an immune defense mechanism if not produced in excess.

[0157] The cell viability test method involved placing RAW 264.7 cells into 1 x 10 wells of a 96-well plate. 4 After inoculating at a cell / well concentration, the cells were cultured for 24 hours. Subsequently, NC-01 was treated at concentrations of 10, 50, 100, and 200 μg / mL per well and cultured for an additional 24 hours. Cells not treated with NC-01 were used as a control.

[0158] In addition, to measure the cell viability of the LPS-treated group, cells were inoculated in the same manner, treated with NC-01 and incubated for 1 hour, then LPS was added to each well at a concentration of 5 ng / mL and cultured for an additional 24 hours. PBS buffer without LPS treatment was used for the control group. Subsequently, WST-1 was added to each well and incubated for 2 hours, after which the absorbance was measured at 450 nm using a microplate spectrophotometer.

[0159] For the analysis of nitric oxide (NO), quantification was performed using Raw 264.7 cells and a Griess reagent system (Promega, USA). 50 μl of culture medium was added to a 96-well plate, and equal volumes of Griess Reagent I (NED, naphthylethylenediamide) solution and Griess Reagent II (sulfanilamide solution) were mixed and added. After incubating for 10 minutes, measurements were taken at 540 nm using a microplate reader (Molecular Devices, USA) within 30 minutes. The concentration of nitric oxide (NO) was calculated using a sodium nitrite standard curve (0–100 μM).

[0161] Figure 15 is a graph showing the cell viability of RAW 264.7 macrophages.

[0162] Referring to Figure 15, NC-01 did not exhibit cytotoxicity in RAW 264.7 cells even at concentrations of up to 200 ng / mL, regardless of the presence or absence of LPS, indicating that it is non-cytotoxic.

[0164] Figure 16 is a graph showing NO production in RAW 264.7 macrophages.

[0165] NO is an important mediator in inflammatory responses, and as a result of confirming the NO production rate, it was found that the NO production rate significantly increased with NC-01 treatment at 50 μg / Ml, 100 μg / Ml, and 200 μg / Ml.

[0167] As described above, although the present invention has been explained by limited embodiments and drawings, the present invention is not limited to the above embodiments, and various modifications and variations are possible from this description by those skilled in the art to which the present invention belongs. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be defined by the claims set forth below as well as equivalents thereof.

Claims

Claim 1 A pharmaceutical composition for the prevention or treatment of cancer characterized by containing Prunella vulgaris extract as an active ingredient. Claim 2 A pharmaceutical composition for the prevention or treatment of cancer according to claim 1, characterized in that the Prunella vulgaris extract is the flower of at least one of Prunella vulgaris var. lilacina and P. vulgaris L. Claim 3 A pharmaceutical composition for the prevention or treatment of cancer according to claim 1, wherein the above-mentioned Prunella vulgaris extract induces at least one of inhibition of cancer cell growth and inhibition of cancer metastasis. Claim 4 A pharmaceutical composition for the prevention or treatment of cancer according to claim 3, wherein the above-mentioned Prunella vulgaris extract induces inhibition of proliferation of at least one of A549 and HCC1937 cancer cell lines. Claim 5 A pharmaceutical composition for the prevention or treatment of cancer according to claim 1, wherein the above-mentioned Prunella vulgaris extract controls the amount of nitric oxide (NO) produced. Claim 6 A pharmaceutical composition for the prevention or treatment of cancer according to claim 1, characterized in that the Prunella vulgaris extract is extracted with a solvent comprising at least one of water, C1 to C4 lower alcohols, an aqueous solution of lower alcohols, hexane, chloroform, and ethyl acetate. Claim 7 A pharmaceutical composition for the prevention or treatment of cancer, characterized in that, in claim 1, the administration concentration of the composition is 25 mg / L to 200 mg / L. Claim 8 A pharmaceutical composition for the prevention or treatment of cancer according to claim 1, characterized in that the composition further comprises at least one of an excipient, a filler, a carrier, a diluent, and a herbal medicine. Claim 9 A pharmaceutical composition for the prevention or treatment of cancer according to claim 1, characterized in that the cancer comprises at least one of lung cancer and breast cancer. Claim 10 A health functional food composition for the prevention or treatment of cancer, characterized by containing Prunella vulgaris extract as an active ingredient. Claim 11 A method for preparing a pharmaceutical composition for the prevention or treatment of cancer, characterized by comprising: a step of preparing a Prunella vulgaris extract by extracting Prunella vulgaris with a solvent comprising at least one of water, a C1 to C4 lower alcohol, an aqueous solution of a lower alcohol, hexane, chloroform, and ethyl acetate; a step of filtering the Prunella vulgaris extract to obtain a filtrate; a step of concentrating the filtrate to prepare a Prunella vulgaris concentrate; and a step of drying the Prunella vulgaris concentrate to prepare a Prunella vulgaris extract.

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