Novel compounds derived from myristic acid and anticancer compositions comprising the same

Abstract

This invention relates to a novel compound derived from nutmeg and an anticancer composition comprising the same. The present invention isolates several novel compounds derived from nutmeg extract and confirms that most of these novel compounds exhibit cytotoxic activity against various cancer cell lines. Five compounds (3, 4, 6, 9, and 11) were identified as having high cytotoxic activity against gastric and colorectal cancers. In particular, the compound represented by chemical formula 3 was confirmed to not only effectively induce apoptosis by regulating the cell cycle of cancer cells but also effectively inhibit tumor growth in xenograft mouse models, thus making it an effective anticancer composition.

Description

Technical Field

[0001] This invention relates to a novel compound derived from nutmeg and an anticancer composition comprising the same. Background Technology

[0002] Normal cells can proliferate and be inhibited in a regular and flexible manner as needed, while cancer cells proliferate without restraint. These clumps of undifferentiated cells are also known as tumors. These cancer cells invade surrounding tissues and metastasize to other organs, causing severe pain and ultimately leading to death. Despite medical advancements, the number of cancer patients in South Korea is reportedly still rising, increasing by approximately 44% over the past decade, and the international market for anti-cancer drugs has grown accordingly, reaching an annual market size of approximately $100 billion.

[0003] However, during or within days of administering anticancer drugs, loss of appetite, nausea, and vomiting may occur. Allergic reactions (rash, angioedema, difficulty breathing) may also occur immediately or within hours of administration. Furthermore, stomatitis, diarrhea, and neutropenia-induced fever may also occur after anticancer drug administration. Therefore, efforts are constantly being made to reduce these side effects, and as part of this effort, research is actively underway using natural products to alleviate the side effects of anticancer treatment.

[0004] On the other hand, nutmeg ( Myristica fragrans Nutmeg is an evergreen tree belonging to the Myristicaceae family. After harvesting the fruit, the seeds are extracted, dried, and then the hard black seed coat is removed. The seeds are then soaked in lime water overnight and dried at room temperature to obtain nutmeg. Nutmeg promotes blood circulation and improves lung function, and has antibacterial effects. Therefore, it is used to improve blood flow, treat respiratory and skin diseases, and is also used as a spice due to its aromatic components. Research on nutmeg has reported cytotoxic effects on cancer cell lines, antibacterial and antioxidant effects, liver detoxification effects, and inhibitory effects on brain cholinesterase associated with dementia (Korean Patent Publication Nos. 10-1999-0034285; 10-2012-0021283; 10-2011-0119483). Summary of the Invention

[0005] Technical issues In view of this, the present invention is dedicated to developing a natural source material that reduces the side effects of anticancer drugs and has excellent anticancer effects. Ultimately, a novel compound derived from nutmeg extract was screened and confirmed to have anticancer activity, thus completing the present invention.

[0006] Therefore, the object of the present invention is to provide a novel compound derived from nutmeg extract.

[0007] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer or a health functional food composition for the prevention or improvement of cancer, comprising a compound derived from nutmeg extract as an active ingredient.

[0008] Technical solution In order to achieve the above objectives, The present invention provides a compound as shown in the following chemical formula A, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

[0009] Chemical formula A (In the chemical formula A, X is or In this case, n is an integer from 1 to 20. At least one of R1 to R5 is In this case, Y is O or -(CH)m- (where m is 0 to 5), and R1' to R6' are each independently hydrogen, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C2-10 allyl, or substituted or unsubstituted C3-10 heterocyclic. The remaining portions of R1 to R5 are each independently hydrogen, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C2-10 allyl, or substituted or unsubstituted C3-10 heterocyclic. R6, R8 and R 10 Each is independently either hydrogen or hydroxyl. R7 and R9 are each independently hydrogen. R 1' To R 6' Each of these groups is independently hydrogen, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C2-10 allyl, or substituted or unsubstituted C3-10 heterocyclic. In a preferred embodiment of the present invention, the compound represented by the chemical formula A may be: the compound represented by the following chemical formula 3; the compound represented by the following chemical formula 4; the compound represented by the following chemical formula 6; the compound represented by the following chemical formula 9; the compound represented by the following chemical formula 10; the compound represented by the following chemical formula 11; the compound represented by the following chemical formula 12; or the compound represented by the following chemical formula 13.

[0010] Chemical formula 3 Chemical Formula 4 Chemical Formula 6 Chemical formula 9 Chemical Formula 10 Chemical Formula 11 Chemical formula 12 Chemical formula 13 In another preferred embodiment of the invention, the compound represented by chemical formula A may be a compound represented by chemical formula 3 below.

[0011] Chemical formula 3 Furthermore, the present invention provides a compound as shown in Chemical Formula 14 below, a pharmaceutically acceptable salt thereof, or an optical isomer thereof; Compounds represented by chemical formula 15 below, their pharmaceutically acceptable salts, or their optical isomers; or Compounds represented by the following chemical formula 16, their pharmaceutically acceptable salts, or their optical isomers.

[0012] Chemical formula 14 Chemical Formula 15 Chemical Formula 16 Furthermore, the present invention provides a compound as shown in Chemical Formula 17 below, a pharmaceutically acceptable salt thereof, or an optical isomer thereof; Compounds represented by the following chemical formula 18, their pharmaceutically acceptable salts, or their optical isomers; Compounds represented by chemical formula 19 below, their pharmaceutically acceptable salts, or their optical isomers; or Compounds represented by the following chemical formula 20, their pharmaceutically acceptable salts, or their optical isomers.

[0013] Chemical Formula 17 Chemical Formula 18 Chemical formula 19 Chemical formula 20 Furthermore, the present invention provides a compound as shown in Chemical Formula 21 below, a pharmaceutically acceptable salt thereof, or an optical isomer thereof; or Compounds represented by the following chemical formula 22, their pharmaceutically acceptable salts, or their optical isomers.

[0014] Chemical Formula 21 Chemical formula 22 To achieve another purpose, The present invention provides a pharmaceutical composition for the prevention or treatment of cancer or a health functional food composition for the prevention or improvement of cancer, comprising the compound as an active ingredient.

[0015] In a preferred embodiment of the present invention, the compound may be: Compounds represented by chemical formula 3, their pharmaceutically acceptable salts, or their optical isomers; Compounds represented by chemical formula 4, their pharmaceutically acceptable salts, or their optical isomers; Compounds represented by chemical formula 6, their pharmaceutically acceptable salts, or their optical isomers; The compound represented by chemical formula 10, its pharmaceutically acceptable salt or its optical isomer; The compound represented by chemical formula 11, its pharmaceutically acceptable salt or its optical isomer; Compounds represented by chemical formula 12, their pharmaceutically acceptable salts, or their optical isomers; The compound represented by chemical formula 14, its pharmaceutically acceptable salt or its optical isomer; The compound represented by chemical formula 15, its pharmaceutically acceptable salt or its optical isomer; Compounds represented by chemical formula 17, their pharmaceutically acceptable salts, or their optical isomers; or Compounds represented by the chemical formula 21, their pharmaceutically acceptable salts, or their optical isomers.

[0016] The effects of the invention This invention isolated several novel compounds derived from nutmeg extract and confirmed that most of these novel compounds exhibit cytotoxic effects against various cancer cell lines. Five compounds (3, 4, 6, 9, and 11) were identified as having high cytotoxic effects against gastric and colorectal cancers. In particular, the compound represented by chemical formula 3 was confirmed to not only effectively induce apoptosis by regulating the cell cycle of cancer cells but also effectively inhibit tumor growth in xenograft mouse models, thus making it a potentially effective anticancer composition. Attached Figure Description

[0017] Figures 1a to 1c Data obtained from the analysis of compounds represented by chemical formulas 1 to 8 using circular dichroism spectroscopy (CD Spectroscopy).

[0018] Figures 2a to 2c Data obtained from the analysis of compounds such as those represented by chemical formulas 10 to 13 using circular dichroism spectroscopy (CD Spectroscopy).

[0019] Figure 3 Data obtained from the analysis of compounds such as those represented by chemical formulas 14 to 16 using circular dichroism spectroscopy (CD Spectroscopy).

[0020] Figure 4 Data showing the main HMBC (heteronuclear multibond correlation spectrum) (→) and COSY (homonuclear correlation spectrum) (red, bold) correlations of compounds as shown in formulas 14 to 16.

[0021] Figure 5 Data obtained from the analysis of compounds such as those represented by chemical formulas 17 to 20 using circular dichroism spectroscopy (CD Spectroscopy).

[0022] Figure 6 Data showing the main HMBC (→) and COSY (red, bold) correlations of compounds such as those shown in Formulas 17 to 20.

[0023] Figure 7Data showing the main HMBC (→) and COSY (red, bold) correlations for compounds such as those shown in Formula 21 or Formula 22.

[0024] Figure 8 A heatmap to confirm the survival rate (%) of cancer cell lines treated with a novel compound (10 μM) derived from nutmeg. The color scale shows the percentage of surviving cells relative to the untreated control group, with green indicating higher cell viability and white indicating lower cell viability.

[0025] Figure 9 To confirm the cell survival (%) data of gastric cancer cell lines and colorectal cancer cell lines treated with the novel compound (10 μM).

[0026] Figure 10a and Figure 10b To confirm the cell cycle distribution data of 23132 / 87 and SW48 cell lines after treatment with 5 μM and 10 μM myricone C and Malabaricone C compounds.

[0027] Figures 11a to 11c To confirm the cell cycle analysis data of 23132 / 87 and SW48 cell lines treated with 5 μM and 10 μM of myrC and malabatone C (MalC) compounds.

[0028] Figure 12a and Figure 12b To confirm the apoptosis patterns of 23132 / 87 and SW48 cell lines after treatment with 5 μM and 10 μM milicocin C and malaboxone C compounds.

[0029] Figure 13a and Figure 13b To confirm the apoptosis distribution (%) data of 23132 / 87 and SW48 cell lines after treatment with 5 μM and 10 μM milicocin C and malaboxone C compounds.

[0030] Figure 14 Data are obtained by comparing non-apoptotic cells (Q4: surviving cells) with the sum of apoptotic and dead cells (Q2 and Q3) in the 23132 / 87 and SW48 cell lines.

[0031] Figure 15 To confirm the caspase-3 and -7 activity data in the 23132 / 87 and SW48 cell lines after treatment with 5 μM and 10 μM milicocin C and malaboxone C compounds.

[0032] Figure 16 shows the data confirming the anticancer effect of mirtazacin C compound in xenograft mouse models. Figure 16a To illustrate the animal experiment process, Figure 16b To confirm tumor volume data after compound administration, Figure 16c To confirm the body weight and major organ (liver, spleen, kidney) weight data of mice after administration of the compound. Detailed Implementation

[0033] The present invention will now be described in detail.

[0034] One aspect of the present invention relates to a compound as shown in the following chemical formula A, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

[0035] Chemical formula A (In the chemical formula A, X is or In this case, n is an integer from 1 to 20. At least one of R1 to R5 is In this case, Y is O or -(CH)m- (where m is 0 to 5), and R1' to R6' are each independently hydrogen, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C2-10 allyl, or substituted or unsubstituted C3-10 heterocyclic. The remaining portions of R1 to R5 are each independently hydrogen, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C2-10 allyl, or substituted or unsubstituted C3-10 heterocyclic. R6, R8 and R 10 Each is independently either hydrogen or hydroxyl. R7 and R9 are each independently hydrogen. R 1' To R 6' Each of these groups is independently hydrogen, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C2-10 allyl, or substituted or unsubstituted C3-10 heterocyclic. In this invention, the compound represented by the chemical formula A can be: the compound represented by the following chemical formula 3; the compound represented by the following chemical formula 4; the compound represented by the following chemical formula 6; the compound represented by the following chemical formula 9; the compound represented by the following chemical formula 10; the compound represented by the following chemical formula 11; the compound represented by the following chemical formula 12; or the compound represented by the following chemical formula 13.

[0036] Chemical formula 3 Chemical Formula 4 Chemical Formula 6 Chemical formula 9 Chemical Formula 10 Chemical Formula 11 Chemical formula 12 Chemical formula 13 More preferably, the compound represented by the chemical formula A can be a compound represented by the chemical formula 3 below.

[0037] Chemical formula 3 Another aspect of the present invention relates to a compound as shown in Chemical Formula 14 below, a pharmaceutically acceptable salt thereof, or an optical isomer thereof; Compounds represented by chemical formula 15 below, their pharmaceutically acceptable salts, or their optical isomers; or Compounds represented by the following chemical formula 16, their pharmaceutically acceptable salts, or their optical isomers.

[0038] Chemical formula 14 Chemical Formula 15 Chemical Formula 16 Another aspect of the present invention relates to a compound as shown in Chemical Formula 17 below, a pharmaceutically acceptable salt thereof, or an optical isomer thereof; Compounds represented by the following chemical formula 18, their pharmaceutically acceptable salts, or their optical isomers; Compounds represented by chemical formula 19 below, their pharmaceutically acceptable salts, or their optical isomers; or Compounds represented by the following chemical formula 20, their pharmaceutically acceptable salts, or their optical isomers.

[0039] Chemical Formula 17 Chemical Formula 18 Chemical formula 19 Chemical formula 20 Another aspect of the invention relates to a compound as shown in Chemical Formula 21 below, a pharmaceutically acceptable salt thereof, or an optical isomer thereof; or Compounds represented by the following chemical formula 22, their pharmaceutically acceptable salts, or their optical isomers.

[0040] Chemical Formula 21 Chemical formula 22 In this invention, the compound is characterized in that it is derived from nutmeg ( Myristica fragrans It is obtained by isolating from nutmeg extract, preferably, the nutmeg extract can be an ethanol extract with a concentration of 70% (v / v) to 100% (v / v).

[0041] In one specific example of the present invention, 35 compounds were isolated from an ethanol extract at a concentration of 75% (v / v) as a nutmeg extract, of which 17 novel compounds, such as those shown in chemical formulas 3, 4, 6, 9 to 22, were isolated.

[0042] Another aspect of the present invention relates to a pharmaceutical composition for the prevention or treatment of cancer or a health functional food composition for the prevention or improvement of cancer, comprising the compound as an active ingredient.

[0043] Preferably, the compound can be a compound as shown in Formula 3, a pharmaceutically acceptable salt thereof, or an optical isomer thereof; Compounds represented by chemical formula 4, their pharmaceutically acceptable salts, or their optical isomers; Compounds represented by chemical formula 6, their pharmaceutically acceptable salts, or their optical isomers; The compound represented by chemical formula 10, its pharmaceutically acceptable salt or its optical isomer; The compound represented by chemical formula 11, its pharmaceutically acceptable salt or its optical isomer; Compounds represented by chemical formula 12, their pharmaceutically acceptable salts, or their optical isomers; The compound represented by chemical formula 14, its pharmaceutically acceptable salt or its optical isomer; The compound represented by chemical formula 15, its pharmaceutically acceptable salt or its optical isomer; Compounds represented by chemical formula 17, their pharmaceutically acceptable salts, or their optical isomers; or Compounds represented by the chemical formula 21, their pharmaceutically acceptable salts, or their optical isomers.

[0044] In this invention, the compound is characterized in that it is derived from nutmeg ( Myristica fragrans It is obtained by isolating from nutmeg extract, preferably, the nutmeg extract can be an ethanol extract with a concentration of 70% (v / v) to 100% (v / v).

[0045] In this invention, the cancer may be pancreatic cancer, liver cancer, lung cancer, stomach cancer, colorectal cancer, prostate cancer, kidney cancer, breast cancer, or ovarian cancer, preferably stomach cancer or colorectal cancer.

[0046] In one specific example of the present invention, various cancer cell lines were treated with novel compounds derived from nutmeg extract. As a result, most of the compounds were confirmed to have anticancer activity, and compounds such as those shown in Formula 3, Formula 4, Formula 6, Formula 10, Formula 11, Formula 12, Formula 14, Formula 15, Formula 17 and Formula 21 were confirmed to have high anticancer activity.

[0047] Furthermore, five compounds (chemical formulas 3, 4, 6, 9, and 11) were identified as having high cytotoxic effects against gastric and colorectal cancers. In particular, the compound represented by chemical formula 3 was found to effectively induce apoptosis by regulating the cell cycle of cancer cells, and the anticancer effect of miracin C compound was confirmed in xenograft mouse models.

[0048] The pharmaceutical compositions of the present invention can be formulated into various dosage forms and used according to conventional methods. For example, they can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, and syrups, as well as into topical preparations, suppositories, and sterile injections. Depending on the dosage form, they may also contain pharmaceutically acceptable carriers, excipients, and diluents. Furthermore, they can be formulated into powders, granules, tablets, capsules, suspensions, emulsions, syrups, topical preparations such as aerosols, and sterile injections according to conventional methods, and used accordingly.

[0049] The carrier, excipients, and diluents include lactose, glucose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, mineral oil, etc. When preparing or formulating the pharmaceutical composition, it is formulated using diluents or excipients, such as commonly used fillers, extenders, binders, humectants, disintegrants, and surfactants.

[0050] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., which are formulated by mixing one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants, such as magnesium stearate and talc, are also used. Liquid dosage forms for oral administration include suspensions, oral solutions, emulsions, syrups, etc., which may contain various excipients, such as wetting agents, sweeteners, flavoring agents, preservatives, etc., in addition to commonly used simple diluents (water and liquid paraffin). Dosage forms for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. Propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable esters (such as ethyl oleate) can be used as non-aqueous solvents and suspensions. As a suppository base, witepsol, macrogol, Tween 61, cocoa butter, laurate, glycerin gelatin, etc. can be used.

[0051] As used in this invention, the term "administration" refers to the delivery of the pharmaceutical composition of the invention to an individual by any suitable method. The pharmaceutical compositions of the invention can be administered at a therapeutically effective dose, which is the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal, or human body, as envisioned by a researcher, veterinarian, physician, or other clinician, i.e., the amount capable of alleviating the symptoms of the treated disease or condition. The therapeutically effective dose and frequency of administration of the pharmaceutical compositions of the invention will vary depending on the desired therapeutic effect, as will be apparent to those skilled in the art. Therefore, those skilled in the art can readily determine the optimal dosage and adjust it according to various factors, including disease type, disease severity, the content of the active ingredient and other components in the composition, the type of formulation, the patient's age, weight, general health condition, sex and diet, timing of administration, route of administration and release rate of the composition, duration of treatment, and concurrent medications. The pharmaceutical compositions of the invention can be administered to an individual via a variety of routes. For example, they can be administered intravenously, intraperitoneally, intramuscularly, intraarterially, intraorally, intracardiacly, intramedullaryly, percutaneously, intraintestinally, subcutaneously, sublingually, or locally, but are not limited thereto. The daily dosage of the pharmaceutical composition of the present invention can be 1 to 10,000 mg / kg, and can be administered once daily or in multiple divided doses.

[0052] The health-functional food composition of the present invention can be used as a health-functional food, a food additive, or a dietary supplement. When the composition of the present invention is used as a food additive, it can be used appropriately according to conventional methods, such as by direct addition or by mixing with other foods or food ingredients.

[0053] Furthermore, the mixing amount of the aforementioned health-functional food composition can be appropriately varied depending on the intended use (prevention, health maintenance, or treatment). As a specific example, when preparing food or beverages, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, relative to the raw materials. However, if long-term intake is intended for health and hygiene purposes or for regulating health, it is possible to add it in amounts lower than the aforementioned range; since there are no safety concerns, the active ingredient can also be used in amounts within the stated range.

[0054] There are no specific limitations on the types of food mentioned, but examples of foods to which the composition of the present invention may be added include meat, sausage, bread, chocolate, candy, snacks, pastries, pizza, ramen, other noodles, chewing gum, dairy products (including ice cream), various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, etc., covering all traditionally considered healthy foods.

[0055] When the health-functional food composition of the present invention is prepared into a beverage, it can contain additional ingredients such as various flavoring agents or natural carbohydrates, just like conventional beverages. The natural carbohydrates can be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; natural sweeteners such as dextrin and cyclodextrin; and synthetic sweeteners such as saccharin and aspartame. The content of the natural carbohydrates is 0.01 to 10% by weight of the total weight of the food composition of the present invention, preferably 0.01 to 0.1% by weight.

[0056] The health-functional food composition of the present invention may include various nutrients, vitamins, electrolytes, flavoring agents, coloring 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., and may include, but is not limited to, fruit pulp used in the preparation of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used alone or in combination. Although there are no significant limitations on the proportions of the above-mentioned additives, preferably, their content is 0.01 to 0.1% by weight of the total weight of the food composition of the present invention.

[0057] The present invention will now be described in more detail through embodiments.

[0058] These embodiments are merely illustrative of the invention, and the scope of the invention should not be construed as being limited to these embodiments, as will be apparent to those skilled in the art.

[0059] Example 1: Preparation and isolation of nutmeg extract 1-1: Preparation of nutmeg extract nutmeg( Myristica fragrans Nutmeg was purchased from Kyunghee Herbpharm, a Korean pharmaceutical company. 5 kg of chopped nutmeg was mixed with 24% 75% ethanol in an extraction vessel and cold-extracted. The mixture was then filtered through extraction solvent filter paper to obtain the extract. The extraction process was repeated twice, followed by concentration under reduced pressure and drying to obtain 205 g of extract.

[0060] 1-2: Isolation of compounds derived from nutmeg extract In this invention, compounds derived from the nutmeg and 75% ethanol extract prepared in Examples 1-1 are isolated.

[0061] The extracts obtained in Examples 1-1 were sequentially partitioned with dichloromethane (MC) and ethyl acetate (EtOAc) to obtain 100 g and 2 g residues, respectively. The 100 g soluble fraction of MC was fractionated by silica gel column chromatography (hexane-EtOAc, 20:1 → 1:10) to obtain 35 fractions (1-35), and then compounds were separated from each fraction by high performance liquid chromatography (HPLC).

[0062] Table 1 HPLC analysis conditions Table 2 Compounds derived from nutmeg extract (1) Table 3 Compounds derived from nutmeg extract (2) Table 4 Compounds derived from nutmeg extract (3) Table 5 Compounds derived from nutmeg extract (4) Table 6 Compounds derived from nutmeg extract (5) Table 7 Compounds derived from nutmeg extract (6) In this invention, as shown in Tables 2 to 7 above, 35 compounds were isolated, among which 18 compounds, as shown in Chemical Formulas 3 to 6 and Chemical Formulas 9 to 22, were identified as novel compounds.

[0063] Furthermore, the HPLC analysis results of the compounds represented by chemical formulas 2 to 22 are shown in Tables 8 to 17 below.

[0064] Table 8 Table 9 Table 10 Table 11 Table 12 Table 13 Table 14 Table 15 Table 16 Table 17 Example 2: Anticancer activity of novel compounds derived from nutmeg extract In this invention, in order to confirm the anticancer activity of the novel compounds isolated in Examples 1-2, gastric cancer cell lines (AGS, 23132 / 87, SNU-719), colorectal cancer cell lines (SNU-81, SNU-C5, SW48), glioblastoma cell lines (SNU201, SNU466), liver cancer cell lines (HepG2), and pancreatic cancer cell lines (MiaPaCa-2) were treated with the novel compounds at a concentration of 10 μM, and then the cell viability was measured.

[0065] AGS, SW48, HepG2, and MiaPaCa-2 were purchased from the American Type Culture Collection (ATCC), SNU cell line from the Korean Cell Line Bank (KCLB), and 23132 / 87 from the Leibniz Institute DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, DSMZ). All cell lines were cultured in RPMI-1640 medium (HyClone, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin (HyClone, USA) at 5% CO2 and 37°C.

[0066] Cells were divided into 5×10 3Cells / well were seeded at a density of 1 / 2 well in 96-well plates and cultured for 24 hours, followed by treatment with an appropriate concentration (10 μM) of the compound for 48 hours. After treatment, 1 / 10 of the treatment volume of Alamar Blue reagent was added, and the cells were incubated at 37°C for 4 hours (avoiding direct light). Cell viability was then determined by measuring absorbance at 570 nm and 600 nm using a microplate reader. Cell viability (%) and the half-maximal inhibitory concentration (IC50) of each natural product were evaluated at specific treatment concentrations. 50 The anticancer effects of the candidate substances were evaluated. Doxorubicin and oxaliplatin were used as positive control groups for gastric and colorectal cancer, respectively.

[0067] All experiments were repeated three times, and results are expressed as mean ± standard deviation (SD). Statistical analysis was performed using R software (version 4.2.2, R Core Team). Differences between groups were assessed using either a student's t-test or one-way ANOVA followed by a post-hoc Tukey HSD test. A p-value < 0.05 was considered statistically significant.

[0068] Table 18 Cancer cell line survival rate (%) after treatment with a novel compound (10 μM) derived from nutmeg. The results show, as shown in Table 18 above, and Figure 8 As shown, most of the novel compounds isolated from nutmeg were confirmed to have anticancer activity. In particular, five compounds (3, 4, 6, 9, and 11) were confirmed to have excellent cytotoxic effects from gastric cancer cell lines and colorectal cancer cell lines.

[0069] Typically, most compounds show efficacy starting at 10 μM treatment, but SNU81 cells exhibit high resistance to these compounds. However, these compounds elicit a superior response in SNU81 cells compared to the positive control group oxaliplatin, while the response in SNUC5 cells was similar to that of oxaliplatin. Figure 9 ).

[0070] Furthermore, in order to determine the IC50 of the compound against six cancer cell lines, including gastric cancer cell lines (AGS, 23132 / 87, SNU-719) and colorectal cancer cell lines (SNU-81, SNU-C5, SW48), further investigation was conducted. 50The IC50 values ​​were determined after treating cells with different concentrations (2.5–40 μM) of the novel compound. 50 value.

[0071] Table 19 The compound's effect on the IC50 of 6 cancer cell lines 50 Value (μM) IC of the compound 50 The values ​​are shown in Table 19. Among the novel compounds, miliconacin C and miliconacin G exhibited the strongest anticancer activity and the lowest IC50 values. 50 Values. As previously mentioned, miricon C and miricon G showed IC50 values ​​for SNU81 and SNUC5 cells. 50 The values ​​were 9.84 μM and 2.54 μM, and 10.30 μM and 4.04 μM, respectively, which were significantly lower than the IC50 values ​​of oxaliplatin. 50 The values ​​were 41.49 μM and 12.55 μM. Conversely, the milico EF mixture showed the weakest effect, with an IC50 value of 41.49 μM and 12.55 μM. 50 The activity levels were 1.5 to 5 times higher than the most potent candidate compounds in all cell lines. Among the existing compounds, gigantoxin A showed superior activity compared to myristone B and malabathone C in most cell lines, demonstrating excellent overall activity. Through this analysis, novel compound Milicocin C and existing compound Malabar ketone C were screened out, and in-depth experiments were conducted on typical cell lines 23132 / 87 (gastric cancer) and SW48 (colorectal cancer) for each cancer.

[0072] Example 3: Confirmation of the effect of miracin C compound on cancer cell cycle 3-1: Confirm cell cycle after treatment with Milicon C One characteristic of cancer is its ability to evade cell cycle regulation and divide rapidly. Therefore, in this invention, the cell cycle of two cancer cell lines was analyzed based on the presence or absence of milicoquinone C or malabathone C.

[0073] DNA staining was performed using FxCycle PI / RNase staining solution (Invitrogen, USA) to analyze cell cycle distribution. The 23132 / 87 and SW48 cell lines were stained with 5×10⁻⁶ ppm of FxCycle PI / RNase staining solution to analyze cell cycle distribution. 5 and 7X10 5Cells were seeded at a density of 10,000 cells / wells in 6-well plates and cultured for 24 hours, followed by overnight starvation with serum-free medium. Cells were then treated with either 5 μM or 10 μM milicon C or malaboxone C, or with the control group dimethyl sulfoxide (DMSO) for 48 hours. Cells were subsequently collected, fixed with 70% ethanol, and stained with PI / RNase solution. Samples were analyzed based on 10,000 events using a flow cytometer (BD FACSVerse Cell Analyzer and FlowJo software, BD Biosciences, Franklin Lake, New Jersey, USA).

[0074] The results, as shown in Figures 10 and 11, indicate that, overall, unlike the 5 μM case, the 10 μM compound significantly interfered with the cell cycle of both tested cell lines.

[0075] Furthermore, the differences between samples treated with each compound and those treated with only the control group DMSO were compared. The results showed that for 23132 / 87 cells, Milicon C (10 μM) caused cell cycle arrest at G1 phase, increasing the proportion by approximately 10% compared to DMSO. Inevitably, the proportions at all other phases decreased, especially at G2 / M phase, where the decrease was approximately 4%.

[0076] Conversely, malathione C had no significant effect on these gastric cancer cells at either concentration. For SW48 cells, treatment with 10 μM milicocin C slightly reduced the proportions of cells in the G1, S, and G2 / M phases, but only the decrease in the G2 / M phase was statistically significant. On the other hand, malathione C increased the proportion of cells in the S phase by approximately 2%, and decreased the proportions in the G1 and G2 / M phases by approximately 8% and 3%, respectively. Notably, compared to the control group DMSO, 10 μM milicocin C and malathione C increased the proportions in the sub-G1 phase by 7% and 11%, respectively.

[0077] 3-2: Confirm cell cycle distribution based on mirtonic C treatment concentration To confirm the cell cycle distribution of milicoquinone C treatment concentrations, the cell cycle distribution of 23132 / 87 and SW48 cell lines was analyzed after treatment with 5 μM and 10 μM milicoquinone C and Malabar ketone C compounds. Student's t-test statistical analysis was also performed between the two concentrations of the same compound.

[0078] Table 20 Cell cycle distribution after treatment with miracone C and malabatone C compounds The results, as shown in Table 20 above, indicated that treatment with the compound at a concentration of 10 μM was more suitable for the SW48 cell line. Specifically, increasing the concentration of milicoquinone C from 5 μM to 10 μM significantly increased the proportion of cells in the sub-G1 phase, from 9.65% ± 1.02% to 24.17% ± 0.98%, while decreasing the proportion in the G2 phase from 8.91% ± 0.24% to 6.13% ± 0.47%. Similarly, compared to 5 μM milicoquinone C, 10 μM milicoquinone C affected all stages of the cell cycle. Therefore, it was confirmed that 10 μM milicoquinone C had a significant effect on the cell cycle of the colorectal cancer SW48 cell line.

[0079] Malabar ketone C increased the proportion of patients in the S and sub-G1 phases and suppressed the proportion of patients in the remaining phases, while milicoquinone C decreased the proportion of patients in the G2 / M phase and caused them to shift to the sub-G1 phase. For 23132 / 87 gastric cancer, both selected compounds showed an increasing trend in the G1 phase, but the opposite trend was observed in the S and G2 / M phases.

[0080] Example 4: Milicon C compound induces apoptosis in cancer cells 4-1: Apoptosis assay In this invention, to confirm whether the miracin C compound can induce apoptosis in cancer cells, apoptosis was measured in cells treated with the compound using Annexin V-FITC / PI staining.

[0081] 23132 / 87 and SW48 cells were respectively treated with 5×10⁻⁶ 5 and 7X10 5 Cells were seeded at a density of 1 / 2 cell / well in 6-well plates and cultured for 24 hours. Subsequently, cells were treated with either 5 μM or 10 μM milicoquinone C or malabathone C, or with the control group DMSO for 20 hours. After treatment, cells were collected and stained with the Annexin V-FITC Early Apoptosis Detection Kit (6592, Cell Signaling Technology, USA). Samples were analyzed based on 10,000 events using flow cytometry (BD FACSVerse Cell Analyzer and FlowJo software, BD Biosciences, Franklin Lake, New Jersey, USA).

[0082] The results, as shown in Figure 12, indicated that the compound treatment induced apoptosis in gastric and colorectal cancer cell lines. This was manifested by an increase in the cell populations of Annexin V-FITC+ / PI+ (Q2, late apoptosis and cell death) and Annexin V-FITC+ / PI- (Q3, early apoptosis), and a decrease in the healthy cell population of Annexin V-FITC- / PI- (Q4).

[0083] 4-2: Comparison of apoptosis-related cell populations (Q2 and Q3) with the control group As shown in Figure 13, the Annexin V-FITC positive cell populations Q2 and Q3 in the treated sample were compared with the control group DMSO. The results showed that in the 23132 / 87 cell line, Malabar ketone C at a concentration of 5 μM significantly increased the Q2 population, and at a concentration of 10 μM, it significantly increased the Q3 population. Similarly, mirtazapine C at a concentration of 5 μM significantly increased the proportion of the Q3 population, and at a concentration of 10 μM, it increased the proportions of the Q2 and Q3 populations by approximately 2% and 6%, respectively. No significant difference was found in the 5 μM treatment group in SW48 cells. On the other hand, in the 10 μM treatment group with increased compound concentration, the Annexin V-FITC+ / PI+ signal was significantly increased; in particular, with mirtazapine C, the proportion of the Q2 population reached 22.43% ± 0.30%, an increase of more than 8% compared to the control group.

[0084] 4-3: Analysis of differences between cells To confirm the cell cycle distribution based on milicoquinone C treatment concentrations, the cell cycle distribution of 23132 / 87 and SW48 cell lines was analyzed after treatment with 5 μM and 10 μM milicoquinone C and Malabar ketone C compounds, respectively. Student's t-test statistical analysis was also performed between the two concentrations of the same compound.

[0085] Table 21 Distribution of apoptosis in cells treated with milicoquinone C and maralabatone C compounds (%) As shown in Table 21 above, when the concentration of the compound was doubled, mirtazapine C increased the proportion of Q3 population in 23132 / 87 cells from 5.76% ± 0.35% to 10.97% ± 0.12%, and increased the proportions of Q2 and Q3 populations in SW48 cells from 13.80% ± 0.65% to 22.43% ± 0.30%, and from 13.73% ± 0.92% to 18.77% ± 1.07%, respectively. The increase of more than 8% in the proportion of Q2 population in SW48 cells was the highest value in the experiment based on all doses, indicating the potent effect of mirtazapine C. It is noteworthy that the changes in cell distribution caused by DMSO were not statistically significant even when the concentration was doubled in both cell lines.

[0086] 4-4: Comparison of apoptosis-related cell populations (Q2 and Q3) with non-apoptotic cell populations (Q4) To more clearly observe apoptosis-related cell populations (Q2 and Q3) and non-apoptotic cell populations (Q4), a side-by-side comparative analysis was performed. The results showed that, Figure 14 As shown, compared with the control group, all treatment groups significantly reduced the proportion of healthy cells in both cell lines. In particular, 10 μM milicon C reduced the Q4 distribution in 23132 / 87 and SW48 cells by 8.27% and 9.43%, respectively.

[0087] Furthermore, the proportion of Q4 decreased with increasing dose, especially in SW48 cells treated with Milicon C, where the proportion of Q4 decreased by approximately 11.23% when the dose was changed from 5 μM to 10 μM.

[0088] Apoptosis-related populations include early apoptotic cells (Q3), late apoptotic cells, and dead cells (Q2). In 23132 / 87 cells treated with milicoquinone C, the proportions of Q2 and Q3 increased significantly with increasing dose. In SW48 cells, malaboxone C showed a trend of increasing the proportions of apoptotic and dead cells, but this increase was not statistically significant. However, 10 μM milicoquinone C increased the proportions of these populations more effectively than DMSO.

[0089] That is, the apoptosis analysis results using Annexin V / FITC-PI double staining confirmed that the compound Milicon C can induce apoptosis in both gastric cancer and colorectal cancer cell lines, with a preferred concentration of 10 μM.

[0090] Example 5: Effect of Milicon C compound on caspase-3 / 7 activity in cancer cells Apoptosis can be activated and proceed through endogenous or exogenous pathways, but in most cases, caspase-3, the effector caspase, is involved. Therefore, the real-time activities of caspase-3 and -7 in cells treated with milicoquinone C and malabathone C were assessed by caspase activity assays. Since this enzyme activity is transient, analyses were performed at two time points, 6 hours and 12 hours later.

[0091] Specifically, Caspase-3 / 7 activity was analyzed by slightly modifying the manufacturer's (Promega, USA) protocol. In short, cells were charged at 5×10⁻⁶... 3 Cells / wells were seeded at a density in 96-well plates and cultured for 24 hours, followed by treatment with an appropriate concentration of the compound for 6 or 12 hours. After treatment, an equal volume of Caspase-Glo 3 / 7 reagent was added to the treated sample. The plate was then shaken and incubated at room temperature for 2 hours. Enzyme activity was determined by measuring relative light units (RLU) using a Varioskan LUX Multimode Microplate Reader (Thermo Fisher Scientific, USA).

[0092] Table 22 caspase-3 / 7 activity after treatment with milicoquinone C and malabatone C compounds like Figure 15 As shown in Table 22, for gastric cancer cells, malathione C showed significantly lower relative light units (RLU) at all concentrations compared to the DMSO control group. 5 μM milicocin C showed a peak after 12 hours, while 10 μM milicocin C showed a peak after only 6 hours. These values ​​were 1.74 times and 1.81 times higher than DMSO, respectively.

[0093] For colorectal cancer cells, low concentrations of malaboxetine C showed signals similar to DMSO at both time points, while 10 μM malaboxetine C showed a significantly low signal after 6 hours and a signal similar to the control group after 12 hours. Milicon C showed a similar trend to gastric cancer cells, with a significant increase in signal at the same concentration as time increased from 6 to 12 hours. However, low concentrations of milicon C only showed a peak signal after 12 hours, while high concentrations showed a peak signal after 6 hours. These values ​​were 1.89 times and 2.37 times higher than the DMSO control group, respectively.

[0094] Based on the above evidence and the fact that RLU values ​​are directly proportional to caspase-3 / 7 activity, it can be concluded that mirtazac C (5μM-10μM) significantly increased caspase activity in 23132 / 87 and SW48 cells within 6 to 12 hours, thereby inducing apoptosis. Malabar ketone C, however, did not affect this enzymatic reaction under the experimental conditions.

[0095] Example 6: Confirmation of the anticancer effect of mirtazacin C compound administration in xenograft mouse models Female BALB / c / nu / nu mice (5 weeks old) were used as an in vivo model.

[0096] like Figure 16a As shown in the schematic diagram, respectively using 5×10 6 Each mouse and 3×10 6 Mice were subcutaneously injected with 23132 / 87 and SW48 cells at a density of 1 mouse / mice. When the tumor reached a minimum width of 3 mm, the mice were divided into three groups and injected intraperitoneally daily on different dates with mirtazapine C (10 mg / kg), malabatone C (10 mg / kg), and a control group (DMSO in 2% saline solution). Tumor volume and body weight were measured twice a week. Mice were sacrificed on day 27, and major organs (liver, spleen, kidney) and tumor tissue were collected and preserved for subsequent analysis. Tumor volume was calculated using the following formula: Volume (mm²) 3 = (length) × (width) 2 Х1 / 2.

[0097] Tumor volume and body weight were monitored throughout the experiment. The results showed that tumor volume gradually increased in all groups. In the gastric cancer model, the smallest tumor volume was confirmed from day 13 of milicocarb C administration. In the colorectal cancer model, tumor growth was also inhibited compared to the control group. Figure 16b Furthermore, the weight of the individual, including body weight and the weight of major organs such as the liver, spleen, and kidneys, remained stable throughout the experiment. Figure 16c ).

[0098] Industrial applicability The novel compounds isolated in this invention have been confirmed to have cytotoxic effects on various cancer cell lines, and five compounds (3, 4, 6, 9, and 11) have been confirmed to have high cytotoxic effects against gastric and colorectal cancer. In particular, it has been confirmed that the compound represented by chemical formula 3 not only effectively induces apoptosis by regulating the cell cycle of cancer cells, but also effectively inhibits tumor growth in xenograft mouse models, and therefore can be effectively used as an anticancer composition.

Claims

1. A compound, characterized in that, It is a compound represented by the following chemical formula A, its pharmaceutically acceptable salt, or its optical isomer. Chemical formula A In the chemical formula A, X is or In this case, n is an integer from 1 to 20. At least one of R1 to R5 is In this case, Y is O or -(CH)m-, where m is 0 to 5, and R1' to R6' are each independently hydrogen, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C2-10 allyl, or substituted or unsubstituted C3-10 heterocyclic. The remaining portions of R1 to R5 are each independently hydrogen, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C2-10 allyl, or substituted or unsubstituted C3-10 heterocyclic. R6, R8 and R 10 Each is independently either hydrogen or hydroxyl. R7 and R9 are each independently hydrogen. R 1' To R 6' Each of them is independently hydrogen, hydroxyl, substituted or unsubstituted C1-6 alkyl, substituted or unsubstituted C1-6 alkoxy, substituted or unsubstituted C3-8 cycloalkyl, substituted or unsubstituted C2-10 allyl or substituted or unsubstituted C3-10 heterocyclic.

2. The compound according to claim 1, characterized in that, The compound represented by chemical formula A is the following compound: Compounds as shown in chemical formula 3 below; Compounds as shown in chemical formula 4 below; Compounds as shown in chemical formula 6 below; Compounds as shown in chemical formula 9 below; Compounds as shown in chemical formula 10 below; Compounds as shown in chemical formula 11 below; Compounds as shown in chemical formula 12 below; or Compounds as shown in chemical formula 13 below Chemical formula 3 Chemical Formula 4 Chemical Formula 6 Chemical formula 9 Chemical Formula 10 Chemical Formula 11 Chemical formula 12 Chemical formula 13 。 3. The compound according to claim 1, characterized in that, The compound represented by chemical formula A is the same as the compound represented by chemical formula 3 below: Chemical formula 3 。 4. A compound, characterized in that, It is: Compounds represented by the following chemical formula 14, their pharmaceutically acceptable salts, or their optical isomers; Compounds represented by the following chemical formula 15, their pharmaceutically acceptable salts or optical isomers thereof; or Compounds represented by the following chemical formula 16, their pharmaceutically acceptable salts, or their optical isomers: Chemical formula 14 Chemical Formula 15 Chemical Formula 16 。 5. A compound, characterized in that, It is: Compounds represented by the following chemical formula 17, their pharmaceutically acceptable salts, or their optical isomers; Compounds represented by the following chemical formula 18, their pharmaceutically acceptable salts, or their optical isomers; Compounds represented by the following chemical formula 19, their pharmaceutically acceptable salts, or their optical isomers; or Compounds represented by the following chemical formula 20, their pharmaceutically acceptable salts, or their optical isomers: Chemical Formula 17 Chemical Formula 18 Chemical formula 19 Chemical formula 20 。 6. A compound, characterized in that, It is: Compounds represented by the following chemical formula 21, their pharmaceutically acceptable salts or optical isomers thereof; or Compounds represented by the following chemical formula 22, their pharmaceutically acceptable salts, or their optical isomers: Chemical Formula 21 Chemical formula 22 。 7. A pharmaceutical composition for the prevention or treatment of cancer, characterized in that, It contains the compound according to any one of claims 1 to 6 as an active ingredient.

8. The pharmaceutical composition for the prevention or treatment of cancer according to claim 7, characterized in that, The cancers mentioned are pancreatic cancer, liver cancer, lung cancer, stomach cancer, colorectal cancer, prostate cancer, kidney cancer, breast cancer, or ovarian cancer.

9. A health-functional food composition for preventing or improving cancer, characterized in that, It contains the compound according to any one of claims 1 to 6 as an active ingredient.

10. The health-functional food composition for preventing or improving cancer according to claim 9, characterized in that, The cancers mentioned are pancreatic cancer, liver cancer, lung cancer, stomach cancer, colorectal cancer, prostate cancer, kidney cancer, breast cancer, or ovarian cancer.

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