KR100836569B1 - A pharmaceutical composition and food additive containing a extract of Styela clava - Google Patents

Abstract

The present invention relates to a pharmaceutical composition and a food additive containing the extract as an active ingredient, by providing an anti-cancer and antioxidant pharmaceutical composition and an antioxidant food additive containing the extract as an active ingredient in the future medical and food industry Mid value can be used as an important resource with very high added value.

Midder extract, Pharmaceutical composition, Food additive, Anticancer, Antioxidant

Description

A pharmaceutical composition and food additive containing a extract of Styela clava}

Figure 1 is a view showing the DNA damage inhibitory effect according to the concentration of the midder acetone extract according to the present invention (A: whole, B: flesh, C: shell).

Figure 2 is a diagram showing the DNA damage inhibitory effect according to the concentration of the extract according to the present invention midderok acetone in a comet image.

Figure 3 is a view showing the cancer cell survival inhibitory effect according to the concentration of the midder acetone extract according to the present invention (A: whole, B: flesh, C: shell).

Figure 4 is a process chart showing an embodiment of the preferred method of extracting midderok of the present invention.

Figure 5 is a view showing the morphological changes of colorectal cancer cells for the solvent-specific middera extract of the present invention (A: control, B: methanol, C: ethanol, D: acetone)

Figure 6 is a view showing the survival inhibitory effect of colorectal cancer cells for the solvent-specific middera extract of the present invention (A: water, B: methanol, C: ethanol, D: acetone).

Figure 7 is a view showing the morphological changes (A) and survival inhibition effect (B) of colorectal cancer cells for the solvent-specific extract fractions of meeduk according to the present invention.

8 is a diagram showing the effect of inhibiting the survival of cancer cells to the diethyl ether extract fraction of the mesophilo according to the present invention (A: colorectal cancer, B: cervical cancer, C: breast cancer).

1. Park HS, Lee YS, Han MJ. 1995. Dietary Life and Health. Hyo-Il Publishing Company, p 262-265.

Nagago M, Sugimura T. 1993. Carcinogenic factor in food with relevance to colon cancer development. Mut Res 290: 45-51.

3. Ames BN, Maron DM. 1983. Revised method for the salmonella typhimurium mutagenicity test. Mut Res 113: 178-215.

4. National Statistical Office. 2004.Annual Report on the Cause of Death Statistics. Seoul, Korea.

5. Kim HJ, Kim MK. 2003. Anticancer effect of persimmon leaf extracts on Korean gastric cancer cell. Korean J Nutrition 36: 133-146.

6. Lee SR. 1993. Food Safety Research. Ewha Woman's University Press.

7. Kang TB, Liang NC. 1997. Studies on the inhibitory effects of quercetin on the growth of HL-60 leukemia cells. Biochem Pharm 54: 1013-1018

8.Park KY, Moon SH, Rhee SH, Baek KY, Lim SY. 1995. Effect of tannin from persimmon leaves on the growth inhibition and the synthesis of mRNA of type IV collagen in AZ-521 human gastric cancer cells. Envirron Mut Carcino 15: 32-37

9. Park JC. 1996. screening of marine natural products on inhibitory effect of the formation of lipid peroxidation. Kor J pharmacogen 27: 117-112.

10. Ministry of Agriculture and Forestry. Ministry of Agriculture and Forestry Statistical Yearbook, p.291. 1993

11. Jo YG. 1978.The sterol composition of Styela clava . Kor Fish Soc 11: 97-101.

12. Lee KH, Park CS, Hong BI, Jung BC, Cho HS, Jea YG. 1995. Seasonal variations of nutrients in warty sea squirt (Styela clava ). J Korean Soc . Food Nutr 24: 268-273.

13. Ahn SH. 2003. Extraction of glycosaminoglycans from Styela clava tunic. Biotechnol Bioproc Eng 18: 180-185.

14. Lehrer RI. 2001.Clavanins and styelins, alpha-helical antimicrobial peptides from the hemocytes of styela clava . Adv Exp Med Biol 484: 71-76.

15. Menzel LP, Lee IH, Sjostrand B, Lehrer RI. 2002. Immunolocalization of clavanins in Styela clava hemocytes. Dev Comp Immunol 26: 505-515.

16. Lee IH, Zhao C, Nguyen T, Menzel, Waring AJ, Sherman MA, Lehrer RI. 2001.Clavaspirin, an antibacterial and haemolytic peptide from Styela clava . J Pept Res 58: 445-456.

17. Taylor SW, Craig AG, Fischer WH, Park M, Lehrer RI. 2000. Styelin D, an extensively modified antimicrobial peptide from ascidian hemocytes. J Biol Chem 275: 38417-38426

18.Brand-Williams W, Cuvelier ME, Berset C. Use of a free radical method to evaluate antioxidant activity. Lebensmittel-Wissenschaft and Technologie 28: 25-30 (1995)

19. Blois MS. Antioxidant determination by the use of a stable free radical. Nature 181: 1199-1200 (1985)

20.Oyaizu M. Studies on products of browning reaction: antioxidative activities of products of browning reaction prepared from glucosamine. Jpn. J. Nutr. 44: 307-315 (1986)

21. Chen, JH, Ho, CT Antioxidant activities of caffeic acid and its related hydroxycinnamic acid compounds. J. Agric . Food Chem . , 45, 2374-2378. (1997)

22. Singh, PN McCoy, MT, Tice RR and Schneider, EL (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp . Cell Res. 175 , 184-191.

23. Fish B. 1984. Clinical trials for the evaluation of cancer therapy. Cancer Res 54: 609-615.

24. 1.Kim JJ, Kim SJ, Kim SH, Park HR, Lee SC. 2006.Antioxidant and anticancer activities of extracts from Styela clava according to the processing methods and solvents. J Korean Soc Food Sci Nutr 35: 278-283.

The present invention relates to pharmaceutical compositions and food additives containing the extract as an active ingredient, more specifically, leukocytes induced by hydrogen peroxide, a cancer cell growth inhibitory effect, an oxidative stress-inducing substance from the extract By proving that the DNA damage inhibiting effect and the antioxidant effect against radical oxygen are very excellent, it is to the pharmaceutical composition for anticancer and antioxidant and the food additive for antioxidant.

In modern society, the cause of cancer is caused by environmental factors rather than genetic factors. Especially, the carcinogenic rate from diet is known as 36% of environmental factors (1-3).

In 2003, cancer accounted for 25.9% of all deaths in Korea, leading the cause of death (4).

If cancer is not detected early, it is almost impossible to cure. As the disease progresses, the pain and economic loss of patients and their families are enormous, and social costs such as financial depletion of medical insurance are incurred (5).

Therefore, the importance of the prevention and early treatment of cancer is very important nowadays, where the quality of life is more important than simply extending the life span.

Recently, new cancer therapeutics are being developed through research on the occurrence and metastasis of cancer, the physiology of cancer cells, the diagnosis and treatment of cancer, and the search for substances having anti-cancer effects including food. If cancer progresses and progresses, intensive treatment through surgery, drug prescription, and radiation therapy should be done, but such drug treatment cannot rule out the sequelae such as toxicity. Because it is required (7,8). In particular, marine life has the potential to explore a variety of new bioactive substances due to its unique metabolic processes and unique environment that are not found in terrestrial organisms. Expectations for the development of unknown natural materials are very high (9).

Autoxidation refers to the reaction of various organic and inorganic compounds to oxidize at room temperature by oxygen in the air. Autooxidation in food and biological systems is not only human health and nutritional status, but also food science and food. It's a big part of technology. Antioxidants play an important role in inhibiting or prolonging automatic oxidation, and synthetic or natural antioxidants are widely used as food additives in most foods.

On the other hand, Styela clava ) is a marine organism belonging to the chromosome and off-white animal, which has contributed to the increase of fishers' income since the farming started in the mid-1980s (10). In addition, midderok is a marine organism found all over Korea, and especially 80% of Korea's consumption is produced in Masan, Gyeongsangnam-do. It is used all year round because of its unique taste and fragrant aroma, and it is the peak of production between April and July. Consumption form of midderk is mainly used in foods such as steamed or miso stew, and others collected from April to May for sashimi.

Most studies on midderk have been conducted on the major components such as sterol content (11) and changes in nutritional composition according to seasons (12, 13). Examples of extracting glucosaminoglycan from the skin as a functional ingredient (14), and recently, reports on hemolytic antimicrobial peptides derived from middodd have been studied and known abroad (15-17), but more research is needed.

Therefore, the present inventors use the lyophilisate of medodeok for the purpose of searching for substances having anti-cancer and antioxidant activity from marine biological resources, prepare extracts for various solvents, and then treat them with various cancer cells derived from humans for cytotoxicity By confirming that the anti-cancer and antioxidant pharmaceutical composition of the extract has been intended.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition and food additives for anticancer and anti-oxidant containing the extract as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for anticancer and antioxidant containing the extract as an active ingredient.

In addition, in order to achieve another object, the present invention provides a food additive for antioxidant containing the extract as an active ingredient.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

The present invention demonstrates that the anticancer and antioxidant properties are excellent by inhibiting the growth of cancer cells, inhibiting DNA damage of leukocytes induced by hydrogen peroxide, which is an oxidative stress-inducing substance, and antioxidant effect against radical oxygen from the extract It is developed as a pharmaceutical composition and food additive for antioxidant.

At this time, the pharmaceutical composition for anticancer in the present invention specifically exhibits an anticancer effect on colorectal cancer, cervical cancer or breast cancer.

The midder used in the present invention is the midder ( Styela In addition, edible midders consisting of two-row, three-row, symmetrical or giraffe-derry can be used.

And, in the present invention, the midderm used to prepare the extract may be a powder prepared by pulverizing the raw or frozen midder, or the powder prepared by grinding the dry meander, but in terms of cost and effectiveness, Use powder of.

In addition, in the present invention, the midder solvent extract refers to an extract in which raw midder ground powder or midder powder is mixed with a solvent at a predetermined ratio and stirred to elute the active ingredient of midder. At this time, in the present invention, it is preferable to use an extract extracted with water, acetone, methanol, ethanol or a mixed solvent thereof.

In addition, the solvent extraction fraction means a solvent fraction obtained by sequentially using a plurality of polar solvents again in the solvent extract. At this time, in the present invention, after extracting the first step with water, acetone, methanol or ethanol, the first extract may be fractions obtained by solvent fractionation with n-hexane, diethyl ether or water, in terms of antioxidant and anti-cancer effect It is preferable to use the fraction obtained by first extracting with ethanol, then solvent fractionation of the primary extract with n-hexane and solvent fractionation of the remaining aqueous solution layer with diethyl ether. Herein, the solvent fraction refers to a process of mixing the mixture and the polar solvent strongly and leaving it to separate into the polar solvent layer and the aqueous layer, and separating the active ingredient dissolved in the polar solvent from the active ingredient contained in the extract into the polar solvent layer.

 However, the extract according to the present invention is not limited by the above-described method.

In addition, as disclosed in the Remington's Pharmaceutical Sciences Handbook (Mack Pub, Co., NY, USA), using the midder extract of the present invention as an active ingredient, a suitable drug according to the administration method, dosage form and therapeutic purpose The mixture may be diluted with a pharmaceutically acceptable carrier in the form of a pharmaceutical composition or encapsulated in a carrier in a container form.

In addition, when the carrier is used as a diluent, saline, buffer, dextrose, water, glycerol, Ringer's solution, lactose, sucrose, calcium silicate, methyl cellulose or ethanol may be used, and oral and parenteral administration And powders, granules, injections, syrups, solutions, tablets, suppositories, and the like. However, the carrier of the present invention is not limited to the above carrier. In this case, parenteral administration refers to the administration of the drug through rectal, intravenous, peritoneal, muscle, arterial, transdermal, nasal, inhalation, in addition to oral.

In addition, the formulation may further include fillers, anti-coagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, and the like, and may be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.

In addition, the extract according to the present invention can be determined according to the type of cancer, the route of administration, the dosage form, the purpose of use, and the severity of the disease of the patient. This is obvious to those skilled in the art.

In addition, the food additive according to the present invention is also used in the same manner as the antioxidant used for antioxidant in food, the composition extracted in the same manner as the pharmaceutical composition described above. At this time, since the effective amount of the food additive varies depending on the type of each food, the description thereof is omitted in the present invention. However, it will be apparent to those skilled in the art having ordinary knowledge in the field of the present invention to use the extract of the present invention according to the food by adding an appropriate effective concentration.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples, and it will be apparent to those skilled in the art that various changes or modifications can be made within the spirit and scope of the present invention.

EXAMPLE

1. Experimental Materials and Reagents

(1) Midduck

Styela used in the present embodiment clava ) was purchased from Gohyeon village, Vibration-myeon, Masan-si, Gyeongsangnam-do.

(2) white blood cells

Blood leukocyte cells were used in this example after only 5 ml of fresh whole blood collected from two healthy adult males were separated from leukocytes using Histopaque 1077.

(3) cancer cells

The cancer cell lines used in this example are human colon carcinoma (HT-29), human colon carcinoma (SW620), cervical cancer cells HeLa (human cervices adenocarcinoma), and breast cancer cells MCF-7 (human breast adenocarcinoma) pleural effusion) was used by the Korea Cell Line Bank (KCLB, Seoul) for the experiment. At this time, the medium for cell culture was RPMI1640 medium for HT-29, SW620, MCF-7 cell line, HeLa cell line was used for DMEM medium, and the culture was 10% FBS, 100 unit / mL penicillin in each medium. , 100 mg / mL of streptomycin was incubated in 37 ℃, 5% CO ₂ incubator (MCO-18AIC, SANYO, Osaka, Japan).

(4) reagent

Reagent MTT (3- [4,5-dimethylthiazol-2-yl] -2, 5-diphenyl tetrazolium bromide) used in experiments on cancer cell growth inhibitory effect and hydrogen peroxide used in comet assay, Histopaque 1077, low melting point agaroses, Normal melting point agaroses Triton X-100, disodium salt ethylenediaminetetraacetic acid, Tris-buffer, sodium chloride, sodium hydroxide, ethidium bromide, potassium chloride, potassium phosphate, sodium hydrogen phosphate, sodium, etc.Reagents such as Sigma Chemical Co. (St. Louis) , MO, USA), and the solvents and reagents used in the other studies all used first grade or higher grades.

2. Sample Preparation

(1) raw midder

The midder was washed three times with tap water to remove salts and sand from the surface, and finally rinsed carefully with clean water. Then, the midder was separated into three parts (whole, flesh, shell), and each part was ground with a mixer (Mixer MC 811C, Nobita, Korea). In the present invention, this was named as a raw midder sample.

(2) midder powder

Wash several times with water to remove foreign substances, and then remove the dried fresh midders with a blender [Mixer MC 811C, Nobita, Korea] by parts (whole, flesh, skin), and then use the Deep Deep freezer. VX 530, Korea) and stored overnight at -70 ℃ and completely dried for 4 days with a freeze dryer (Freeze Dryer FD 5512, Ilsin Lab, Korea). The dried sample was made into powder using a grinder again, and the size of the powder was uniformly filtered by 27 mesh sieve. In the present invention, this was named as a midder powder sample.

4. Antioxidant and Anticancer Effects of Solvent Extracts from Raw and Middera Powders

First, the solvent extract of the raw midderm sample was mixed with 100 ml of the raw midderm sample and 1 L of a solvent (methanol, ethanol, acetone, water [distilled water]), and the extract was obtained by stirring (100 rpm) for 24 hours at room temperature. This is called FR.

In addition, the solvent extract of the midder powder sample was dissolved in 100 ml and a solvent (methanol, ethanol, acetone, water) and 3.616g of the midder powder sample, and then used to store the extracted by stirring (100 rpm) for 24 hours. This is called FD.

Each extract was filtered with a filter paper (Whatman No. 1), and then concentrated at 37 ° C. with a rotary pressure reducer (EYELA N-1000, Tokyo Rikakikai Co., Tokyo, Japan). Each concentrate was placed in a glass bottle and replaced with nitrogen and stored at 4 ° C. It was dissolved in DMSO to adjust the final concentration and used for the experiment.

(One) DPPH Radical  Scavenging activity effect

DPPH radical scavenging activity of the extracts was performed according to the method of Lee et al, 18-19. 0.1 mL of the extract (1 mg / mL, 5 mg / mL, 10 mg / mL) was mixed with 0.9 mM ethanol solution of 0.041 mM DPPH for 25 minutes, and then centrifuged at 12,000 rpm for 5 minutes. At this time, the extract was used for the raw midderm samples and midderm powder samples for each part.

The supernatant was then absorbed at 517 nm using a spectrophotometer (Sspectrophotometer, Shimadzu UV-1601, Tokyo, Japan). As a control, a mixture of 0.1 mL of DMSO and 0.9 mL of DPPH was used. Radical scavenging activity was calculated as in the following formula 1 and expressed as the inhibition rate (%). At this time, all experiments were performed in three iterations, and the analysis of the variables was processed by the general preceding model method using SAS software. Student-Newman-Keul's multi-range experiments used totests for the difference in measured mean values (p <0.05).

[Equation 1]

% DPPH radical scavenging activity = [1- (sample OD / control OD)] × 100

The results are as shown in Table 1 below.

It was found that free radical scavenging activity increased with increasing concentration of the extract. Among them, the activity of methanol in the FR extract and acetone in the FD extract was very high in the entire region of melon. In the case of peeling, no solvent was clearly high in the FR extract and high in acetone in the FD extract. In the flesh site, acetone was high in the FR extract and D.W in the FD extract.

From the above results, overall, DPPH radical scavenging activity was the highest in the extract of flesh (distilled water) in the case of FD extract, and the extract of methanol in the whole region was the best in extract of FR. .

(2) reducing power effect

Reducing power of the extracts of the midderm was carried out according to the Oyaizu method (20). 1 mL of 1% potassium ferricyanide was added to 1 mL of 0.2 M sodium phosphate buffer (pH 6.6) and 1 mL of midder extract (1 mg / mL, 5 mg / mL, 10 mg / mL). It reacted at 20 degreeC for 20 minutes. 1 ml of 10% TCA was added to stop the reaction. The reaction stopper was centrifuged at 12,000 rpm, 4 ° C. for 5 minutes, 1 ml of supernatant was added with 1 ml of distilled water and 0.1 ml of 0.1% ferric chloride, and then absorbance was measured at 700 nm. The higher the absorbance value, the higher the reducing power). At this time, all experiments were performed in three iterations, and the analysis of the variables was processed by the general preceding model method using SAS software. Student-Newman-Keul's multi-range experiments used totests for the difference in measured mean values (p <0.05).

The results are shown in Table 2 below.

The above results showed the same tendency as the results of DPPH radical scavenging activity.

(3) Lancemat  Way( rancimat  antioxidative effect

According to the method of Chen et al, 21, the oxidation period of lard due to the addition of antioxidant was measured.

First, the degree of oxidation of lard oil with or without the addition of midderm extract was measured using a Metrohm 793 Rancimat instrument (Herisan, Switzerland). Oxidation was induced with an air flow rate of 20 L / h at 120 ° C. In addition, 2.5 g of lard was added to 1.0 mL of each midderm extract sample (1 mg / mL, 5 mg / mL, 10 mg / mL) immediately before the measurement of the lancet mat, and vigorously mixed for 8 seconds using a vortex machine.

At this time, all experiments were performed in three iterations, and the analysis of the variables was processed by the general preceding model method using SAS software. Student-Newman-Keul's multi-range experiments used totests for the difference in measured mean values (p <0.05).

(There are no experimental results. If you do not have experimental data, please delete this part.)

5. Inhibition of DNA Damage and Anticancer Effect of Solder Extract

First, 3.616 g of the midderm powder sample of (2) was added to 100 ml of acetone solvent, and extracted with shaking culture machine (HB-201s, Hanbaek, Korea) at 25 ° C. and 100 rpm for 24 hours. Each extract was filtered through filter paper (Whatman No. 1), and then concentrated at 37 ° C. with a rotary pressure reducer (EYELA N-1000, Tokyo Rikakikai Co., Tokyo, Japan). Each concentrate was stored in a glass bottle at 4 ° C after nitrogen replacement, and dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 5, 10, 50, 100, 500 µg / ml.

In addition, each sample ( Styela) was prepared on leukocytes prepared to induce oxidative stress on leukocytes. clava - whole, flesh, skin part) was treated at 5, 10, 50 ㎍ / ㎖ concentration for 30 minutes at 37 ℃, leukocytes washed with PBS and then 200 μM to artificially induce oxidative stress H ₂ O ₂ was treated with white blood cells and reacted at 4 ° C. for 5 minutes, and then washed with PBS again. At this time, the positive control (Positive control) was treated with the same amount of DMSO and 200 μM H ₂ O ₂ in place of the midderk sample, the negative control (negative control) was treated with the same amount of DMSO.

(1) Effect of DNA assay

First, leukocytes were treated with the extract of Midodeok and the following method was performed to induce oxidative stress.

The above-mentioned (1) to the white blood cells treated with the sample (all, flesh, peel) per Styela clava portion of the powder at a concentration of 5, 10, 50 ㎍ / ㎖ was 30 min at 37 ℃ ¹⁵ prepared ^in). After the reaction, the white blood cells were washed with PBS and 200 μM of H ₂ O ₂ was treated with leukocytes for 5 minutes at 4 ° C. in order to cause oxidative stress, and then washed with PBS. At this time, the positive control (Positive control) was treated with 200 μM H ₂ O ₂ after treating the solvent DMSO instead of the sample of the extract, and the negative control was used only solvent (DMSO). At this time, the sample was extracted for 24 hours at 25 ℃, 100rpm condition in a shake incubator to add 3.616g of the medium powder powder sample 3.616g each site, each extract was filtered by filter paper (Whatman No. 1) and then rotated The concentrate was concentrated at 37 ° C using a immersion concentrator, and each concentrate was used in a glass bottle, nitrogen-substituted, and then stored at 4 ° C.

Then, to perform a Comet assay (22) on the oxidative stress-induced white blood cells, 75 μl of 0.7% low melting agarose gel (LMA) was mixed with 1.0% normal melting agarose (NMA) precoating normal slide The cell suspension and the suspension of the LMA were evenly dispersed, and then covered with a cover glass and stored in a 4 ° C refrigerator. Then, when the gel was hardened, the cover glass was peeled off and again covered with 75 μl of 0.7% LMA solution. In addition, 1% Triton X-100 was mixed with cold alkali lysis buffer (2.5 M NaCl, 100 mM Na ₂ EDTA, 10 mM tris), and the slide was immersed in low temperature and dark for 1 hour. Loosen the double strand of. After the lysis, slides were placed in an electrophoresis tank and filled with cold electrophoresis buffer (300 mM NaOH, 10 mM Na ₂ EDTA, pH> 13) at 4 ° C for unwinding for 40 minutes to reveal alkali labile sites of DNA. Electrophoresis was performed for 20 minutes under a voltage of 25 V / 300 ± 3 mA. In order to prevent additional damage of DNA by light, the above procedure was performed with the electrophoretic tank covered with a dark cloth. After the electrophoresis, the slide was dried by repeating the process of soaking in 0.4 M Tris buffer (pH 7.4) for 10 minutes three times. Nuclei were stained with ethidium bromide at a concentration of 20 μl / ml, covered with a cover glass, and observed on a fluorescence microscope (Leica, Germany). Each nucleus image sent through a CCD camera (Nikon, Japan) was analyzed on a computer equipped with a Komet 5.0 comet image analyzing system (Kinetic Imaging, UK). The degree of DNA damage caused by hydrogen peroxide of leukocytes and the inhibition of damage caused by the extracts were measured by measuring the% DNA content in the tail portion, which migrated from the nucleus to the tail portion. Two slides were made at each treatment to measure the DNA damage of 100 cells, and each treatment was repeated at least three times. In this case, to calculate the percentage and mean ± standard error (SE) according to each item, and to compare the degree of DNA damage inhibition of each substance, one-way analysis of variance (ANOVA) was performed to find the F value and Duncan's multiple range test. The significance difference of each section was verified. Statistical significance was evaluated at the 5% level.

As shown in FIG. 1, the acetone extract was treated with leukocytes at concentrations of 5, 10, and 50 μg / ml, and then induced DNA damage at a concentration of ₂₀₀ μM of H ₂ O _2, followed by Comet assay. of was the the DNA content han% fluorescence in tail measured significantly lower than the 63.9% of H ₂ O ₂ treatment positive control to 26.9, 27.0 and 23.8%, respectively, as a result of this out by calculating it as a DNA suppressing the damaging effects respectively 57.8 , 57.7 and 62.8%. Similarly, the acetone extract showed the effect of inhibiting DNA damage of more than 50% in each treatment group.

In particular, in the case of midderk acetone extract, the DNA damage inhibitory effect was 30.6, 54.0, 78.3% concentration-dependently and significantly increased when treated at the same concentration (see Fig. 1C). Accordingly, the DNA damage inhibitory effect of the acetone extract was the highest when 50 ㎍ / ㎖ treated with the middle peel fraction.

Meanwhile, the comet image observed with the fluorescence microscope is as shown in FIG. 2.

(We want you to express opinion of result about Figure 2)

These results indicate that acetone extracts in each part of MID may effectively inhibit DNA damage induced by hydrogen peroxide, an oxidative stressor.

(2) Anticancer Effect of Midderm Extracts Extracted with Acetone Solvent

As a result of the above, 3.616 g of the midderok powder sample prepared in the above (2) was added to 100 ml of acetone solvent, and then stirred for 24 hours at 25 ° C. and 100 rpm in a shake incubator [HB-201s, Hanbaek, Korea]. Extracted. Each extract was filtered through filter paper (Whatman No. 1), and then concentrated at 37 ° C. with a rotary pressure reducer (EYELA N-1000, Tokyo Rikakikai Co., Tokyo, Japan). In addition, each concentrate was stored in a glass bottle at 4 ° C. after nitrogen replacement, and was dissolved in dimethyl sulfoxide (DMSO) at a final concentration of 5, 10, 50, 100, 500 μg / ml and used in the experiment.

Then, the MTT assay was performed in the same manner as the cancer cell growth inhibitory effect measurement experiment of (10). At this time, the statistical processing of all data was processed using the SPSS-PC + statistical package.

The anti-cancer effects of the extracts of different parts were compared with those of the control, and as shown in FIG. 3, when the extracts of whole, flesh, and skin parts of midderm were added at concentrations of 100 μg / ml, respectively, 90.5%, 82.0%, and 75.2, respectively. when showed relatively low activity in%, treated with 500 μg / m l it could be seen to rapidly increase the activity to 26.9%, 30.6% and 12.0%, respectively. In particular, the acetone extract of the skin part showed a strong cancer cell growth inhibitory effect. At this time, Figure 3 shows the growth when treated with cancer cells in the concentration of 10, 50, 100, 500 ㎍ / ㎖, respectively, the acetone extract extracted by the whole (A), flesh (B) and skin (C) of each part Inhibitory effect is shown.

6. Solvent Extract and Solvent Extraction Fraction  Cancer cell growth inhibitory effect

First, 5 g of the powdered powder prepared in step (2) was added with 100 mL of water, methanol, ethanol, and acetone solvent in a shake incubator (HB-201s, Hanbaek, Korea) at 25 ° C. and 100 rpm. 48 hours extraction. Each extract was filtered through filter paper (Whatman No. 1), and then concentrated at 37 ° C. with a rotary pressure reducer (EYELA N-1000, Tokyo Rikakikai Co., Tokyo, Japan). Among them, solvent fractionation was sequentially performed according to the polarity of the solvent for the purpose of confirming the cancer cell growth inhibitory effect by increasing the purification yield by using the ethanol extract having the strongest activity. In other words, n-hexane (n-hexane) and water in the same ratio to fractionate the n-hexane layer from the fractional filter, and in the same way to the remaining aqueous layer diethyl ether (ethyl ether), ethyl acetate (ethyl acetate) ), Fractions were separated with a water layer, and each solvent was extracted and concentrated under reduced pressure (see FIG. 4). Each extract and extract fractions were dissolved in DMSO (dimethyl sulfoxide) at a concentration of 50 mg / mL, diluted to an appropriate concentration and used in the experiment.

(One) Morphological Observation of Cancer Cells

For the morphological observation of the HT-29 cell line, the solvent of the medium powder powder dissolved in DMSO was incubated for 24 hours at 37 ° C, 5% CO ₂ incubator at 1 × 10 ⁵ cells / well in 6 well plates. Each star extract was treated at 500 mg / mL concentration, and after 24 hours, the cell morphology of each well was observed with an inverted microscope (TS 100-F, Nikon, Tokyo, Japan), and photographs were taken 100 times.

The results are shown in FIG.

In other words, the morphology of HT-29 cell line was examined by the inverted microscope to determine the effect of the extract on each solvent. Cells treated at the concentration of were reduced in binding force, and the cells were disturbed and the cells were condensed and killed. Among them, the extract of ethanol (Methanol extract) (hereinafter referred to as ethanol extract) has the highest activity against cancer cell growth inhibitory effect as the cell number decreases due to the large condensation of cells compared to the extracts of control and other solvents. Could confirm.

(2) Effect of Midderm Extract on Inhibition of Cancer Cell Growth

Cancer cell growth inhibitory effect of the extracts of each part according to the present invention was performed by MTT assay (23).

First, the cell line was adjusted to a concentration of 1 × 10 ⁵ cells / ml, and 100 μl of each was added to a 96 well plate, incubated in 37 ° C. and 5% CO ₂ incubator for 24 hours, and then the extract fraction samples for each solvent were dissolved in DMSO. each was treated with concentrations of 10, 50, 100, 500 μg / ㎖ 18). After incubation for 24 hours, 10 μl of MTT (5 μg / mL) solution dissolved in PSB buffer was added to each well, followed by incubation for 1 hour, and the supernatant was removed so that the formazan formed at the bottom of the well was not dispersed. After the addition of ㎕l was dissolved, the absorbance was measured at 540 nm using an ELISA reader (Model 680, BioRad, USA) to determine the relative inhibition of cell growth when the control cell number was 100%. At this time, all the experiment was made of three iterations, the experimental results were expressed as the mean ± standard deviation. All treatment differences were analyzed by comparison with a confidence level of 95% (p <0.05).

The results are shown in FIG.

In other words, as a result of confirming the cancer cell growth inhibitory effect compared to the control group without the extract using the MTT reduction assay method (24), each solvent was treated with a concentration of 10, 50, 100, 500 mg / mL When acetone and water extracts showed weak activity of 52.6% and 66.1% at the concentration of 500 mg / mL, ethanol and methanol extracts showed strong anticancer activity of 45.0% and 46.3%. In particular, it was found that ethanol extract has the highest anticancer activity among various solvent extracts as a result of morphological changes. In addition, ethanol extract was the highest activity, methanol, acetone extract in the order of high activity.

(3) By solvent Fraction  Anticancer effect

In addition, the effect of the solvent extraction fraction of the ethanol extracts of the midderm was compared and analyzed in the same manner as in the above 6 (1) to (2).

As a result, cell morphology observation and MTT reduction assay method for the cancer cell growth inhibitory effect of each solvent fraction is shown in FIG. At this time, the solvent-specific fraction (a) in Figure 7 (A) is a control, (b) shows n-hexane, (c) diethyl ether and (d) ethyl acetate, and in Figure 7 (B) (a) n-hexane, (b) diethyl ether, (c) ethyl acetate and (d) water.

As a result of cell morphology, the control group grew cancer cells densely and normally, whereas the cells treated with each fraction at the concentration of 500 mg / mL decreased the binding force, disturbed the cells, condensed and died. Among them, the diethyl ether fraction was found to have the highest activity for inhibiting cancer cell growth by decreasing the number of cells as the cells condensed significantly compared to the control and other extracts (see FIG. 7A). ).

The fractions of each solvent were treated at concentrations of 50, 100, 250, and 500 mg / mL, and the MTT reduction assay showed that the diethyl ether fraction had the highest anticancer activity among the various solvent fractions. When treated, the cancer cell survival rate was 25.9%, and at 500 mg / mL, the survival rate was 11.0% or less. This is similar to the result of morphological observation.

In addition, the ethyl acetate fraction showed a lower cancer cell growth inhibition effect of less than 50% at a concentration of 250 mg / mL although lower than that of the diethyl ether fraction.

(3) diethyl ether (Diethyl ether), tumor growth inhibitory effects of the fractions

On the other hand, in order to confirm the anticancer activity spectrum of the diethyl ether fraction showing the highest anticancer activity among the solvent fractions, anticancer activity was obtained by treating human colon cancer cell lines SW620, cervical cancer cell HeLa, and breast cancer cell MCF-7 such as HT-29. It was confirmed.

As a result, as shown in FIG. 8, HeLa cells showed the best inhibitory effect on cancer cell growth compared to other cells at 26.7% even at 50 mg / mL concentration, and other cells had good activity of 50% or less than HT-29. Showed. At the concentration of 250 mg / mL, SW620, HeLa, and MCF-7 cells showed high anticancer activity of 13.1%, 9.5%, and 7.5%. Therefore, it was confirmed that the extracts of the midderm showed strong anticancer activity not only in human colon cancer cell lines but also in several other cancer cell lines.

As described above, the extract according to the present invention is very effective in inhibiting cancer cell growth, inhibiting DNA damage of leukocytes induced by hydrogen peroxide, which is an oxidative stress-inducing substance, and antioxidant effect against radical oxygen. It can be provided as a composition and a food additive.

Therefore, the present invention can be used as an important resource with a very high added value in the medical and food industry in the future.

Claims (8)

A pharmaceutical composition for anticancer and anti-oxidation, which comprises the extract of medo-deok, which is a fraction obtained by first extracting medo-deok with water, acetone, methanol or ethanol, and then extracting the first extract with n-hexane, diethyl ether or water as an active ingredient. The method of claim 1, The midder is anti-cancer and antioxidant pharmaceutical composition, characterized in that it is extracted from the freeze-dried midder powder. The method of claim 2, The midder is Styela clava ( Styela clava) characterized in that the pharmaceutical composition for anticancer and antioxidant. delete delete The method of claim 1, The midder extract, After extracting the first step in ethanol, solvent fractionation of the primary extract with n-hexane, the remaining aqueous layer is a fraction obtained by solvent fractionation with diethyl ether. The method according to any one of claims 1 to 3 and 6, The cancer is anticancer and antioxidant pharmaceutical composition, characterized in that the colorectal cancer, cervical cancer or breast cancer. Food additive for antioxidant containing the extract of claim 1 as an active ingredient.

Images