Composition for preventing, alleviating or treating liver cancer, comprising extract of melosila nummuloides as active ingredient

Abstract

The present invention relates to a composition for preventing, alleviating or treating liver cancer, containing, as an active ingredient, a Melosila nummuloides extract, and, more specifically, to an excellent cell death and metastasis inhibitory effect of a Melosila nummuloides extract on liver cancer cells, the Melosila nummuloides extract being extracted using a mixed solvent of chloroform and methanol.

Description

Composition for preventing, improving or treating liver cancer containing Melosira nummuloid extract as an active ingredient

[0001] The present invention relates to a composition for preventing, improving or treating liver cancer, containing an extract of Melosila nummuloides as an active ingredient.

[0002]

[0003] This work was supported by the National Research Foundation of Korea's Local Government-University Cooperation-Based Regional Innovation Project (Project Number: 2023RIS-009).

[0004] Research and technology development of microalgae strains are shifting from fuel applications like biodiesel and bioethanol to high-value-added products like colorants, cosmetic ingredients, and health functional / medicinal substances. While research and technology development for pharmaceutical production using microalgae is still in its infancy, the aging population and growing awareness of wellness are expected to lead to an expansion of the natural product-derived pharmaceutical market and potential for growth.

[0005] Melosira nummuloides, a marine diatom, has been reported to be a resource for animal and fish nutrition as it contains polyunsaturated fatty acids (PUFA), monounsaturated fatty acids (MUFA), omega-3 (ω-3) EPA (eicosapentaenoic acid), and polyphenols. Melosira nummuloides is a diatom isolated from Jeju lava seawater, and is capable of mass production. It is a source of silicon (Si(OH)4) and magnesium (Mg) dissolved in Jeju lava seawater. 2+ ) is mainly used to form cell walls (skeletons), and the content of silicon is known to be the highest among all plants and animals at 200,000 ppm / tissue.

[0006] Although Melosira nummuloides is known to possess potential pharmacological effects, including anti-inflammatory, antioxidant, and anti-obesity activities, its anticancer effects have not yet been reported. In the present invention, the anticancer effects of Melosira nummuloides extract on liver cancer cells were evaluated for the first time.

[0007] Meanwhile, Korean Patent Publication No. 2023-0032193 discloses a 'composition having an anti-obesity effect containing a Melosila nummuloides extract as an active ingredient', and Korean Patent Publication No. 2023-0032194 discloses a 'composition having an anti-inflammatory effect containing a Melosila extract as an active ingredient', but there is no description of the 'composition for preventing, improving, or treating liver cancer containing a Melosila nummuloides extract as an active ingredient' of the present invention.

[0008] The present invention was derived from the above-mentioned needs, and the present inventors prepared hot water (HW), ethanol (EtOH), methanol (MeOH), supercritical fluid, and chloroform:methanol (2:1 v / v; CM) extracts of Melosira nummuloides, and analyzed the proliferation inhibitory activity of each extract against liver cancer cells. As a result, it was confirmed that the CM extract exhibited superior cell proliferation inhibitory activity compared to other extracts, and at low concentrations, the CM extract inhibited the migration and invasion ability of cancer cells, reduced the expression of EMT markers, and promoted the accumulation of sub-G1 phase and caspase-dependent apoptosis in liver cancer cells, thereby completing the present invention.

[0009] To solve the above problem, the present invention provides a pharmaceutical composition for preventing or treating liver cancer containing an extract of Melosila nummuloides as an active ingredient.

[0010] In addition, the present invention provides a health functional food composition for preventing or improving liver cancer containing an extract of Melosira nummuloide as an active ingredient.

[0011] The Melosira nummuloides extract of the present invention is derived from a natural product and has excellent anticancer activity against liver cancer cells, and thus can be usefully utilized in related industries such as pharmaceuticals and life sciences.

[0012] Figure 1 shows the results of confirming the proliferation inhibition ability of Melosira nummuloides extracts on liver cancer cells, a is the MTT result analyzing the cell death ability by type of extract, and at the same treatment concentration, each column represents HW, EtOH, MeOH, CM, and Supercritical from left to right, b is the result analyzing the colony formation inhibition ability of the chloroform:methanol (CM) extract, and c is the result analyzing the toxicity of the CM extract on fibroblasts (normal cells). HW: hot water extract, EtOH: ethanol extract, MeOH: methanol extract, CM: chloroform:methanol (2:1) extract, Supercritical: supercritical extract, *p < 0.05.

[0013] Figure 2 shows the results of confirming the cancer metastasis inhibition effect of CM extract in liver cancer cells. a is the cytotoxicity confirmation result according to the extract treatment concentration, b is the wound-healing assay result, and c is the transwell migration assay result. ***p < 0.001.

[0014] Figure 3 shows the results of analyzing the changes in the expression of metastasis-related proteins by CM extract in liver cancer cells. *p < 0.05, **p < 0.01.

[0015] Figure 4 shows the results confirming the apoptosis-promoting effect of CM extract on liver cancer cells. (a) is the result of Hoechst 33342 staining, (b) is the result of cell cycle analysis of liver cancer cells treated with CM extract, and (c) is the result of analyzing the activity of caspase, a protein that promotes apoptosis. C-Caspase 3 and C-PARP represent cleaved Caspase 3 and PARP, respectively. *p < 0.05, **p < 0.01.

[0016] Figure 5 shows the results of analyzing the changes in the expression of MAPK signaling pathway (a) and JAK2 / STAT3 signaling pathway (b) proteins by CM extract treatment in liver cancer cells. NS: non-significant, *p < 0.05, ***p < 0.001.

[0017] In order to achieve the purpose of the present invention, the present invention provides a pharmaceutical composition for preventing or treating liver cancer containing an extract of Melosila nummuloides as an active ingredient.

[0018] In the present invention, the above-mentioned M. nummuloides is an attached diatom collected together with Jeju lava seawater.

[0019] In the pharmaceutical composition of the present invention, the Melosira nummuloides extract may be extracted using an organic solvent, preferably, it may be extracted using a mixed solvent of chloroform and methanol, more preferably, it may be extracted using a mixed solvent of chloroform:methanol mixed in a volume ratio of 1.5 to 2.5:1, and even more preferably, it may be extracted using a mixed solvent of chloroform:methanol mixed in a volume ratio of 2:1, but is not limited thereto. The extraction method may use any conventional method known in the art, such as ultrasonic extraction, filtration, immersion extraction, and reflux cooling extraction, and in the present invention, an ultrasonic extraction method is used, but is not limited thereto.

[0020] In one embodiment of the present invention, the Melosira nummuloides extract extracted using a mixed solvent of chloroform:methanol mixed in a volume ratio of 1.5 to 2.5:1 is characterized in that it has a significantly superior proliferation inhibition ability against liver cancer cells compared to the Melosira nummuloides extract extracted using water, ethanol or methanol, and supercritical. In addition, the Melosira nummuloides extract extracted using a mixed solvent of chloroform:methanol (2:1) according to the present invention is characterized in that it effectively inhibits the metastasis of liver cancer cells by inhibiting the migration and invasion ability of liver cancer cells and reducing the expression of an epithelial-to-mesenchymal transition (EMT) marker.

[0021] In addition, the pharmaceutical composition comprising the Melosira nummuloide extract of the present invention may further comprise an appropriate carrier, excipient or diluent commonly used in the manufacture of pharmaceutical compositions.

[0022] The pharmaceutical dosage form of the pharmaceutical composition of the present invention can be used alone or in combination with other pharmaceutically active compounds as well as in appropriate combinations.

[0023] The pharmaceutical composition according to the present invention can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and injections, respectively, according to conventional methods. Carriers, excipients, and diluents that can be included in the pharmaceutical composition include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0024] When formulated, they are usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can include withepsol, macrogol, Tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0025] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and the excretion rate, the duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition 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 singly or in multiple doses. It is important to take all of the above factors into consideration and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art. In addition, the pharmaceutical composition of the present invention may be administered to mammals such as rats, mice, livestock, and humans via various routes. Any route of administration may be envisaged, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine, epidural or intracerebroventricular injection.

[0026] The present invention also provides a health functional food composition for preventing or improving liver cancer containing an extract of M. nummuloides as an active ingredient.

[0027] In the health functional food composition of the present invention, the Melosira nummuloid extract may be extracted using an organic solvent, preferably extracted using a mixed solvent of chloroform and methanol, more preferably extracted using a mixed solvent of chloroform:methanol mixed in a volume ratio of 1.5 to 2.5:1, and even more preferably extracted using a mixed solvent of chloroform:methanol mixed in a volume ratio of 2:1, but is not limited thereto.

[0028] The active ingredient of the present invention can be added directly to food or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the active ingredient mixed can be appropriately determined depending on its purpose of use (prevention or improvement). Generally, the content of the active ingredient included in a health functional food can be added in an amount of 0.1 to 90 parts by weight based on the total weight of the health functional food. However, in case of long-term intake for the purpose of health and hygiene or health control, the amount can be below the above range, and since there is no problem in terms of safety, the active ingredient can also be used in an amount exceeding the above range.

[0029] The health functional food composition of the present invention may be in one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto.

[0030] There are no specific restrictions on the types of the above health functional foods. Examples of foods to which the above health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages, and vitamin complexes, and include all health foods in the conventional sense.

[0031] In addition, the health functional food composition of the present invention can be manufactured into a food, particularly a functional food. The functional food of the present invention includes ingredients commonly added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured into a drink, a natural carbohydrate or flavoring agent may be included as an additional ingredient in addition to the active ingredient. The natural carbohydrate is preferably a monosaccharide (e.g., glucose, fructose, etc.), a disaccharide (e.g., maltose, sucrose, etc.), an oligosaccharide, a polysaccharide (e.g., dextrin, cyclodextrin, etc.), or a sugar alcohol (e.g., xylitol, sorbitol, erythritol, etc.). The flavoring agent can be a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) or a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

[0032] In addition to the above health functional food composition, various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. may be further contained. The ratio of these added components is not particularly important, but is generally selected within the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health functional food composition of the present invention.

[0033]

[0034] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0035]

[0036] Materials and Methods

[0037] 1. Microalgae

[0038] The Melosila nummuloides used in the present invention was provided by JDK Bio. Melosila was cultured using Jeju lava seawater.

[0039]

[0040] 2. Preparation of microalgae extract

[0041] 1) Hot water extract

[0042] 5 g of Melosira powder sample was mixed with 200 ml of distilled water, then heated using high-pressure steam at 125°C for 15 minutes in an autoclave, cooled at 25°C for 24 hours, and filtered through a 150 mm filter paper. The filtered extract was concentrated using a reduced pressure rotary evaporator and freeze-dried at -55°C for 72 hours to obtain Melosira hot water (HW) extract.

[0043]

[0044] 2) Ethanol, methanol extracts

[0045] A 5 g sample of Melosira powder was mixed with 400 ml of 70% ethanol or 400 ml of 80% methanol, and then sonicated (at 20°C for 45 minutes). The mixture was then left to stand at 25°C for 24 hours and filtered using a 150 mm filter paper. The filtered extract was concentrated using a reduced pressure rotary evaporator and freeze-dried at -55°C for 72 hours to obtain Melosira ethanol (EtOH) or methanol (MeOH) extracts.

[0046]

[0047] 3) Chloroform:methanol (2:1, v / v) extract

[0048] A 5 g sample of Melosira powder was mixed with 200 ml of chloroform:methanol (2:1, v / v), and then sonicated (at 20°C for 45 min). The mixture was then left to stand at 25°C for 24 h and filtered using a 150 mm filter paper. The filtered extract was transferred to a separatory funnel, 200 ml of distilled water was added, mixed, the aqueous layer was discarded, and the extract was collected and concentrated using a rotary evaporator under reduced pressure. The resulting extract was freeze-dried at -55°C for 72 h to obtain a Melosira chloroform:methanol (CM) extract.

[0049]

[0050] 4) Supercritical extract

[0051] 80 g of Melosira sample was extracted using a CO2 extraction system (cat. no. 06RA06-104010-A0031; ISA-SCFE system; Ilshin Autoclave Co., Ltd) at a pressure of 500 bar, a temperature of 50°C, and a ratio of 0.6 ml x 240 min with ethanol as an auxiliary solvent.

[0052]

[0053] Example 1. Effect of Melosira nummuloid extract on liver cancer cell viability

[0054] Cancer is characterized by uncontrolled cell proliferation, and numerous studies have demonstrated the ability of natural and synthetic compounds to inhibit the growth of cancer cells. The cell proliferation inhibitory effect of the Melocira nummuloides extract was evaluated in HepG2 cells (human liver cancer cell line) using the MTT assay. As shown in Fig. 1a and Table 1, the hot water extract (HW) showed a relatively low cell proliferation inhibitory effect in HepG2 cells compared to the other extracts, and the cell proliferation inhibitory effect was confirmed in the order of CM > EtOH > MeOH > Supercritical extract. The CM extract had an IC of 104.8±12.3 μg / mL and 68.1±16.78 μg / mL in HepG2 cells after 24 and 48 hours of treatment, respectively. 50 The CM extract exhibited the strongest antiproliferative effect, demonstrating a significant anti-proliferative effect. No cytotoxicity was observed in fibroblasts (normal cells) at the same concentration (Fig. 1c). Therefore, further experiments were conducted, as the CM extract exhibited a higher inhibition of cell proliferation in liver cancer cells than the other extracts.

[0055] Colony formation assays assess the ability of individual cells to develop into colonies, a key criterion for assessing cell death induced by extract treatment. As shown in Figure 1b, CM extract treatment decreased colony formation in a dose-dependent manner in HepG2 cells. These results support the potent anticancer efficacy of CM extract against HepG2 cells.

[0056] IC for inhibition of hepatoma cell growth by Melosira nummuloid extract 50 (㎍ / ㎖)24 hr48 hrHot water extract (HW)NDNDethanol extract (EtOH)306.3 ± 73.32 b 308.4 ± 32.78 b Methanol extract (MeOH) 395.1 ± 56.4 c 315.5 ± 16.44 bChloroform:methanol extract (CM) 104.8 ± 12.3 a 68.1 ± 16.78 a Supercritical extract 1275 ± 369.6 d 780.1 ± 157.0 c

[0057]

[0058] Example 2. Inhibition of liver cancer cell migration and invasion by CM extract

[0059] Wound-healing assay (Kim, JS, et al., 2022, Appl Biol Chem. 65, 34) is a widely used method for observing cell migration. The present inventors evaluated whether the CM extract could inhibit cell migration in hepatoma cells at a non-toxic concentration. The analysis results showed that the Melocira nummuloides CM extract decreased cell migration in HepG2 cells in a concentration- and time-dependent manner (Fig. 2b). Subsequently, the invasion ability of cancer cells was analyzed using a trans-well migration assay (Kim, JS, et al., 2022). As a result, it was observed that the invasion ability of HepG2 cells was significantly reduced in a concentration-dependent manner when treated with the CM extract (Fig. 2c).

[0060]

[0061] Example 3. Inhibition of the expression of metastasis-related proteins in liver cancer cells by CM extract.

[0062] Epithelial-mesenchymal transition (EMT) plays a crucial role in tumor invasion and metastasis. In EMT, epithelial cells lose intercellular adhesion, promoting their migration to distant sites. Slug, Snail, Vimentin, and MMP9 have been widely used as EMT markers in hepatocellular carcinoma cells. In this study, we investigated whether Melocira nummuloides CM extract affects the expression of EMT markers in HepG2 cells. The expression levels of EMT markers were confirmed by Western blotting using Vimentin (#3932), Snail (#3879S), Slug (#9585), MMP9 (#3852), and β-actin (#4967) as primary antibodies. All antibodies used in this study were from Cell Signaling Technology.

[0063] As a result, it was shown that the expression levels of Slug, Snail, Vimentin, and MMP9 proteins decreased in a concentration-dependent manner when CM extract was treated in HepG2 cells (Fig. 3), and these results demonstrated that CM extract inhibits migration and invasion by suppressing the EMT process in liver cancer cells.

[0064]

[0065] Example 4. Effect of CM extract on promoting apoptosis in liver cancer cells

[0066] Although avoiding apoptosis is a key characteristic of cancer cells, numerous natural anticancer compounds have been identified as promising agents that induce apoptosis. In this context, the present inventors investigated whether the Melocira nummuloides CM extract could induce apoptosis in hepatoma cells. First, the apoptotic effect of CM extract treatment on hepatoma cells was verified microscopically using Hoechst 33342 staining. As shown in Figure 4a, a dose-dependent increase in apoptotic bodies was observed in HepG2 cells treated with CM extract.

[0067] Since previous studies have shown that apoptosis induction is associated with the cell cycle in cancer cells, the effect of CM extract on the cell cycle in HepG2 cells was evaluated. The cell cycle was measured using a CytoFLEX flow cytometer (BECKMAN COULTER Life Sciences, USA). As shown in Figure 4b, the experimental group treated with 100 μg / mL of CM extract showed a significant increase in the sub-G1 phase, from 3.63% in the untreated control group to 15.59%.

[0068] The well-established mechanism of programmed cell death is closely linked to the sustained activation of caspases. Among caspases, the activation of Caspase-3 plays a role in cleaving other apoptosis-related substrates, such as Poly (ADP-ribose) polymerase (PARP). Western blot analysis using antibodies against Caspase-3 (#9662S), Cleaved Caspase-3 (#9664), PARP (#9542), and Cleaved PARP (#9541) (Fig. 4c) revealed that exposure to CM extract increased the expression of cleaved-caspase-3 and cleaved-PARP in HepG2 cells. The increase in the ratio of C-Caspase-3 / Caspase-3 and C-PARP / PARP with increasing CM extract concentration suggests that CM extract can induce caspase-dependent apoptosis in hepatocellular carcinoma cells.

[0069]

[0070] Example 5. Inhibition of MAPK and JAK2 / STAT3 signaling pathways in liver cancer cells by CM extract.

[0071] Previous studies have shown that MAPK signaling plays a central role in controlling cellular responses to external stimuli and affects processes such as proliferation, differentiation, survival, and apoptosis of liver cancer cells. To determine whether Melosira nummuloides CM extract can affect the MAPK signaling pathway in liver cancer cells, the inventors treated the cells with the CM extract and examined the changes in protein expression levels of each signaling pathway using Western blotting. Antibodies used were ERK (#4695), p-ERK (#9101), p38 (#9212), p-p38 (#4631), JNK (#9252), p-JNK (#9251), JAK2 (#3230), p-JAK2 (#3776), STAT3 (#30835), and p-STAT3 (#9145) from Cell Signaling Technology.

[0072] As a result of Western blot analysis, it was confirmed that the p-ERK / ERK, p-P38 / P38, and p-JNK / JNK ratio levels were significantly reduced in a concentration-dependent manner with CM extract treatment (Fig. 5a), and the p-JAK2 / JAK2 and p-STAT3 / STAT3 levels were significantly reduced (Fig. 5b).

[0073]

[0074] Example 6. Analysis of fucoxanthin content in CM extract

[0075] Melocira nummuloides has been reported to be a microalga capable of producing fucoxanthin, and fucoxanthin is known to possess anticancer activity. Accordingly, the present inventors analyzed the correlation between the cell proliferation inhibitory activity of Melocira nummuloides CM extract against liver cancer cells and fucoxanthin.

[0076] The fucoxanthin content in the Melosira nummuloides extracts was quantified using high-performance liquid chromatography (HPLC; DGU-20A 3R degassing unit, SHIMADZU Inc., Japan). Each extract (1 mg) was diluted in 1 ml of methanol, filtered through a 0.2 mm filter, and used for HPLC analysis. The mobile phase gradient was set up using 100% water (A) and 100% methanol (B) as follows: starting with 100% solvent A, maintaining it for 5 min, and gradually decreasing to 50% within 3 min, then decreasing from 50% to 25% solvent A in 4 min, and then decreasing from 25% to 10% in 6 min. Afterwards, it was further reduced from 10% to 5% in 8 minutes, and finally, solvent A was reduced to 0% and maintained for 15 minutes, then increased again to 100% and maintained for 10 minutes. The sample injection volume was set to 20 μl, the flow rate was 1 ml / min, and the absorbance was measured at 445 nm. The fucoxanthin content was determined using a standard curve with five different concentrations using a standard (CAS no. 3351-86-8 with purity ≥ 95%, Sigma-Aldrich) and expressed as milligrams per gram of dry weight powder extract. All experiments were performed in triplicate.

[0077] The analysis results showed that the CM extract contained 3.22 μg / mg of fucoxanthin, and based on this, the calculation results showed that 100 μg / ml of CM extract contained 0.322 μg / mg of fucoxanthin. The inventors treated HepG2 cells with up to 10 μM of fucoxanthin (6.7 μg / ml) and analyzed cell viability using the MTT assay, but the inhibitory effect of fucoxanthin on liver cancer cell proliferation was not confirmed up to the corresponding concentration range. In addition, as a result of performing a Pearson correlation analysis on the correlation between the content of fucoxanthin in the Melocira nummuloides CM extract and anticancer activity, no significant correlation was found between the content of fucoxanthin and the anticancer activity.

Claims

1. A pharmaceutical composition for the prevention or treatment of liver cancer containing an extract of Melosila nummuloides as an active ingredient.

2. A pharmaceutical composition for preventing or treating liver cancer, characterized in that the extract of Melosira nummuloides in the first paragraph is extracted using a mixed solvent of chloroform and methanol.

3. A pharmaceutical composition for preventing or treating liver cancer, characterized in that in the second paragraph, the Melosira nummuloid extract is extracted by adding a mixed solvent of chloroform:methanol mixed in a volume ratio of 1.5 to 2.5:1 to a Melosira nummuloid sample.

4. A pharmaceutical composition for preventing or treating liver cancer, characterized in that the extract of Melosira nummuloides induces death of liver cancer cells and inhibits metastasis of liver cancer cells in the first paragraph.

5. A pharmaceutical composition for preventing or treating liver cancer, characterized in that in paragraph 1, the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient.

6. A health functional food composition for preventing or improving liver cancer containing Melosila nummuloides extract as an active ingredient.

7. A health functional food composition for preventing or improving liver cancer, characterized in that the Melosira nummuloide extract in paragraph 6 is extracted using a mixed solvent of chloroform and methanol.

8. A health functional food composition for preventing or improving liver cancer, characterized in that the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candies, syrups, and beverages in the 6th paragraph.

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