TWI458486B - Cancer cell growth inhibiting, cancer cell apoptosis inducing and cancer cell dna damage causing pharmaceutical composition, and uses of gracilaria spp. alcohol extract - Google Patents

Description

Pharmaceutical composition for inhibiting cancer cell growth, inducing apoptosis of cancer cell cells and/or causing DNA damage, and use of red algae asparagus alcohol extract

The present invention relates to an algae extract, and in particular to a red algae Gracilaria spp . alcohol extract having the effects of inhibiting cancer cell growth, inducing apoptosis of cancer cells, and causing DNA damage to cancer cells. .

Natural products (BMC Cancer 2006, 6: 119; BMC Cancer 2010, 10: 210; BMC Cancer 2008, 8: 310; BMC Cancer 2011, 11: 360; BMC Cancer 2010, 10: 46) offer many treatments that can be developed Pathways for cancer drugs, especially marine sources (Clinical advances in hematology & oncology: H&O 2009, 7(6): 383-385; Nat Rev Drug Discov 2009, 8(1): 69-85; Biochem Pharmacol 2009, 78 ( 5): 440-448; Trends Pharmacol Sci 2010, 31(5): 225-265) is even more so. Algae contain many biologically active primary or secondary metabolites (Nat Prod Rep 2002, 19(1): 1-48; Mar Drugs 2011, 9(7): 1273-1292) and accounts for about 9% in marine biopharmaceutical compositions (Mar Drugs 2004, 2: 123-146). Among them, the crude drug composition of the red genus Asparagus has been integrated (J Appl Phycol 1995, 7: 231-243; Int J Mol Sci 2011, 12 (7): 4550-4573) and is divided into: water extraction by extraction method ( Food Chem 2010, 123: 395-399; Plant Foods Hum Nutr 2009, 64(3): 218-223; Fish Shellfish Immunol 2010, 28(5-6): 887-894) and A/Ethanol Extract (Bioresour Technol 2008) , 99(8): 2717-2723; J Med Food 2010, 13(6): 1494-1499; J Agric Food Chem 2011, 59(10): 5589-5594). Most of these studies focus on their health-promoting effects, such as anti-inflammatory, anti-hypercholesterolemic, antioxidant and antibacterial properties.

Previous studies have also shown that seaweed derivatives have anti-cancer potential that inhibits the growth of some human cancer cell lines, including the inhibition of promyelocytic leukemia cell line HL-60 (Queiroz, KC; Assis, CF; Medeiros, VP; Rocha, HA; Aoyama, H.; Ferreira, CV; Leite, ELCytotoxicity effect of algal polysaccharides on HL60 cells), erythromyeloblastoid leukemia cell line K562 (Int J Mol Med, 2004, 14, 925-929), colon adenocarcinoma cell line HT-29 (J Med Food, 2007, 10, 587-593) and breast adenocarcinoma cell line MCF7 (J Agric Food Chem, 2009, 57, 8677-8682). Seaweed extract has also been shown to protect against hypercholesterolemia and free radical stress (Phytother Res, 2005, 19, 506-510) and can promote the growth of probiotics (Pak J Pharm Sci, 1991, 4, 49-54).

The main seaweed species found in the surrounding areas of Taiwan include Gracilaria tenuistipitata, Gracilaria coforvoides, Gracilaria gigas, and Gracilaria chorda. ) and Gracilaria compressa. Algae extracts from some of these species have been reported to have anti-aging activity (Journal of Plant Diseases and Protection, 2009, 116, 263-270) and contain amino acids, fatty acids, vitamins, minerals, polyphenolic compounds (poly phenolic) Compound) and a rich source of carbohydrates (Food Control, 2007, 18, 639-645). It is edible and can be used as a component of food and cosmetics. It is the main type of asparagus cultivated in Taiwan (Lin Zhiyuan, Master's thesis of the Institute of Aquatic Food Science, National Kaohsiung University of Marine Science and Technology, 2009). Previous reports have shown that Asparagus has a free radical scavenging activity sensitive to autoclaving (Plant Foods Hum Nutr, 2009, 64, 218-223).

The present invention provides a pharmaceutical composition for inhibiting the growth of cancer cells, which has an ability to inhibit the growth of cancer cells and which comprises an effective amount of an alcohol extract of Gracilaria spp . as an active ingredient.

The present invention also provides a use of a red algae asparagus extract for the preparation of a pharmaceutical composition for inhibiting the growth of cancer cells.

The present invention further provides a pharmaceutical composition for inducing apoptosis of cancer cell cells and/or causing DNA damage to cancer cells, which has the ability to induce apoptosis of cancer cells and cause DNA damage to cancer cells and includes an effective amount. The alcohol extract of the red genus Gracilaria spp . is the active ingredient.

The present invention further provides a use of a red algae asparagus extract for the preparation of a pharmaceutical composition for inducing apoptosis of cancer cell cells and/or causing DNA damage to cancer cells.

Figure 1A shows that different concentrations (0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) of red algae asparagus methanol extract inhibits the growth of Ca9-22 cells and cultured 24 After the hour, the cell viability data measured by the WST-1 reagent group were presented as mean ± standard deviation (n = 3), and the difference was not significant between the groups containing the same letter.

Figure 1B shows that different concentrations (0mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5mg/ml) of red algae asparagus ethanol extract inhibits the growth of Ca9-22 cells. In the case, the cell viability data measured by the MTS reagent group were presented as mean ± standard deviation (n=6), and the strips containing the same letter between the groups were not significantly different (p<0.05 to 0.0001). ).

Figure 1C shows the 24 hours of H2299 cell growth in different concentrations (0.5 mg/ml, 1 mg/ml, 2 mg/ml, 3.5 mg/ml) of red algae asparagus methanol extract, which is based on WST- 1 reagent group measured cell storage The survival rate data is presented as mean ± standard deviation (n = 6).

Figure 2 shows the changes in cell cycle distribution of Ca9-22 cells in different concentrations (0mg/ml, 0.1mg/ml, 0.25mg/ml, 0.5mg/ml, 1 mg/ml) of red algae asparagus methanol extract. The effect (at the 24th hour), the flow cytometry data was presented as mean ± standard deviation (n = 3), and the strips containing the same letter between the groups, the difference was not significant.

Figure 3A shows annexin-V-FITC at 24 hours after Ca9-22 cells were treated with different concentrations (0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) of methanol extract. A flow cytometric analysis of the fluorescent marker content, "% positive" is included in all graphs.

Figure 3B shows a quantitative analysis of the intensity of annexin-V fluorescence. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant.

Figure 4A shows the 24th hour of annexin- treatment of Ca9-22 cells treated with different concentrations (0 mg/ml, 0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml) of ethanol extract. A flow cytometric analysis of the V-FITC fluorescent label content, "% positive" is included in all graphs.

Figure 4B shows a quantitative analysis of the intensity of annexin-V fluorescence. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant.

Figure 5A shows the gamma-H2AX fluorescence of Ca9-22 cells treated with different concentrations (0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) of methanol extract at 24 hours. A flow cytometric analysis of the labeled content, "% positive" is included in all charts. Figure 5B shows a quantitative analysis of the fluorescence intensity of γ-H2AX. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant.

Figure 6A shows that Ca9-22 cells were treated with different concentrations (0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) of methanol extract and H ₂ O ₂ (100 μM as positive). Control group) CMF-DA fluorescent marker content map at 24 hours after treatment, "% positive" is indicated in all charts.

Figure 6B shows a quantitative analysis of the fluorescence intensity of CMF-DA. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant.

Figure 6C shows Ca9-22 cells with different concentrations (0mg/ml, 0.1mg/ml, 0.25mg/ml, 0.5mg/ml, 1mg/ml) methanol extract and H ₂ O ₂ (100 μ M as positive control) Group) The H2DCF-DA fluorescent marker content map at the 24th hour after treatment, "% positive" is indicated in all the charts.

Figure 6D shows a quantitative analysis of the H2DCF-DA fluorescence intensity. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant.

Figure 7A shows Ca9-22 cells with different concentrations (0mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5mg/ml) ethanol extract and H ₂ O ₂ (100 μ The CMF-DA fluorescent marker content map at the 24th hour after the treatment of M as the positive control group, "positive %" is indicated in all the charts.

Figure 7B shows the quantitative analysis of the fluorescence intensity of CMF-DA. Mean ± standard deviation (n = 3) to present, the strips between the groups contain the same letter, the difference is not significant.

Figure 7C shows Ca9-22 cells with different concentrations (0mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5mg/ml) ethanol extract and H ₂ O ₂ (100 μ The H2DCF-DA fluorescent marker content map at the 24th hour after the treatment of M as the positive control group, "positive %" is indicated in all the charts.

Figure 7D shows a quantitative analysis of the H2DCF-DA fluorescence intensity. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant.

Figure 8A shows ethanol extract of red algae asparagus with different concentrations (0mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5mg/ml) and 500X of 1 μl The effect of Pan-Caspase activation on Ca9-22 cells was observed by TF2-VAD-FMK treatment (24th hour), and "% positive" was included in all the graphs.

Figure 8B shows a quantitative analysis of the fluorescence intensity of TF2-VAD-FMK. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant.

Figure 9A shows DiOC ₂ (3) at 24 hours after treatment with different concentrations (0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) of methanol extracts of Ca9-22 cells. The fluorescent marker content map, "% positive" is marked in all charts.

Figure 9B shows a quantitative analysis of DiOC ₂ (3) fluorescence intensity. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant.

FIG. 10A cells display Ca9-22 of different concentrations (0mg / ml, 0.5mg / ml , 1mg / ml, 1.5mg / ml, 2mg / ml, 2.5mg / ml) 24 h after ethanol extraction was treated DiOC ₂ (3) Fluorescent mark content map, "% positive" is marked in all charts with horizontal lines.

Figure 10B shows a quantitative analysis of DiOC ₂ (3) fluorescence intensity. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant.

In one aspect of the invention, the invention provides a red genus Gracilaria spp . alcohol extract having the ability to inhibit the growth of cancer cells.

The above red algae asparagus may include Gracilaria tenuistipitata , Gracilaria coforvoides , Gracilaria gigas , Gracilaria chorda, Gracilaria lichenoides or Gracilaria compressa . The cancer cells may include oral cancer cells, lung cancer cells, breast cancer cells, liver cancer cells, colorectal cancer cells, renal cancer cells, or gastric cancer cells, but are not limited thereto.

The step of obtaining the red algae asparagus alcohol extract of the present invention may comprise subjecting the red algae asparagus to a first extraction step to obtain a first extract of red algae asparagus extract. The first extraction step can include extracting red algae asparagus with an alcohol.

The alcohol used to extract the red algae asparagus may include, but is not limited to, methanol, ethanol, propanol or a combination thereof. In one embodiment, the red algae asparagus used in the first extraction step is a fine base asparagus, and the alcohol used is methanol. In another embodiment, the red algae asparagus used in the first extraction step is a fine base of asparagus, and the alcohol used is ethanol.

In one embodiment, in the first extraction step, the ratio of red algae to asparagus to alcohol is about 10:1000 (w (g) / v (ml)) to 50: 1000 (w (g) / v (ml)), preferably about 50:1000 (w (g) / v (ml)).

In one embodiment, the temperature of the first extraction step is from about 15 to 35 ° C, preferably from about room temperature or from about 24 to 26 ° C. In still another embodiment, the first extraction step is for a period of from about 15 to about 30 hours, preferably about 24 hours.

In another embodiment, the step of obtaining the red algae asparagus extract may further comprise: after obtaining the first extract, filtering the first extract to obtain a filtrate and a filter residue, wherein the filtrate is a first The second extract is used as the red algae asparagus extract of the present invention.

Further, in another embodiment, the step of obtaining the red algae asparagus extract may further comprise the following steps. First, after filtering the first extract to obtain the filtrate and the residue, a second extraction step is performed on the residue to obtain an extract of the residue, wherein the second extraction step comprises extracting the residue with an alcohol. Next, the extract of the residue is filtered to obtain a second filtrate. Finally, the second filtrate is mixed with the above second extract to obtain a third extract, and the third extract is used as the red algae asparagus alcohol extract of the present invention.

The alcohol used in the second extraction step may include, but not Limited to methanol, ethanol, propanol or a combination thereof. The alcohol used in the first extraction step and the second extraction step may be the same or different. In one embodiment, the alcohol used in the second extraction step is methanol. In another embodiment, the alcohol used in the second extraction step is ethanol. In still another embodiment, the temperature of the second extraction step is from about 15 ° C to 35 ° C, preferably from about room temperature or from about 24 ° C to 26 ° C. Additionally, in one embodiment, the second extraction step is for a period of from about 15 to about 30 hours, preferably about 24 hours.

In still another embodiment, the red algae asparagus alcohol extract obtained above may be further subjected to evaporation drying, concentration under reduced pressure, and/or freeze drying. In one embodiment, the red algae asparagus alcohol extract obtained above is subjected to a vaporization treatment at 35 ° C to 45 ° C. In another embodiment, the aforementioned temperature is preferably about 40 °C.

In one aspect of the invention, the invention provides a pharmaceutical composition for inhibiting the growth of cancer cells, which has the ability to inhibit the growth of cancer cells, and the pharmaceutical composition may comprise an effective amount of the above-mentioned red algae dragon The vegetable alcohol extract is the active ingredient. Further, the present invention can also provide a use of the above-described red algae asparagus alcohol extract for the preparation of a pharmaceutical composition for inhibiting the growth of cancer cells.

In one embodiment, the above pharmaceutical composition for inhibiting the growth of cancer cells may further comprise a pharmaceutically acceptable carrier or salt. The pharmaceutically acceptable carrier or salt may comprise from 0.01 wt% to 99.99 wt%, preferably from 0.1 wt% to 99.9 wt%, of the above pharmaceutical composition.

In one embodiment, the red algae asparagus alcohol extract also has the effect of inducing apoptosis of cancer cell cells and/or causing DNA damage to cancer cells. The effect. DNA damage can include DNA damage within the cell. In one embodiment, intracellular DNA damage can include damage to DNA in the nucleus, such as damage to the genomic DNA, or damage to other DNA within the cell, but is not limited thereto.

Further, in another aspect of the present invention, the present invention provides a pharmaceutical composition for inducing apoptosis of cancer cell cells and/or DNA damage to cancer cells, which has an effect of inducing apoptosis of cancer cell cells And/or the ability to cause DNA damage to cancer cells, and the medicament may comprise an effective amount of the above red algae asparagus alcohol extract as an active ingredient. Further, the present invention can also provide a use of the above-described red algae asparagus alcohol extract for the preparation of a pharmaceutical composition for inducing apoptosis of cancer cell cells and/or for causing DNA damage to cancer cells.

In one embodiment, the above-described pharmaceutically acceptable composition for inducing apoptosis of cancer cell cells and/or causing DNA damage to cancer cells may further comprise a pharmaceutically acceptable carrier or salt. The pharmaceutically acceptable carrier or salt may comprise from 0.01 wt% to 99.99 wt%, preferably from 0.1 wt% to 99.9 wt%, of the above pharmaceutical or health food.

The above pharmaceutically acceptable carrier may include, but is not limited to, a solvent, a dispersion medium, a coating, an antibacterial and antifungal agent, an osmotic pressure and an absorption delaying agent, and the like. Vote for the compatible person. For different modes of administration, the pharmaceutical compositions can be formulated into a dosage form using conventional methods.

The aforementioned pharmaceutically acceptable salts may include, but are not limited to, salts including inorganic cations, for example, alkali metal salts such as sodium, potassium or amine salts, Alkaline earth metal salts, such as magnesium, calcium salts, salts containing divalent or tetravalent cations, such as zinc, aluminum or zirconium salts. In addition, it may also be an organic salt such as dicyclohexylamine salt, methyl-D-glucosamine, an amine acid salt such as arginine, lysine, histidine, glutamine. .

Administration of the aforementioned agents or drugs can be by oral, parenteral, by inhalation spray or by implantation of an implanted reservoir. Non-oral may include subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal. Intrathecal, intraleaional injection, and perfusion techniques.

The form of the pharmaceutical oral ingredient or health food may include, but is not limited to, tablets, capsules, emulsions, aqueous suspensions, dispersions, and solutions.

[Examples] Materials and Methods 1. Untreated material

Gracilaria tenuistipitata was collected in the spring of 2009 from a farm located in the seaside of Kouhu Township, Yunlin County, Taiwan, and shipped to the laboratory at 0 °C. In the laboratory, seaweed is washed with tap water to remove epiphytes and salts, impurities and sand. The seaweed is then immersed in distilled water twice and then lyophilized. Dry the dried sample And through a 60 mesh screen. The lyophilized sample was ground to a fine powder and stored at -40 °C.

2. Extraction and separation

Methanol extract: The freeze-dried powder (50 g) obtained in the previous step was wetted three times with 250 ml of methanol at room temperature, and extracted with 1000 ml of 99.9% methanol in a mechanical shaker for 24 hours. . The extract was filtered on Whatman No. 1 filter paper. The filtrate was placed in a rotary evaporator (Buchi Laboratoriums-Technik, Switzerland) and evaporated to dryness at 40 ± 2 ° C, and then lyophilized. The freeze-dried red algae asparagus extract is stored in a sealed container at -20 ° C until use.

Ethanol extract: The freeze-dried powder (50 g) obtained in the previous step was wetted three times with 250 ml of ethanol at room temperature, and extracted with 1000 ml of 99.9% ethanol in a mechanical shaker for 24 hours. . The extract was filtered on Whatman No. 1 filter paper. The filtrate was placed in a rotary evaporator (Buchi Laboratoriums-Technik, Switzerland) and evaporated to dryness at 40 ± 2 ° C, and then lyophilized. The freeze-dried red algae asparagus extract is stored in a sealed container at -20 ° C until use.

3. Cell culture

Human oral cancer cell lines (oral squamous cell carcinoma, OSCC) , Ca9-22 cells were cultured in 10% fetal calf serum, 100 U / ml penicillin, 100 μ g / ml streptomycin, and 1mM glutamic acid 0.03% Sodium pyruvate in complete DMEM medium (Gibco, Greenland, NY, USA). The cells were maintained in a humidified atmosphere containing 5% CO ₂ at 37 °C.

The human lung adenocarcinoma cell line, H1299 cells were cultured in 10% fetal calf serum, 100 U / ml penicillin, 100 μ g / ml streptomycin complete RPMI-1640 medium and 0.03% of glutamic acid. The cells were maintained in a humidified atmosphere containing 5% CO ₂ at 37 °C.

4. Cell viability analysis

Treatment of Ca9-22 cells with methanol extract: The red algae asparagus methanol extract was dissolved in dimethyl hydrazine (DMSO) and added to the medium with a final concentration of DMSO of less than 1%. The cell proliferation was examined by the WST-1 reagent group (Roche). Ca9-22 cells were plated onto 96-well plates at a density of approximately 1 x 10 ⁵ cells/disk, and were dosed at 0 mg/ml, 0.01 mg/ml, 0.25 mg/ml, 0.5 mg/ml, respectively. The 1 mg/ml red algae asparagus methanol extract was treated for 24 hours. After treatment and culture for 24 hours, WST-1 proliferation reagent (Roche) was added in an amount of 10 μl per well and cultured at 37 ° C for 1-2 hours. The 96-well plate will observe the color difference with the naked eye to distinguish whether the cells have been treated with the red algae asparagus methanol extract.

Treatment of H1299 cells with methanol extract: The red algae asparagus methanol extract was dissolved in dimethyl hydrazine (DMSO) and added to the medium with a final concentration of DMSO of less than 1%. 3-MTS analysis (3-(4,5-dimethylthiazol-2-yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) (CellTiter 96 Aqueous One Solution, Promega, Madison, WI, USA). Briefly, H1299 cells were plated onto 96-well plates at a density of approximately 1 × 10 ⁵ cells/dish, and were dosed at 0 mg/ml, 0.5 mg/ml, 1 mg/ml, 2, respectively. The methanol extract of red algae asparagus, mg/ml, 3.5 mg/ml, was treated for 24 hours. After 24 hours of treatment and culture, the MTS solution was added in a manner of 10 μl per well and cultured at 37 ° C for 1-2 hours. The absorbance was finally measured at a wavelength of 595 nm using a 96-well disc reader (Dynex MRX Model 96, MTX Lab Systems, Inc.).

Treatment of Ca9-22 cells with ethanol extract: The red seaweed asparagus ethanol extract was dissolved in dimethyl hydrazine (DMSO) and added to the medium with a final concentration of DMSO of less than 1%. 3-MTS analysis (3-(4,5-dimethylthiazol-2-yl)-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium) (CellTiter 96 Aqueous One Solution, Promega, Madison, WI, USA). Briefly, Ca9-22 cells were plated onto 96-well plates at a density of approximately 1 x 10 ⁵ cells/dish and were dosed at 0 mg/ml, 0.5 mg/ml, 1 mg/ml, respectively. The 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml red algae asparagus methanol extract was treated for 24 hours. After 24 hours of treatment and culture, the MTS solution was added in a manner of 10 μl per well and cultured at 37 ° C for 1-2 hours. The absorbance was finally measured at a wavelength of 595 nm using a 96-well disc reader (Dynex MRX Model 96, MTX Lab Systems, Inc.).

5. Apoptosis analysis

Apoptosis was tested in the annexin-V reagent group (Pharmingen, San Diego, Ca, USA) as described in Free Radic . Biol . Med . 1999, 27, 612-616. Briefly, red earth algae alcohol extracts in vehicle or increasing concentrations (methanol extracts: 0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) Ethanol extracts: 0 mg/ml, 0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml) (1×10 ⁶ cells/petri-dish), and Cultivate for 24 hours. Then, 10 μg/ml of annexin V-fluorescein isothiocyante (FITC) was added and analyzed by FACSCalibur flow cytometry. The fluorescent labeling content of annexin-V represents the degree of cell membrane eversion, that is, apoptosis.

6. Cell cycle distribution analysis (cell cycle distribution)

The cell culture medium treated with the above-mentioned red algae methanol extract of 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml was cultured for 24 hours. After trypsin treatment, cells were harvested and washed twice with PBS buffer solution and immersed in 70% ethanol overnight to fix them. The cells were then centrifuged with 70% ethanol to obtain a cell pellet. The cells were resuspended in a solution containing propidium iodide (10 μ g / ml) ( Sigma, St Louis, MO) with RNase A (10 μ g / ml ) of PBS buffer for staining. After incubation for 15 minutes at room temperature in the dark, analysis was performed with a FACSCalibur flow cytometer (Becton-Dickinson, Mansfield, MA) and Win-MDI software version 2.9 (http://facs.scripps.edu/wm29w98.exe). cell.

7. Flow cytometric analysis with γ-H2AX antibody labeling

The cell culture medium treated with 0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml red algae asparagus alcohol extract was fixed in 70% ethanol and BSA-PBST solution (1% bovine serum albumin in PBS of polyethylene glycol octylphenyl acid 0.2%; Sigma) were washed twice, and containing 2 μ g of p-Histone H2A.X monoclonal antibody (Santa Cruz Biotechnology, CA, USA) BSA-PBST solution was incubated overnight at 4 ° C, washed twice with BSA-PBST solution, and suspended in a 100-fold diluted Alexa Fluor 488-tagged secondary antibody (Jackson Laboratory, Bar) After 1 hour in Harbor, ME, USA), it was washed twice with BSA-PBST solution, resuspended in PBS, and analyzed using a FACSCalibur flow cytometer. The γ-H2AX marker can be regarded as an indicator of DNA double strand breakage.

8. Detection of intracellular reactive oxygen species (ROS) by flow cytometry

Flow cytometry was performed after calibrating reactive oxygen species using a specific marker of 2,7'-dihydrodichlorofluorescein diacetate (H2DCF-DA). Briefly, cells treated with 0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml red algae asparagus methanol extract, and 0 mg/ml, 0.5, respectively mg / ml, 1 mg / ml , 1.5 mg / ml, 2 mg / ml, 2.5 mg / ml of kainic cells treated Gracilaria ethanol extracts were washed twice with PBS and the solution containing 10 μ M of H2DCF-DA The PBS was incubated in a 37 ° C environment of CO ₂ for 30 minutes, and the cells were collected, washed twice with PBS, centrifuged, resuspended in PBS, and immediately analyzed by FACSCalibur, and the wavelength of the excitation light was set to 480 nm, and the wavelength of the divergent light was set. It is 525 nm.

9. Detection of intracellular glutathione (GSH) by flow cytometry

Flow cytometry was performed after measuring intracellular glutathione using a specific marker of 5-chloromethylfluorescein diacetate (CMF-DA). In short, methanol extracts of 0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml red algae, and 0 mg/ml, 0.5 mg/ml, respectively. , 1 mg / ml, 1.5 mg / ml, 2 mg / ml, 2.5mg / ml of kainic Gracilaria ethanol extract of treated cells 5 μ M of CMF-DA cultured at 37 [deg.] C CO ₂ environment of 20 minutes The cells were collected, washed with PBS, and the cells were collected by centrifugation and analyzed by FACSCalibur.

10. Pan-Caspase activity assay

The activation of caspases (Caspase 1, 3, 4, 5, 6, 7, 8, 9) and red were measured by the Pan-Caspase active reagent group (Abeam, Cambridge, UK). The association of the ethanol extract of the genus Algae. Most of the caspase has a substrate selectivity for the Val-Ala-Asp (VAD) peptide sequence. Thus, TF2-VAD-FMK, which is capable of passing through cells and irreversibly binding to activated caspase, can be used as a reporter sequence which is non-toxic and fluorescent. Briefly, cells treated with 0 mg/ml, 0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml red algae asparagus ethanol extract were suspended at 37 °C. In a medium of 0.5 ml, the cell density was about 1×10 ⁶ cells/ml, and another 1 μl of 500×TF2-VAD-FMK was added, and the cells were cultured in a CO ₂ atmosphere at 37 ° C for 1 hour, and washed twice with PBS. Immediately thereafter, it was suspended in a buffer solution of the reagent group to perform flow cytometry analysis of an excitation light wavelength of 480 nm and a divergent light wavelength of 525 nm.

11. Granulocyte membrane potential analysis

Using MitoProbe ^TM DiOC ₂ (3) group reagent (Invitrogen, San Diego, CA, USA) analyzing mitochondrial membrane potential. In short, methanol extracts of 0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml red algae, and 0 mg/ml, 0.5 mg/ml, respectively. The cells treated with 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml red algae ethanol extract were suspended in 37 ml of 1 ml medium to make the cell density about 1×10 ⁶ cells. /ml and then add 10 μl of MDiOC _{2 for} 5 μl . After incubation for 20-30 minutes at 37 °C in CO ₂ , collect the cells and wash them with PBS, then resuspend in PBS for excitation wavelength 480 nm, divergent wavelength. Flow cytometric analysis at 525 nm.

12. Statistical analysis

All data are presented as mean ± standard deviation. The mean difference between the groups was tested using one-way ANOVA and Tukey's HSD Post Hoc Test (software JMP ^® 9). Different letters between the groups represent significant differences (p < 0.05), and if there is at least one letter identical, there is no significant difference.

result 1. The increase in the concentration of alcohol extracts of sea bream reduces the viability of cancer cells

As shown in Figures 1A, 1B and 1C, the test results show that the alcohol extract is effective in reducing the survival rate of cancer cells and has a concentration-dependent effect. Figure 1A shows Ca9-22 cells treated with methanol extracts at concentrations of 0 mg/ml, 0.01 mg/ml, 0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml. Cell viability after 24 hours. 100±2.1%, 106.1±4.3%, 55.5±4.1%, 37.4±2.5%, 14.2±1.1% (n=5; mean±SD), which decreased significantly with increasing methanol extract concentration, which has a concentration effect (p < 0.05), the IC ₅₀ value at 24 hours was 0.326 mg/ml. Figure 1B shows Ca9-22 cells treated with ethanol extracts at concentrations of 0 mg/ml, 0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, and cell viability after 24 hours, respectively. 100.0±2.8%, 106.7±2.2%, 85.5±1.2%, 57.5±0.4%, 16.8±1.1% (n=6), which decreased significantly with increasing methanol extract concentration, which had a concentration effect (p<0.0001). ), the IC ₅₀ value at 24 hours is 1.632 mg/ml. Figure 1C shows H1299 cells treated with methanol extracts at concentrations of 0.5 mg/ml, 1 mg/ml, 2 mg/ml, and 3.5 mg/ml. After 24 hours, the cell viability was 80.5 ± 4.1%, 66.8 ± 2.2%, 53.6 ± 4.9%, 37.1 ± 5.2% (n = 6), and the IC ₅₀ value at the 24th hour was 2.346 mg/ml. The results showed that the cancer cell survival rate decreased as the concentration of methanol extract of A. sinensis increased.

2. The increase of the concentration of Alcohol extracts increases the content of Sub-G1 cells.

In the cell cycle, cells grow mainly during the G1 period. When the S phase, DNA begins to be synthesized, and G2 is ready to divide. In the G1 period, there is another period called Sub-G1. If the cell cycle stays in the Sub-G1 period, It is in a state of being apoptotic. Therefore, when Sub-G1 is increased in flow cytometry, it means that part of the cell population of the assay has tended to apoptosis. Flow cytometry analysis of 0-1 mg/ml red algae asparagus methanol extract (0 mg/ml, 0.01 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) Cell cycle distribution of Ca9-22 cells at 24 hours. As shown in Figure 2, cells treated with 0-0.25 mg/ml had a significant increase in the G1 ratio and resulted in G1 arrest, whereas at concentrations greater than 0.5 mg/ml, cells of G1 Significantly reduced (p < 0.0005). In addition, Sub-G1 was slightly reduced at 0.25 mg/ml treatment, Sub-G1 was moderately increased at 0.5 mg/ml treatment, and Sub-G1 was significantly increased at 1 mg/ml treatment. With the increase of the concentration of methanol extract of A. sinensis, the content of Sub-G1 in the cell cycle also increased, and the content of other cell stages decreased at the same time, which had a concentration-dependent effect (p<0.0005). This result indicates that the red algae dragon must The alcoholic alcohol extract has the ability to inhibit the progression of cancer cells.

3. Hailongchool alcohol extract induces apoptosis

Annexin-V is a phospholipid-binding protein, so if it is used as a marker, the degree of cell membrane eversion of the cell population can be determined, and cell membrane eversion is an indicator of apoptosis. Figure 3A shows annexin-V-FITC at 24 hours after Ca9-22 cells were treated with different concentrations (0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) of methanol extract. Flow cytometric analysis of fluorescently labeled content, positive % is included in all charts. Figure 3B shows a quantitative analysis of the intensity of annexin-V fluorescence. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant. Figure 4A shows the 24th hour of annexin- treatment of Ca9-22 cells treated with different concentrations (0 mg/ml, 0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml) of ethanol extract. Flow cytometric analysis of V-FITC fluorescent labeling content, positive % is included in all charts. Figure 4B shows a quantitative analysis of the intensity of annexin-V fluorescence. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant. Further, after treatment with methanol and ethanol extracts at various concentrations for the above 24 hours, it was found that the fluorescence content of annexin-V had a significant concentration effect (methanol: p < 0.001; ethanol: p < 0.05 to 0.0001). It is shown that the sea cockroach alcohol extract has the ability to induce apoptosis.

4. The DNA double strand break of cancer cells induced by sea dragon alcohol extract

The double-stranded helix break was determined using the γ-H2AX antibody as a label, and the higher the fluorescence content, the more the double-stranded helix breaks. Figure 5A Fluorescent labeling of γ-H2AX antibody at 24 hours after treatment with different concentrations (0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) of methanol extract Flow cytometric analysis, positive % is included in all charts. Figure 5B shows the quantitative analysis of γ-H2AX fluorescence intensity. The data are presented as mean ± standard deviation (n = 3). The bars with the same letters between the groups have no significant difference (p < 0.05). , shows the statistical results of Ca24-22 cells at 24 hours after treatment with vehicle control group and different concentrations of methanol extract. The content of γ-H2AX fluorescent label increased with the concentration of extract (0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml, n=3), and also had a concentration-dependent effect (p<0.05). This result shows that the sea dragon alcohol extract has the ability to cause DNA damage to cancer cells.

5. The extract of sea wormwood alcohol affects the growth and decline of ROS and GSH in cancer cells

Reactive oxygen species (ROS) are caused by cellular metabolism or environmental free radical stress and are considered to be contributing factors to cell aging; glutathione (GSH) is the most important antioxidant in human body. Therefore, when ROS increases and GSH decreases, it will promote cell aging and apoptosis; when ROS decreases and GSH increases, it means that the aging rate of cells slows down and survives. Using specific fluorescent markers ROS, GSH and flow cytometry, the results shown in Figure 6A show that Ca9-22 cells are treated with different concentrations (0mg/ml, 0.1mg/ml, 0.25mg/ml, 0.5mg/ml, 1). The MMF-DA fluorescent label content map at the 24th hour after treatment with methanol/ml and H2O2 (100 μM as positive control group), positive % is included in all the graphs. Figure 6B shows a quantitative analysis of the fluorescence intensity of CMF-DA. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant. Figure 6C shows Ca9-22 cells with different concentrations (0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) of methanol extract and H ₂ O ₂ (100 μM as positive Control group) H2DCF-DA fluorescent marker content map at 24 hours after treatment, positive % is included in all graphs. Figure 6D shows a quantitative analysis of the H2DCF-DA fluorescence intensity. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant. Figure 7A shows Ca9-22 cells with different concentrations (0mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5mg/ml) ethanol extract and H ₂ O ₂ (100 μ The CMF-DA fluorescent marker content map at the 24th hour after treatment with M as the positive control group, the positive % is included in all the charts. Figure 7B shows a quantitative analysis of the fluorescence intensity of CMF-DA. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant. Figure 7C shows Ca9-22 cells with different concentrations (0mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5mg/ml) ethanol extract and H2O2 (100 μM as positive Control group) H2DCF-DA fluorescent marker content map at 24 hours after treatment, positive % is included in all graphs. Figure 7D shows a quantitative analysis of the H2DCF-DA fluorescence intensity. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant. The results showed that the content of ROS in Ca9-22 cells was increased after 24 hours of different doses of methanol extract from sea bream (0 mg/mL, 0.1 mg/mL, 0.25 mg/mL, 0.5 mg/mL, 1 mg/mL). Significantly increased (p<0.0001), while GSH content decreased significantly (p<0.05), while different doses of ethanol extract from seaweed (0 mg/ml, 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, After 24 hours at 2 mg/mL and 2.5 mg/ml, the ROS content of Ca9-22 cells was significantly increased (p<0.0001), while the GSH content was significantly decreased (p<0.01). This result shows that the sea dragon alcohol extract has the ability to promote the aging of cancer cells.

6. Algae extract from sea squid induces activation of caspase

Pan-Caspase is highly associated with apoptosis, which in the activated state leads to a series of procedures required for apoptosis. Here, a general-purpose caspase inhibitor was used as a marker for flow cytometry to examine the relationship between the seaweed alcohol extract and the pan-caspase class. Results, as shown in Figure 8A, ethanol extraction of red algae asparagus with different concentrations (0mg/ml, 0.5mg/ml, 1mg/ml, 1.5mg/ml, 2mg/ml, 2.5mg/ml 0-2.5 mg/ml) as well as 1 μ l of 500X of processing TF2-VAD-FMK, was observed to affect the degree of activation of the Pan-Caspase Ca9-22 cells (24 h),% positive for all standard chart. Figure 8B shows the quantitative analysis of the fluorescence intensity of TF2-VAD-FMK. The data are presented as mean ± standard deviation (n = 3). The bars with the same letters between the groups have no significant difference. -2 mg/ml of red algae asparagus ethanol extract (0 mg/ml, 0.5 mg/ml, 1 mg/ml, 1.5 mg/ml, 2 mg/ml, 2.5 mg/ml) for the 24th hour of Ca9- The caspase-like activation in the 22 cells had a significant concentration-dependent effect on the extract concentration (p < 0.0001) and showed a positive growth. This result indicates that the sea dragon alcohol extract has the ability to induce apoptosis of cancer cells.

7. The decrease of mitochondrial membrane potential in cancer cells caused by sea dragon alcohol extract

The interstitial material in the mitochondria has a variety of substances such as cytochrome c and apoptosis-inducing factor (AIF), which can activate caspase and cause a series of apoptosis. When the huge channel of mitochondrial permeability transition pore (PT pore) on the surface of the mitochondria is opened, the H+ concentration gradient on both sides of the inner membrane of the granular line disappears, the membrane potential of the mitochondria decreases, and the interstitial osmotic pressure increases, while the granular line After the body is enlarged and the outer membrane is broken, cytochrome c and AIF are released into the cell matrix to cause apoptosis. Therefore, the decrease in mitochondrial membrane potential is an important indicator of apoptosis. Here, a lipophilic fluorescent dye DiOC ₂ (3) was used as a marker for flow cytometry to examine the relationship between the extract of the sea bream and the mitochondrial membrane potential. Results, as shown in Figure 9A, DiOC ₂ at 24 hours after treatment with different concentrations (0 mg/ml, 0.1 mg/ml, 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml) of methanol extracts of Ca9-22 cells. 3) Fluorescent mark content map, positive % is marked in all charts. Figure 9B shows a quantitative analysis of DiOC ₂ (3) fluorescence intensity. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, and the difference is not significant. FIG. 10A cells display Ca9-22 of different concentrations (0mg / ml, 0.5mg / ml , 1mg / ml, 1.5mg / ml, 2mg / ml, 2.5mg / ml) 24 h after ethanol extraction was treated DiOC ₂ (3) Fluorescent marker content map, positive % is marked in all charts with horizontal lines. Figure 10B shows the quantitative analysis of DiOC ₂ (3) fluorescence intensity. The data are presented as mean ± standard deviation (n = 3). The bars in the group contain the same letter, the difference is not significant when extracting The higher the concentration of the substance, the lower the content of DiOC ₂ (3), that is, the greater the decrease of the mitochondrial membrane potential, and the concentration effect (methanol: p<0.05; ethanol: p<0.005). This result indicates that the sea dragon alcohol extract has the ability to promote apoptosis of cancer cells.

discuss

In the present invention, the ability of the red algae asparagus alcohol extract to inhibit the growth of cancer cells is studied, and the ability of the red algae asparagus alcohol extract to induce apoptosis of cancer cells and cause DNA damage of cancer cells is also included. In addition, in the present invention, the concentration of the red algae asparagus alcohol extract and the inhibition of cancer cell growth or apoptosis and DNA damage are also studied.

In the present invention, the ability of the red algae asparagus alcohol extract to inhibit the growth of cancer cells is observed by cell survival rate; the ability of such extracts to induce apoptosis of cancer cell cells is evaluated by cell cycle distribution and annexin-V; γ-H2AX antibody was investigated for its DNA damage; ROS and GSH were used to estimate the free radical pressure; DiOC ₂ (3) dye was used to explore the depolarization of this extract and mitochondrial membrane potential The relationship between. As can be seen from the results of the foregoing examples, the red algae asparagus alcohol extract has an effect of inhibiting cancer by inducing apoptosis, DNA damage, and free radical pressure of cancer cells.

In general, the concentration of the red algae asparagus alcohol extract in the current embodiment is similar to the concentration range of the natural extract used in other cancer treatment studies. In addition, the range of IC ₅₀ values (50% inhibitory concentration) of the red algae asparagus alcohol extract for Ca9-22 cells, such as the red algae asparagus methanol extract is 326 μ g / ml (24 hours), also approximate Usually used in cancer treatment of natural products, such as leaves of Cassia tora methanol extract of HeLa cells the IC ₅₀ of 191 μ g / ml (48 hours) (Toxicol in Vitro 2009,23 (6 ): 1034-1038). Therefore, from the above results, it can be seen that the red algae asparagus alcohol extract has potential for cancer treatment and has become a natural medicine for chemotherapy.

In view of the rapid growth rate of seaweed and the widespread use of seaweed extracts in the food and cosmetic industry, the red algae asparagus alcohol extract of the present invention exhibits an attractive multifunctional choice in these applications.

While the present invention has been described in its preferred embodiments, the present invention is not intended to limit the invention, and the present invention may be modified and modified without departing from the spirit and scope of the invention. The scope of protection is subject to the definition of the scope of the patent application.

Claims (16)

A pharmaceutical composition for inhibiting the growth of cancer cells, which has the ability to inhibit the growth of cancer cells and comprising an effective amount of a methanol or ethanol extract of Rhizia spp . as an active ingredient, wherein the red algae fine-yl Gracilaria Gracilaria (Gracilaria tenuistipitata), the cancer cell is a lung cancer or oral cavity cancer, wherein the concentration of the methanol or ethanol extract 0.5 ~ 3.5mg / ml. The pharmaceutical composition for inhibiting the growth of cancer cells according to claim 1, wherein the step of obtaining the red algae asparagus extract comprises performing a first extraction step on the red algae asparagus to obtain a The first extract, and the first extraction step comprises extracting the red algae asparagus with methanol or ethanol. The pharmaceutical composition for inhibiting the growth of cancer cells according to claim 2, wherein the first extraction step is carried out at room temperature. The pharmaceutical composition for inhibiting the growth of cancer cells according to claim 1, wherein the extraction step is for about 15 to 30 hours. The pharmaceutical composition for inhibiting the growth of cancer cells according to the second aspect of the invention, wherein the step of obtaining the red algae asparagus extract further comprises: filtering the first extract to obtain a filtrate and a filter residue wherein the filtrate is a second extract. The pharmaceutical composition for inhibiting the growth of cancer cells according to the second aspect of the invention, wherein the step of obtaining the red algae asparagus extract further comprises: Performing a second extraction step on the filter residue to obtain a residue of the filter residue, wherein the second extraction step comprises extracting the filter residue by using methanol or ethanol at room temperature; filtering the extract of the filter residue to obtain a second a filtrate; and mixing the second filtrate with the second extract to obtain a third extract. The pharmaceutical composition for inhibiting the growth of cancer cells according to claim 6, wherein the step of obtaining the red algae asparagus extract further comprises performing a third extraction step: performing the second extract Filtration to obtain a filtrate and a filter residue, wherein the filtrate is a third extract; and drying the third extract, which is evaporated to dryness at 35 ° C to 45 ° C. A use of a methanol or ethanol extract of red algae asparagus for preparing a pharmaceutical composition for inhibiting the growth of cancer cells, wherein the red algae asparagus is Gracilaria tenuistipitata , the cancer cell is oral cancer Cell or lung cancer cells. A pharmaceutical composition for inducing apoptosis of cancer cell cells and/or causing DNA damage to cancer cells, which has the ability to induce apoptosis of cancer cells and cause DNA damage to cancer cells and includes an effective amount of red The methanol or ethanol extract of Gracilarias spp. is an active ingredient, wherein the red algae asparagus is Gracilaria tenuistipitata , which is an oral cancer cell or a lung cancer cell. The concentration of the methanol or ethanol extract is 0.5 to 3.5 mg/ml. The pharmaceutical composition for inducing apoptosis of cancer cell cells and/or causing DNA damage to cancer cells according to claim 9, wherein the step of obtaining the red algae asparagus extract comprises the red algae dragon The first extraction step is performed to obtain a first extract, and the first extraction step comprises extracting the red algae asparagus with methanol or ethanol. The pharmaceutical composition for inducing apoptosis of cancer cell cells and/or causing DNA damage to cancer cells according to claim 10, wherein the first extraction step is performed at room temperature. The pharmaceutical composition for inducing apoptosis of cancer cell cells and/or causing DNA damage to cancer cells according to claim 9, wherein the extraction step is for about 15-30 hours. The pharmaceutical composition for inducing apoptosis of cancer cell cells and/or causing DNA damage to cancer cells according to claim 10, wherein the step of obtaining the red algae asparagus extract further comprises: The first extract is filtered to obtain a filtrate and a filter residue, wherein the filtrate is a second extract. The pharmaceutical composition for inducing apoptosis of cancer cell cells and/or causing DNA damage to cancer cells according to claim 10, wherein the step of obtaining the red algae asparagus extract further comprises: The filter residue is subjected to a second extraction step to obtain a residue of the filter residue, wherein the second extraction step comprises extracting the filter residue at a room temperature with methanol or ethanol; and filtering the extract of the filter residue to obtain a second filtrate; And mixing the second filtrate with the second extract to obtain a third extraction Things. The pharmaceutical composition for inducing apoptosis of cancer cells and/or causing DNA damage to cancer cells according to claim 14, wherein the step of obtaining the red algae asparagus extract further comprises performing a first a third extraction step: filtering the second extract to obtain a filtrate and a filter residue, wherein the filtrate is a third extract; and drying the third extract, which is evaporated to dryness at 35 ° C - 45 ° C . A use of a methanol or ethanol extract of red algae asparagus for the preparation of a pharmaceutical composition for inducing apoptosis of cancer cells and/or causing DNA damage to cancer cells, wherein the red algae asparagus is a fine base ( Gracilaria tenuistipitata ), the cancer cell is an oral cancer cell or a lung cancer cell.