KR20130030971A - Pharmaceutical composition having anticancer and antioxidant comprising lutein from isolated soybean - Google Patents

Abstract

PURPOSE: A pharmaceutical composition is provided to ensure antioxidation activity and to suppress proliferation of various cancer cell lines. CONSTITUTION: A pharmaceutical composition contains 0.01-80 wt% of lutein isolated from soybeans, as an active ingredient. A method for preparing lutein comprises: a step of germinating seeds of soybeans; a step of drying and pulverizing the germinated soybeans; a step of dipping the soybean powder in an organic solvent; a step of filtering the soybean powder solution and concentrating by compression to collecting germinated soybean extract; and a step of isolating and purifying lutein from the extract.

Description

Pharmaceutical composition having anticancer and antioxidant comprising lutein from isolated soybean}

The present invention relates to a pharmaceutical composition having anticancer and antioxidant activity comprising lutein isolated from soybean as an active ingredient.

In recent years, dietary phytochemicals in crops have been shown to be potent antioxidants and play an important role in the prevention and treatment of various diseases. Specifically, various anti-carcinogens and anti-mutants have been shown to reduce the proliferation and frequency of biomarkers, which are mediators of tumors and cancers in vivo , and short-term genotoxicity in vitro . It is confirmed that the test shows anticancer efficacy.

Various animal experiments have demonstrated anti-cancer efficacy at or above the level of phytochemicals ingested by the human body.However, pigments such as chlorophyll and carotenoids found in nutrients such as leaves and fruits of crops Consideration is very poor. In addition, it is unclear whether phytochemicals such as vitamins C, E, and polyphenols or pigment compounds such as chlorophyll and carotenoids contribute to disease treatment and health promotion due to the antioxidant activity of crops. Most of the research is limited to other phytochemicals, such as polyphenols and saponins, except for these pigment compounds.

Lutein, one of the family of carotenoids present in plants, is one of the antioxidants present in green vegetables, and among the about 700 carotenoids isolated from natural materials, it is the most effective and important hydride that can eliminate free radicals. Hydroxyl carotenoids.

Zeaxanthin, a compound associated with lutein, is a dihydroxy xanthophyll family of carotenoids that contains two hydroxyl groups, while hydrocarbon carotenoids such as β-carotene or lycopene. ) Does not have an oxygen atom. The hydroxy group of lutein and zeaxanthin has higher polarity than hydrocarbon carotenoid, a carotenoid that does not contain it, and contributes to the beneficial effect in the visual system of the human body.

Lutein is known to act as the only filter for high energy blue light or to remove photocatalytic radicals or reactive oxygen species (Krinsky, 1989). Lutein intake is known to be effective in eye diseases such as age-related macular degeneration or cataract. In addition, lutein protects intracellular DNA by scavenging free radicals and reactive oxygen species or protecting other antioxidants as antioxidants, and can be expected to have therapeutic and prophylactic effects on clinical diseases.

Based on these characteristics, carotenoids have been reported to exhibit antiproliferative and differentiating effects of transformed cells during the course of carcinogenesis (Gross et al., 1997; Kim et al., 1998; Nishino et al. , 2000).

Soybean ( Glycine max L.) is a major food crop in Korea, containing a large amount of protein (about 40%) and lipids (about 20%), and is an excellent food in terms of nutrition. Soybean is processed in the form of fermented products such as soybean paste, cheonggukjang, red pepper paste, soy sauce, germinated products such as bean sprouts, and other products such as soymilk, tofu, cooking oil, etc. It is also widely used as.

Soybeans are evaluated to contain various bioactive substances that are beneficial to the human body in addition to the main nutritional functions. Therefore, the establishment of analytical methods for these nutrients and bioactive substances and systematic review of useful resources at home and abroad will strengthen the competitiveness of domestic food crops in the future. It will act as a key factor for

Conventionally, researches focusing on proteins or isoflavones have been mainstream among the components of the soybean, and technology development using the same has been mainly performed, and research on various other functional ingredients has been hardly made.

Krinsky NI. (1989). Carotenoids and cancer in animal model. Journal of Nutrition. 119: 123-126 Gross MD, Bishop TD, Belcher JD, Jacobs DR Jr. (1997). Induction of HL-60 cell differentiation by carotenoids. Nutrition and Cancer. 27: 169-173. Kim JM, Araki S, Kim DJ, Park CB, Takasuka N, Baba-Toriyama H, Ota T, Nir Z, Khachik F, Shimidzu N, Tanaka Y, Osawa T, Uraji T, Murakoshi M, Nishino H and Tsuda H. (1998). Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhydrazine initiation. Carcinogensis. 19 (1): 81-85. Nishino H, Tokuda H, Murakoshi M, Satomi Y, Masuda M, Onozuka M, Yamaguchi S, Takayasu J, Tsuruta J, Okuda M, Khachik F, Narisawa T, Takasuka N and Yano M. (2000). Cancer prevention by natural carotenoids. Biofactors. 13: 89-94. Nariswa T, Fukaura Y, Hasebe M, Ito M, Aizawa R, Murakoshi M, Uemura S, Khachik F and Nishino H. (1996). Inhibitory effects of natural carotenoids, alpha-carotene, beta-carotene, lycophene and lutein, on colonic aberrant crypt foci formation in rats. Cancer Lett 107: 137-142 Chew BP, Brown CM, Park JS and Mixter PF. (2003). Dietary lutein inhibits mouse mammary tumor growth by regulating angiogenesis and apoptosis. Anticancer Reserch. 23 (4): 3333-3339 Park JS, Chew BP and Wong TS. (1998). Dietary lutein from marigold extract inhibits mammary tumor development in BALB / c mice. Journal of Nutrition. 128: 1650-1656.

In order to solve the problems of the prior art as described above, an object of the present invention is to provide a pharmaceutical composition having anticancer and antioxidant activity, including lutein isolated from soybean.

It is another object of the present invention to provide a dietary supplement for enhancing anticancer and antioxidant effects comprising lutein isolated from soybeans.

It is another object of the present invention to provide a cosmetic composition having an antioxidant activity including lutein isolated from soybean.

In addition, the present invention can suggest a new source as a composition having anti-cancer and antioxidant activity while revealing a new physiological activity of soybeans, and anti-cancer and anti-cancer effect of assaying the anti-cancer and antioxidant effects of lutein isolated from soybeans and It is an object to provide a pharmaceutical composition, dietary supplement and cosmetic composition having an antioxidant activity.

In order to achieve the above object, the present invention provides a pharmaceutical composition having anticancer and antioxidant activity, comprising lutein isolated from soybean as an active ingredient.

The lutein is preferably included in the pharmaceutical composition 0.01 to 80% by weight.

The lutein can be separated from soybeans, germinated beans or bean sprouts.

Specifically, the lutein is a step of germinating the seeds of the beans, drying and grinding the germinated beans, soaked in an organic solvent and concentrated under reduced pressure to obtain a germinated soybean extract under reduced pressure and separating the lutein from the germinated soybean extract It is good to separate in the step of purification.

The pharmaceutical composition comprising lutein isolated from the beans as an active ingredient shows excellent anticancer activity against prostate cancer, lung cancer, kidney cancer, colon cancer or breast cancer.

In another aspect, the present invention provides a dietary supplement for anticancer and antioxidant effects, comprising lutein isolated from soybean as an active ingredient.

The lutein is preferably included in the dietary supplement 0.01 to 50% by weight.

In another aspect, the present invention provides a cosmetic composition having an antioxidant activity comprising lutein isolated from soybean as an active ingredient.

The lutein is preferably contained in 0.01 to 40% by weight in the cosmetic composition.

According to the present invention, the antioxidant activity against lutein isolated from soybeans and the proliferation inhibitory effect against various cancer cell lines are verified, thereby revealing a new biological activity of soybean, thereby suggesting a new source as a composition having anticancer and antioxidant activity. You can expect the effect.

Figure 1 shows the ¹ H-NMR spectrum of lutein isolated from the soybean extract germinated according to an embodiment of the present invention.
Figure 2 shows the DPPH radical scavenging activity of lutein and α-tocopherol isolated from soybeans according to one embodiment of the present invention. α-tocopherol was used as a positive control, absorbance was measured at 517 nm, and the results are expressed as mean ± SD (n = 3).
Figure 3 shows the reducing power of lutein and BHA isolated from soybeans according to one embodiment of the present invention. BHA was used as a positive control and the results were expressed as mean ± SD (n = 3).
Figure 4 shows the results of measuring the superoxide radical scavenging ability according to the concentration of lutein, ascorbic acid and β-carotene isolated from soybeans according to an embodiment of the present invention by ESR spectrum. The left panel shows the inhibitory activity (%) of lutein, ascorbic acid and β-carotene isolated from soybeans by concentration (n = 3), and the right panel shows lutein (20 mM), ascorbic acid (20 mM) and Figure shows the ESR signal strength of superoxide radicals with the addition of β-carotene (20 mM). The superoxide radical formed by the reaction of 10 mM xanthine and 0.25 U xanthine oxidase was reacted with DMPO (0.3M) as a negative control, and β-carotene and asad were reacted to the superoxide radical formed by the reaction of xanthine and xanthine oxidase. The reaction of DMPO (0.3M) with the amount of superoxide radicals that were eliminated by adding corvinic acid was used as a positive control.
Figure 5 shows the superoxide radical production inhibition rate for NBT reduction of lutein isolated from soybeans according to one embodiment of the present invention. α-tocopherol was used as a positive control and the results were expressed as mean ± SD (n = 3).
Figure 6 shows the result of measuring the hydroxy radical scavenging ability of each concentration of lutein, ascorbic acid and β-carotene isolated from soybeans by ESR spectrum according to an embodiment of the present invention. The left panel shows the scavenging activity of lutein, ascorbic acid and β-carotene by concentration (%), and the right panel shows lutein (20 mM), ascorbic acid (20 mM) and β-carotene ( Figure shows the ESR signal strength of superoxide radicals with addition of 20 mM). The reaction of hydroxy radicals produced by the Fenton reaction of ₁₀ mM H ₂ O ₂ with 10 mM FeSO ₄ with DMPO (0.3M) was used as a negative control, and β was added to the hydroxyl radical (OH ·) formed by the Fenton reaction. The reaction of DMPO (0.3M) with the amount of superoxide radicals that were eliminated by addition of carotene and ascorbic acid was used as a positive control.
Figure 7 shows the hydroxy radical scavenging activity of lutein isolated from soybeans according to one embodiment of the present invention. BHA and α-tocopherol were used as positive controls, and negative controls were acetone without addition of antioxidants. Results are expressed as mean ± SD (n = 3).
FIG. 8 shows the effect of carotenoids (β-carotene, lycopene and lutein isolated from soybean) on PCNA (36kDa) protein expression in LNCaP prostate cancer cells cultured for 24 hours according to one embodiment of the present invention. β-Actin (43kDa) was used as an internal control, and the results were measured by the Student 't-test with mean ± SD (n = 3) values, with “*” being 0 μM. The data show a significant difference from the treatment (p <0.05).
Figure 9 shows the effect of carotenoids (β-carotene, lycopene and lutein isolated from soybean) on the expression of cyclinD ₁ (36kDa) protein in LNCaP prostate cancer cells cultured for 24 hours according to an embodiment of the present invention. β-Actin (43kDa) was used as an internal control, and the results were measured by the Student 't-test with mean ± SD (n = 3) values, with “*” being 0 μM. The data show a significant difference from the treatment (p <0.05).

Hereinafter, the present invention will be described in detail.

The present inventors obtained the carotenoid pigment lutein (purine) from soybeans in purified form in order to investigate the bioactive activity of lutein for human health and the characteristics of the use of such pigment-based compounds. As a result of assaying for antioxidant activity and anticancer activity, it was confirmed that lutein isolated from soybean had high antioxidant and anticancer activity, and thus completed the present invention.

Soybean described in the present invention means all the plant seeds belonging to the legumes, germinated soybean (shrinkage is 1cm or less) and bean sprouts (shrinkage is 5 ~ 10cm) obtained by germinating soybeans in a conventional manner in addition to pure soybeans Of course, it includes all). In addition, lutein described in the present invention means all lutein isolated from soybean, pure bean, germinated soybean, cotyledon of sprouts, hypocotyl or all lutein isolated from cotyledon and hypocotyl of soybean sprouts.

The pharmaceutical composition of the present invention is characterized by comprising lutein isolated from soybean as an active ingredient.

Lutein included in the pharmaceutical composition of the present invention can be separated from soybean by conventional methods. For example, 1 to 30 times acetone as the extraction solvent, preferably 5 to 15 times the volume of 10 to 50 ℃, preferably cold extraction for 0.5 to 48 hours, preferably 20 to 30 hours at room temperature, Extraction methods such as hot water extraction, ultrasonic extraction, reflux cooling extraction, etc., preferably by cold extraction, or 1 to 30 times, preferably 5 to 15 times the volume of alcohols including methanol or ethanol as the extraction solvent. Extraction solution after extraction by extraction method such as 0.5-10 hours, preferably 1-5 hours deposition, cold sewage extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction at 4 ~ 80 ℃, preferably 30 ~ 80 ℃ extraction temperature The lutein may be separated from the soybean by centrifugation, filtration and concentration.

Specifically, an example performed to separate lutein from soybean in the present invention is as follows.

First, after germinating the seeds of soybean, drying and grinding the germinated soybeans, immersing them in an organic solvent, and concentrating the filtrate under reduced pressure to obtain a germinated soybean extract, and then separating and purifying lutein from the germinated soybean extract. Is done.

Germination of soybeans is accomplished by immersing the seeds of soybean in water and then intermittently spraying them in a cancerous state where room temperature and humidity are maintained. It is preferable that the immersion is carried out in water at a temperature of 18 to 25 DEG C for 4 to 12 hours for smooth germination. After sufficient immersion, germination is performed at room temperature and high humidity, that is, in a dark state in which relative humidity is maintained at 50 to 100% at room temperature. The soybean can be applied to any of the seeds of the plant belonging to the legume.

The intermittent sprinkling can be made by sprinkling for 1 to 10 minutes at intervals of 4 to 6 hours, and cultivation is convenient when using a device equipped with a timer. Germination is 20 days from the cotyledon and hypocotyl separation time, preferably 4 to 15 days.

Then, the germinated beans are dried, pulverized, immersed in an organic solvent, and the filtrate is filtered under reduced pressure to obtain a germinated bean extract.

The germinated soybeans include cotyledons and hypocotyls, and the germinated soybeans can be used from the time point at which the cotyledons and hypocotyls are separated after germination. Preferably, lutein is used for 2-20 days after germination. Preferred in terms of obtaining.

Lutein has 40 carbons and 56 hydrogens, but it shows fat solubility due to its structural features including only a few hydroxyl groups (OH). Therefore, since it is not dissolved at all in a completely polar solvent such as water, it is preferable to use a polar or non-polar organic solvent as the extraction solvent.

As the polar or non-polar organic solvent, lower alcohols having 2 to 5 carbon atoms, acetone, acetonitrile, ethyl acetate, chloroform, dichloromethane, ethyl ether, xylene, hexane and the like may be used.

The fat-soluble lutein is relatively well soluble in polar organic solvents having a polarity of 5 to 6, such as alcohol or acetonitrile, but these solvents have excellent solubility in various polar substances including sugars. In the case of using a polar organic solvent, there is a tendency to comprehensively extract other polar substances including lutein, sugars, flavonoids, glycosides, and proteins.

In addition, the use of organic solvents such as ethyl acetate, chloroform, butanol, propanol, dichloromethane, ethyl ether, xylene and hexane, which show relatively meso or non-polar characteristics, can effectively extract fat-soluble lutein, but lower polarity than lutein. The solubility of substances having lower polarity than lutein, such as chlorophyll compounds, pheophitin derivatives, carotene compounds including alpha and beta-carotene, lipids, etc., tends to be extremely increased, leading to greater incorporation of nonpolar substances.

On the other hand, acetone shows a level of 5.1 similar to alcohols in terms of polarity, but sugar, glycoside, and protein components present in a large amount in plants are extremely poor in solubility in acetone, and thus acetone is not easily extracted when used as a solvent. In addition, even if a small amount of extraction, cold soaking is extremely low solubility is precipitated in the form of precipitates can be removed by filtration has the advantage of extracting a relatively high purity lutein.

The solvent may be used at a weight of 5 to 20 times the dry powder weight of the germinated soybean. The extraction may be carried out by various methods such as low temperature dark condition extraction, room temperature extraction, and ultrasonic extraction.

The germinated soybean extract from which the acetone was removed was concentrated, followed by column chromatography to obtain a fraction of lutein, each fraction was concentrated to dryness and redissolved, and then purified and purified by preparative HPLC to separate lutein from soybean. have.

On the other hand, the present invention was able to obtain a free form of active lutein in the form of lutein activity immediately in the body by a simple method by the extraction operation using a solvent as described above.

In the present invention has been described in the present invention a method for ideally obtaining lutein from soybean, it will be apparent that the present invention is not limited to the method for separating lutein. Lutein included in the anticancer composition of the present invention can be separated and obtained by a method for separating lutein from plants to be improved and developed in the past or in the future.

In the present invention, the scavenging activity and reducing power assay for active oxygen species such as DPPH radical scavenging activity, superoxide anion scavenging activity, and hydroxy radical scavenging activity by applying ESR (Electron Spin Resonance) and chemical method to lutein isolated from soybean The inhibition of cancer cell proliferation by MTT assay and Western blotting of prostate cancer cell lines were examined.

The present invention provides a pharmaceutical composition having an anticancer and antioxidant activity comprising lutein isolated from soybean as an active ingredient, wherein the lutein is preferably included in the composition in an amount of 0.01 to 80% by weight, more preferably. Preferably 0.01 to 50% by weight. If the content is less than 0.01% by weight, the anticancer and antioxidant effects of the pharmaceutical composition may be insignificant, and if the content exceeds 80% by weight, the anticancer and antioxidant effects may be relatively low.

Pharmaceutical compositions comprising lutein isolated from the soybean of the present invention as an active ingredient can be administered orally or parenterally. The route of administration of the pharmaceutical compositions of the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, gingiva, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, , Sublingually or topically.

For such clinical administration, the pharmaceutical compositions of the present invention can be formulated into suitable formulations such as oral administration, parenteral administration, injection, etc. using known techniques. For example, upon oral administration, it may be admixed with an inert diluent or edible carrier, sealed in a hard or soft gelatin capsule, or pressed into tablets. For oral administration, the active compound may be formulated as an ingestible tablet, buccal tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, suspensions, Hard or soft capsules, elixirs, suspensions, syrups, wafers, and the like. At this time, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin for formulation into tablets, capsules and the like; Excipients such as dicalcium phosphate; Disintegrants such as corn starch or sweet potato starch; Magnesium stearate, calcium stearate, sodium stearyl fumarate, or a lubricant such as polyethylene glycol wax may be mixed. In the case of a capsule formulation, a liquid carrier such as fatty oil may be mixed in addition to the above-mentioned substances.

Parenteral administration is also applied to subcutaneous injections, intravenous injections, intramuscular injections, intra-thoracic injection injections, mucous membranes, or topical application, including dispersing agents, suppositories, powders, aerosols (nasal sprays or inhalants), gels, , Or a non-aqueous liquid suspension, an oil-in-water emulsion or a water-in-oil emulsion), a solution, and the like. For formulation into a parenteral dosage form, the composition may be formulated into a solution by mixing with a stabilizer or buffer in water to form a unit dosage form of ampoule or vial.

Effective dosage of the pharmaceutical composition of the present invention as described above may be adjusted to the necessary range in accordance with the clinical judgment in consideration of the age, physical conditions, weight, etc. of the patient. In general, the effective dosage of the pharmaceutical composition is 1 to 20 mg / day per 1 kg of adult patient weight, preferably 5 to 10 mg / day, several times a day at regular time intervals according to the judgment of the doctor or pharmacist, Preferably it may be administered in divided doses 2 to 5 times a day.

In addition, the present invention provides a dietary supplement for anticancer and antioxidant effect enhancement comprising lutein isolated from soybean as an active ingredient, the dietary supplement of the present invention includes a lutein separated from a widely edible soybean as an active ingredient. Because it is safe for human body, it is suitable for adding to food.

The lutein is preferably contained in 0.01 to 50% by weight, more preferably 0.01 to 30% by weight in the dietary supplement for improving anticancer and antioxidant effects. If the content is less than 0.01% by weight, the anticancer and antioxidant enhancement effects of the dietary supplement may be insignificant. If the content exceeds 50% by weight, the anticancer and antioxidant enhancement effects may be relatively low compared to the concentration.

The type of health supplement is not limited, but, for example, in addition to the usual palatability food, that is, noodles, tofu, cereals, breads, chewing gum, candy, confectionary, etc. It can manufacture by various foods, and can also apply as an edible pigment. In addition, it can be formulated into a common formulation such as tablets, granules, pills, hard capsules, soft capsules or liquid formulations, and can be prepared as a juice, a pouch, a drink, or the like. It goes without saying that components other than the above-mentioned components can be appropriately selected and blended by those skilled in the art according to the formulations.

In addition, lutein contained in the dietary supplement of the present invention is excellent in antioxidant activity, it can effectively prevent the smell or flavor change of the food caused by oxidation, rancidity of fat and oil, and discoloration of food. Therefore, the lutein separated from the soybean of the present invention can be used to preserve these foods or to maintain the freshness and quality of foods for a long time by blending them with ordinary foods.

Such foods include not only typical foods but also beverages (including alcoholic beverages), fruit and processed foods thereof (e.g., canned fruits, jars, jam, maare marlade, etc.), fish, meat and processed foods Ham, sausage, corn oil, etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, Vegetable protein, retort food, frozen food, various seasonings (e.g., soybean paste, soy sauce, soy sauce, etc.), and the like.

In addition, the present invention provides a cosmetic composition having an antioxidant activity comprising lutein isolated from soybean as an active ingredient.

The lutein is preferably contained in an amount of 0.01 to 40% by weight, more preferably 0.01 to 20% by weight in the cosmetic composition having an antioxidant activity. When the content is less than 0.01% by weight, the antioxidant activity may be insignificant, and when the content exceeds 40% by weight, the antioxidant effect may be relatively low compared to the input concentration.

The cosmetic composition of the present invention can be used as it is, or diluted as necessary. The lutein can be prepared in liquid or solid form using bases, adjuvants and additives commonly used in the cosmetic field. Cosmetics in liquid or solid form may include, but are not limited to, for example, cosmetics, creams, lotions, bathing agents, and the like.

Bases, adjuvants and additives commonly used in the cosmetic field are not particularly limited and include, for example, water, alcohols, propylene glycol, stearic acid, glycerol, cetyl alcohol, liquid paraffin and the like.

The cosmetic composition of the present invention as described above is very useful for preventing damage caused by oxidation of the skin, for example, spots (brown spots), freckles, skin cracks, UV damage (sunburn), etc. It can be very useful to maintain the quality of cosmetics by preventing the.

Hereinafter, the present invention will be described in more detail with reference to examples. These embodiments are for purposes of illustration only and are not intended to limit the scope of protection of the present invention.

Example 1. Preparation of soybean (germinated bean, bean sprouts) extract

Poongsan herb 500 g of soybeans were placed in a plastic cultivation vessel of 6 cm (width) × 6 cm (length) × 14 cm (height), soaked in water at 20 ° C. for 4 hours, HB-301L, Seoul) for 10 days at a temperature of 20 ° C and a humidity of 80%. During soybean germination, water was sprinkled on the stomach for 3 minutes every 4 hours using an automatic watering device equipped with a timer.

The cotyledons of the germinated sprouts (bean sprouts) were taken, frozen at −80 ° C., lyophilized and ground to 60 mesh.

Acetone 1L was added to 100 g of the crushed soybeans, and extracted for 30 minutes in an ultrasonic extractor at 40 ° C., and extracted three times in the same manner. The extracted solutions were added together to form Whatman No. It was filtered using 2 filter paper and left for 24 hours at a low temperature of -20 ° C to refilter insoluble precipitate. The filtered extract was concentrated in a vacuum concentrator at 40 ℃ to obtain a yellow soybean extract from which the acetone solvent was removed.

Example 2. Isolation and Identification of Lutein from Soybeans (Sprouted Beans, Sprouts)

Lutein was isolated from the soybean extract of Example 1 using preparative HPLC (Agillent 1200 series, USA). At this time, the column was separated by preparative HPLC using HiQ sil C18-10 (21.0 × 250mm, KYA TECH, Japan), analytical wavelength 430 nm, flow rate 10ml / min, solvent A as solvent, 75% methanol, For solvent B, ethyl acetate was used as the concentration gradient (0 min: 70% solvent A, 25 min: 15% solvent A, 26 min: 70% solvent A, 35 min: 70% solvent A), and the sample injection amount was 2 ml. High purity separation was carried out by adjusting to.

NMR and UV-VIS spectroscopy and mass spectrometry were performed on lutein among several yellow and green compounds obtained by separation, and chemical shift of lutein's ¹ H-NMR (300MHz, CDCl ₃ ) result The value analysis results are shown in Table 1, and the ¹ H-NMR spectrum is shown in FIG.

Figure ^{1 H-NMR (300MHz, CDCl} 3) shown in Figure 1 Referring to the spectral measurement result, ¹ H- ¹ H correlation between Me-18 '(1.63ppm) and H-3' when viewing the (correlation) (4.25ppm) Assuming a correlation with the proton H-2 '(eq, 1.86 ppm and ax, 1.38 ppm) protons in this alicyclic compound region, a peak of 5.55 ppm could be determined as H-4' and H-7 '(5.44ppm) is H-8' could be identified by (6.15ppm) and ^{H-6 '(2.42ppm) 1} H- 1 H -related signals of the peak (correlation signal). In addition, protons of two secondary hydroxy groups corresponding to H-3 and H-3 'were found in the 4.00 ppm and 4.25 ppm regions, respectively, and H-16 / H-2ax by 1D selective NOESY. viewed as a corresponding to the H-16 / H-17, H-18 / H-17 peak indicating the correlation _{CH 3 -16, CH 3 -18,} H-2ax, H-3 are both present in the same ring I could see. The NOESY correlations of the H-19 / H-7, H-19 / H-10 and H-20 / H-14 peaks were also able to identify CH ₃ bound to the hydrocarbon group. 19 'and H-20' were identified. The proton peak of the olefin group was found to be between 5 and 7 ppm. In particular, the multiplet signal up to 6.65 ppm was H-15 / H-15 '(6.65 ppm) and H-11 / H-. 11 '(6.63ppm), H-7 / H-7' (6.44ppm), H-12 / H-12 '(6.36ppm), H-14 / H-14' (6.25ppm), H-8 / Peaks were identified by pairs of H-8 ′ (6.12 ppm) protons.

On the other hand, protons of H-2 (1.48ppm and 1.79ppm) and H-2 '(1.38ppm and 1.86ppm) were identified in different regions from protons of other olefin groups. It was found around 1.3-1.8 ppm because it paired with the surrounding methyl group instead of pairing with the protons of the group. Other methyl groups (CH ₃ , H-16-H-20, H-16'-H-20 ') were identified in the vicinity of 1-2 ppm, which is a region where a typical methyl peak appears.

Mass spectrometry and UV-VIS. Analysis of the spectrum showed m / z = 569.3, and UV-VIS. Absorbance spectra showed λ (nm) = 426, 448, 476.

More than ¹ H-NMR, MS and UV-VIS. As a result of comprehensive analysis of the spectrum, the isolate was yellow and was identified as ( all-trans ) -lutein with the formula C ₄₀ H ₅₆ O ₂ = 569.3.

Position 隆_H Position 隆_H One One' 2 ax 1.48 (m) 2' ax 1.38 (dd, 13, 7) eq 1.79 (m) eq 1.86 (m) 3 4.00 (m) 3 ' 4.25 (m, br) 4 ax 20.5 (m) 4' 5.55 (m, br) eq 2.38 (m) 5 5 ' 6 6 ' 2.42 (d, 9.5) 7 6.12 ov ^a 7 ' 5.44 (dd, 15.5, 10.1) 8 6.12 ov ^a 8' 6.15 ov ^a 9 9 ' 10 6.17 ov ^a 10 ' 6.14 ov ^a 11 6.63 (dd, 14.3, 11.4) 11 ' 6.61 (dd, 15.0, 11.4) 12 6.36 (d, 14.7) 12 ' 6.36 (d, 14.7) 13 13 ' 14 6.25 (m) 14 ' 6.25 (m) 15 6.65 (m) 15 ' 6.65 (m) 16 1.07 (s) 16 ' 1.00 (s) 17 1.07 (s) 17 ' 0.85 (s) 18 1.74 (s) 18 ' 1.63 (s) 19 1.97 (s) 19 ' 1.91 (s) 20 1.97 (s) 20 ' 1.97 (s)

Experimental Example  3. Lutein Isolated from Soybeans DPPH Radical  Scavenging activity

The DPPH radical scavenging activity assay of lutein isolated from soybean in Example 2 was assayed by the method by Schimada et al. (1992). 1 ml of 0.5 mM DPPH ethanol solution and 2 ml of 0.1 M acetate buffer were mixed and treated with concentration-specific (10-50 μg / ml) isolated lutein. Subsequently, after standing in a dark state at room temperature for 30 minutes, DPPH radical scavenging activity was assayed by measuring absorbance at 517 nm using a UV-1200 UV / VIS spectrometer (spectrometer, Shimadzu, Kyoto, Japan). The results are shown in FIG. At this time, 10-50 μg / ml α-tocopherol (VE) was used as a positive control.

As shown in FIG. 2, lutein isolated from soybean showed 79% DPPH radical scavenging activity at 20 μg / ml, whereas the same level of inhibition was about 40 μg in the case of α-tocopherol used as a positive control. Observed at / ml. The DPPH radical scavenging activity of lutein is thought to be due to the double bonds that are alternately structurally linked to the hydrophobic properties of lutein itself.

Experimental Example 4. Comparison of Reducing Power of Lutein Isolated from Soybean

In Example 2, the reducing power of lutein isolated from soybean was measured according to the method of Oyaizu (1986), and the reducing power of iron ions to the antioxidant material was measured. 1 ml of 0.2 M sodium phosphate buffer (pH 6.6), 1 ml of concentration-specific sample (10-100 µg / ml), 1 ml of 1% potassium ferricyanide, and the mixture was After reacting for 20 minutes at 1 ° C., 1 ml of 10% TCA (triobarbituric acid) was added. 2 ml of methanol was added to 2 ml of the supernatant obtained by centrifuging the reaction mixture at 13,000 × g for 5 minutes, and 0.1 ml of 0.1% iron chloride was added. The absorbance was measured at 700 nm using a UV / VIS spectrophometer. It was. In this case, BHA was used as a positive control, and the elimination rate was calculated by the following Equation 1, and the results are shown in FIG. 3.

[Equation 1]

(%) = [1- (A ₀ / A ₁ )] × 100 (A ₀ = blank, A ₁ = extract)

As shown in FIG. 3, the lutein isolated from the soybean showed a reducing power of about 89% at 20 µg / ml, which was significantly higher than that of BHA, a positive control, and 90% even at a concentration of 20 µg / ml or more. It was confirmed that the level of reducing power tends to be kept constant. On the other hand, BHA, a positive control, showed about 8% reducing power at 20µg / ml, increased slightly to 28% up to 60µg / ml, and reduced 48% at 80µg / ml and 86% at 100µg / ml. In terms of reducing power, BHA 100 μg / ml treatment level was similar to the level of lutein 20 μg / ml treatment.

The reducing power of lutein is thought to be due to the hydrogen donating ability. Through these results, lutein not only acts as an electron donor, but also reacts with radicals to transform into a stable compound, thus exhibiting the function of terminating the radical chain reaction. I could see that.

Experimental Example 5 Assay of Superoxide Anion Scavenging Activity of Lutein Isolated from Soybean

The superoxide anion scavenging activity of the lutein superoxide anion scavenged from soybean in Example 2 was rapidly reacted with the superoxide anion generated by the reaction of xanthine and xanthine oxidase and nitrospin. It was measured using DMPO (5,5-dimethyl-1-pyrroline-N-oxide) which is a trap (nitrone spin trap).

That is, 20 μl, 3M DMPO for each sample of lutein, ascorbic acid and β-carotene concentrations (10, 20, 30, 40 μM) isolated from soybeans in 120 μl of 0.1 M phosphate buffer (pH 7.4). Add 20 µl, 20 µm 10 µm xanthine and 20 µl 0.25U xanthine oxidase, mix to a total volume of 200 µl, leave for 2.5 minutes at room temperature, transfer to a quartz capillary tube and transfer to an ESR spectrometer (JES- FA ESR spectrometer, JEOL, Tokyo, Japan). A blank was used to add 20 µl of acetone instead of a sample. The magnetic field of the ESR was 336.5 mT, the power was 20 mW, the frequency was 9.8 GHz, and the amplitude modulation was 1.0 gauss, gain 200, scan time 0.5 minutes, scan width 10 mT, time constant 0.03 seconds, temperature 25 It measured by fixing to ° C. Calculation of superoxide anion scavenging activity for the sample was calculated according to the following Equation 2 using the difference in the average height of the signal intensity (signal intensity) of the treatment and control, and the results are shown in FIG.

&Quot; (2) &quot;

[(A _blank -A _sample ) / A _blank ] × 100%

The superoxide anion scavenging activity of lutein isolated from soybean was assayed by ESR spectroscopy and compared with β-carotene and ascorbic acid. As shown in FIG. 4, the superoxide anion scavenging activity was positive control β-carotene. Lutein isolated from soybean than in or ascorbic acid was highest in all concentrations. Lutein showed 38% superoxide anion scavenging activity at 40 μM, and ascorbic acid showed 34.5% and β-carotene at 11.6% superoxide anion scavenging activity. For the superoxide anion ESR signal intensity of the control group without antioxidant treatment, the reduction rate was about 69% when lutein was added, about 42% for ascorbic acid and 10% for β-carotene. .

These results indicate that the superoxide anion scavenging activity of lutein is superior to ascorbic acid at the same concentration and about 5 times higher than β-carotene.

Meanwhile, superoxide radical scavenging activity of lutein isolated from soybean using ESR was assayed using nitro-blue tetrazolium (NBT) reduction method (Nagai et al. , 2005). That is, 0.02 ml of 3 mM xanthine, 0.48 ml of 0.05 mM sodium carbonate buffer (pH 10.5), 0.02 ml of 3 mM ethylenediaminetetraacetic acid disodium salt (EDTA) and 0.02 ml of 0.15% bovine serum albumin (0.75 mM). Lutein isolated from soybean was treated at a concentration of 10, 20, 40, 60 µg / ml in a mixed solution composed of 0.02 ml of NBT. The mixture was incubated at 25 ° C. for 10 minutes, and then 1.0 mL of XOD (6 mU / mL) was added to start the reaction, followed by reaction at 25 ° C. for 20 minutes. Thereafter, 0.02 ml of 6 mM CuCl was added to terminate the reaction, and the absorbance was measured at 560 nm. The results are shown in FIG. 5. 5 shows the inhibition rate for NBT reduction according to the amount of sample added by concentration, and compared using α-tocopherol as a positive control.

The superoxide radical scavenging activity of lutein isolated from soybean using ESR was confirmed by nitro-blue tetrazolium (NBT) reduction. As shown in FIG. 5, lutein isolated from soybean was about 87% scavenged at 20 µg / ml. Activity was shown, and it was confirmed that the results were kept constant at higher concentrations. On the other hand, in the case of α-tocopherol, the superoxide radical scavenging rate when treated with 60 µg / ml was similar to that of 20 µg / ml treated with lutein. It was confirmed that it has about three times more activity than tocopherol.

These results showed that lutein had higher scavenging activity of superoxide radicals than any antioxidant at the same concentration.

Experimental Example 6 Hydroxy radical scavenging activity assay of lutein isolated from soybean

The inhibitory activity of the production of hydroxy radicals using ESR was assayed for hydroxy radical scavenging activity assay for each concentration of lutein isolated from soybean in Example 2. Hydrogen radical generation was performed using a fenton reaction (H ₂ O ₂ + FeSO ₄ ), and DMPO capable of rapidly reacting with hydroxy radicals was used. 20 μl and 0.3 M DMPO 0.2 samples of lutein, β-carotene and ascorbic acid (10, 20, 30 and 40 μM) isolated from soybean in 0.1 M phosphate buffer (pH 7.4), respectively ㎖, 0.2 ml of 10 mM FeSO _{4 and} 0.2 ml of 10 mM H ₂ O ₂ were added and mixed. The mixture was allowed to stand at room temperature for 2.5 minutes and then transferred to a quartz capillary tube. The ESR spectrometer (JES-FA ESR spectrometer, JEOL, Tokyo, Japan). A control was used to add 20 µl of acetone instead of the sample. The magnetic field of the ESR was 336.5 mT, the power was 20 mW, the frequency was 9.8 GHz, the amplitude modulation was 1.0 gauss, the gain was 200, and the scan time was 0.5 minutes. The scan width was 10mT, the time constant was 0.03 seconds and the temperature was fixed at 25 ° C. Hydroxy radical scavenging activity calculation for the sample was calculated and tested according to the following equation 3 using the difference in the average height of the signal intensity of the treatment and control, and the results are shown in FIG.

&Quot; (3) &quot;

[(A _control -A _blank )-(A _blank -A _sample ) / A _control -A _blank ] × 100%

Hydroxy radicals are known to be most reactive with all reduced forms of oxygen and are known to be the most toxic radicals that cause early damage to cells. As a result of assaying hydroxy radical scavenging activity of lutein, β-carotene and ascorbic acid concentration samples isolated from soybeans through ESR, DMPO-OH signal was obtained when lutein was treated at 10 μM level as shown in FIG. While the inhibition rate of 18.6% was shown, ascorbic acid and β-carotene showed the inhibition rate of less than about 2.5% at the same concentration. In addition, as the concentration increased, the hydroxy radical generation inhibition rate of lutein was 58.9% at 40μM, while β-carotene was 10.6% at the same concentration, and ascorbic acid was about 8.5%. there was.

From these results, lutein isolated from soybean showed a hydroxy radical scavenging activity significantly better than β-carotene or ascorbic acid.

Meanwhile, in order to confirm the results of the hydroxy radical scavenging activity of lutein isolated from soybean by ESR, 2-deoxyribose by Fenton reaction was oxidized by hydroxy radical and malon. It was confirmed by measuring the degree of conversion to aldehyde (malonaldehyde) to form chromagen (chromagen). To a solution containing 2.8 mM 2-deoxyribose, 100 μM FeCl ₃ , 104 μM EDTA, 1 mM H ₂ O ₂ was added 20 mM sodium phosphate buffer (pH 7.4) to prepare 1 ml of the mixture. The lutein concentration sample was placed in an Eppendorf tube and reacted at 37 ° C. for 1 hour in a dark state. Then, 0.75 ml of 1.0% (w / v) TBA was added to terminate the reaction. The reaction solution was treated with water at 98 ° C. for 20 minutes, cooled at room temperature, and stabilized by adding 1 ml of acetone. The reaction solution was 535 using a UV-1200 UV / VIS spectrometer (Shimadzu, Kyoto, Japan). Absorbance at nm was measured and the results are shown in FIG. 7. The hydroxy radical scavenging activity assay was calculated at the rate of inhibiting the oxidation of 2-deoxyribose by the hydroxy radical with the addition of lutein isolated from soybean, and the existing antioxidants α-tocopherol (VE) and BHA were determined. Comparison was made with the control.

In order to confirm the hydroxy radical scavenging activity of lutein isolated from soybean by ESR, the hydroxy radical scavenging activity of lutein was confirmed by Fenton reaction. As shown in FIG. Inhibition rate of 79.6% in ㎖ (concentration of about 10 μM), while similar control hydroxy radicals at 90 μg / ml (concentration of about 400 μM) and α-tocopherol in 180 μg / ml (concentration of about 400 μM) It was confirmed that the scavenging activity. In addition, it was confirmed that lutein showed 18-fold hydroxy radical scavenging activity for BHA and 36-fold for α-tocopherol in view of the treatment concentration necessary to exhibit the same level of antioxidant efficacy.

In general, hydroxy radicals are derived from H ₂ O ₂ and are often produced by plants under severe stress and are most reactive and toxic in the mechanism of formation of reactive oxygen species and in the reaction of these reactive oxygen species with other reduced forms of molecules. This is known to be strong (Foyer et al., 1994; Asada, 2000). Lutein isolated from soybeans in scavenging and elimination of reactive oxygen species has a relatively higher scavenging activity against hydroxy radicals than other antioxidants, and it is an antioxidant that can be used to treat lutein and various diseases caused by oxidative damage. The prophylactic effect is very high.

Experimental Example 7 Assay of Cancer Cell Proliferation Inhibition of Lutein Isolated from Soybean

In order to assay the cell growth inhibition rate of cancer cells (cnacer cell line) of lutein isolated from soybean in Example 2, human lung cancer cell line A549, kidney cancer cell line ACHN, prostate cancer cell line LNCaP, colon cancer cell line HCT15, breast cancer cell line MCF Lutein isolated from soybean was treated at -7 concentrations (50, 100, 200 µg / ml), and cell viability was assayed through MTT analysis. After incubating each cancer cell line in a 96 well plate (1 × 10 ⁴ / well), the samples were treated by concentration and further incubated for 36 hours. Cell viability was assayed using a commercially available measurement kit (Cell Titer 96 non-radioactive cell proliferation assay kit, Promega, Madison, Wis.), And tetrazolium compound MTS (3- (4,5-dimethylthiazol-2-). 20 μl of a mixture containing yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-PMS (tetrazolium, inner salt) and PMS (electron coupling reagent phenazine methosulfate) is added to each well and again After incubation at 5% CO ₂ for 1 hour at 37 ° C., the absorbance was measured at 490 nm using an enzyme-linked immunosorbent assay plate reader (ELISA), and the results are shown in Table 2 below.

Concentration
(Μg / ml) Viable Cell (% of Control) / Human Cancer Cell line ^* A549 ACHN HCT15 LNCaP MCF-7 200 22.0 ± 1.0 28.0 ± 3.0 15.0 ± 3.0 28.0 ± 1.0 19.0 ± 2.0 100 35.0 ± 2.0 37.0 ± 2.0 26.0 ± 2.0 38.0 ± 2.0 28.0 ± 2.0 50 46.0 ± 3.0 51.0 ± 2.0 38.0 ± 2.0 57.0 ± 2.0 39.0 ± 2.0

* After incubation with different concentrations of camellia extracts and isolated pigments at 37 ° C for 36h, the effect on cell growth was examined by MTT assay. The percentage of viable cells was compared with that of the vehicle-only control. This experiment was repeated three times. Result shown as mean ± S.D. (N = 3)

To test the growth and proliferation inhibitory effects of lutein isolated from soybean on human cancer cell lines, lung cancer cell line (A549), kidney cancer cell line (ACHN), prostate cancer cell line (LNCaP), colon cancer cell line (HCT15) and breast cancer cell line (MCF) -7) As a result of performing the MTT assay, as shown in Table 2, the lutein isolated from the soybeans, the survival rate of less than about 50% compared to the control at the level treated with 50㎍ / ㎖ for all cancer cell lines It was confirmed that the highest cytotoxicity was shown. In addition, it was confirmed that the cancer cell proliferation inhibitory effect in a concentration-dependent manner, in particular, it was confirmed that higher activity for the prostate cancer cell line (LNCaP) and breast cancer cell line (MCF-7).

Experimental Example 8. Western blot assay for prostate cancer cell proliferation inhibitory activity of lutein isolated from soybean

Isolation from soybeans against LNCaP cancer cell lines to identify cancer cell proliferation inhibitory mechanisms of lutein which have specificity for prostate cancer cell line (LNCaP) in the assay of growth and proliferation inhibitory effect on human cancer cell line and showed high cancer cell proliferation inhibitory activity One lutein was treated by concentration and Western blot was performed to assay the expression of PCNA (Proliferating cell nuclear antigen) and CyclinD ₁ expression in cancer cell lines, and the results are shown in FIGS. 8 and 9. Respectively.

PCNA is a coprotein of DNA polymerase σ, which is a key protein that regulates the cell cycle or promotes cell proliferation or differentiation. In normal cellular state, its expression is normal, but its expression is increased in the pre-cancerous stage or malignant, and when the expression of PCNA is increased, metaplasia of general mucosal tissues or squamous epithelial cells occurs. Squamous cells cause dysplasia, forming cancer. CyclinD ₁ is an important nuclear protein that acts between the G1 / S phases of the cell cycle. CyclinD ₁ overexpression and gene amplification are observed in many cancer patients during carcinogenesis. PCNA / Cyclin also regulates early cell proliferation via DNA polymerase and increases the rate of G1 / S phase in the cell cycle.

To determine the expression of genes related to cell proliferation in LNCaP prostate cancer cell lines, LNCaP cell lines were 1.5 cell density for 1.5 hours in DMEM F-12 medium containing 2.5% charcoal-stripped FBS. After culturing to 10 ⁵ cells / ml, lutein isolated from DMSO and soybeans was added at 0, 10, 20, 50, and 100 μM levels and incubated for 24 hours. As a control, β-carotene and lycopene were added at the same concentration and cultured. Radioimmunoprotein assay (RIPA) buffer (50 mM Tris-HCl pH 7.2, 150 mM NaCl, 1%) containing 1% proteinase inhibitor cocktail (Sigma-Aldrich) to extract proteins from each cultured cell After adding IGEPAL CA-630, 0.05% SDS and 1% sodium deoxycholate (Ohnishi et al., 1996), the mixture was centrifuged (12,000 × g, 10 minutes at 4 ° C.) Repeated twice: Protein quantification was performed by Lowry et al. (1951).

Protein electrophoresis was performed on a 12.5% polyacrylamide gel, and 50 μg per lane was introduced in immunoblotting of the protein. SDS-PAGE was developed for 3-4 hours at 120V and transferred to a 0.2 μm PVDF membrane (Polyvinylidene difluoride, BioRad, CA). Each membrane was blocked for 30 minutes in TBST buffer containing 5% milk (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) and then the primary antibody in TBST buffer containing 5% milk. (primary antibody) was added, incubated at 4 ° C. for one day, and secondary antibody was added to TBST buffer containing 5% milk and incubated for 90 minutes. PCNA protein was used as a primary antibody by diluting anti-PCNA human monoclone (IgG _2α , Santa Cruz Biotechnology, USA) at 1: 2000, and cyclinD1 protein was anti-cyclinD ₁ human monoclone (IgG1, Santa Cruz Biotechnology, USA) Was diluted 1: 500, and anti-β-actin human clone (IgG _1k , Santa Cruz Biotechnology, USA) was diluted 1: 1000, and each membrane was a secondary antibody, goat anti-mouse IgG. -HRP (200 μg / 1 mL, Santa Cruz, USA) was used diluted 1: 1000. In order to identify the immunoreactive bands, peroxidase BMB (peroxidase BMB, BM blue POD substrate precipitating, Roche, Germany) was added and a densitometer (densitometer, GC-710, Bio-Rad, USA) ) Was observed.

As shown in Fig. 8 and 9, Western blotting confirmed the ratio of PCNA expression inhibition between lutein, lycopene, and β-carotene isolated from soybean, and visually confirmed that the inhibition of PCNA expression with increasing concentration of lycopene However, there was no significant difference between lutein and β-carotene. On the other hand, as a result of analysis using a densitometer, lycopene statistically inhibited PCNA expression in LNCaP cancer cell lines at a high concentration of 20-100 μM, but PCNA at 50 μM or more in lutein and β-carotene. Expression inhibition was not large, 2 ~ 19% level, showed a statistically insignificant results and did not show a concentration-dependent trend (Fig. 8). On the other hand, lutein and β-carotene inhibited the expression of cyclinD ₁ in a concentration-dependent manner, and at a high concentration of 100 μM, lutein exhibited very high inhibition rate of 81% and β-carotene 72%, and lutein was higher than β-carotene. Although excellent expression inhibition was shown, lycopene did not inhibit the expression of cyclinD ₁ (FIG. 9).

In phenotypic observation of tumorigenesis, cell-to-cell interactions through gap junctions are very important, as they become communication pathways between cell membranes for intercellular signaling (Kumar and Gilula, 1996). It is believed that the inhibitory activity of cancer cell proliferation inhibitors may be closely related to the rate and rate of absorption into the cells.

Lutein is a type of xnathophyll, each of which has a hydroxyl group on each of the two terminal β-ion rings having eight conjugated carbon double bonds with a closed ring at the end of each structure. hydroxy group), which allows lutein to exhibit more hydrophilic properties than other hydrocarbon carotenoids such as α-carotene, β-carotene, and lycopene. The property is reported to make it easier to react with singlet oxygen from water than other nonpolar carotenoids (Ojima et al., 1993). Lycopene is a nonpolar carotenoid, which is attached to the cell membrane in parallel with the hydrophobic region of the lipid bilayer, making it difficult to introduce into the cell (Volk et al., 1984), but the hydroxy group possessed by lutein is hydrophilic. Compared with reports that it is attached perpendicular to the cell membrane surface and can be easily introduced into the cell (Sundqvis et al., 1989), lutein is associated with cancer cell proliferation and rapid growth at lower concentrations than lycopene or β-carotene. Inhibiting the expression of related cyclinD ₁ shows higher hydrophilicity than other carotenoids due to the hydroxy group of lutein, which makes it easier to access the cell membrane and to penetrate the cell membrane into the cell. It is judged because

In addition, in the present invention, the treatment of lycopene having hydrophobic properties significantly decreases the expression of PCNA, but the expression of cyclinD ₁ is less likely to be decreased, and when treating lutein having hydrophilic properties among carotenoids, the PCNA expression is decreased. The results showed that cyclinD ₁ expression was not significantly affected, but carotenoids with different polarities were thought to exhibit alternative activities and mechanisms of action in the proliferation of cancer cells and the expression of related genes.

Although the present invention has been described as the preferred embodiment mentioned above, it is possible to make various modifications or variations without departing from the spirit and scope of the invention. The appended claims also cover such modifications and variations as fall within the spirit of the invention.

Claims (10)

Pharmaceutical composition having anticancer and antioxidant activity, comprising lutein isolated from soybean as an active ingredient. The method of claim 1,
The lutein is a pharmaceutical composition having anticancer and antioxidant activity, characterized in that contained in 0.01 to 80% by weight in the pharmaceutical composition. The method of claim 1,
The lutein is a pharmaceutical composition having anticancer and antioxidant activity, characterized in that isolated from soybeans, germinated beans or sprouts. The method of claim 1,
The lutein is a step of germinating the seeds of the beans, drying and grinding the germinated beans, soaked in an organic solvent and concentrated under reduced pressure to obtain a germinated soybean extract under reduced pressure, and separating and purifying lutein from the germinated soybean extract. Pharmaceutical composition having anticancer and antioxidant activity, characterized in that it is separated into. The method of claim 1,
The pharmaceutical composition has anticancer and antioxidant activity, characterized in that it exhibits anticancer activity against any one or more selected from prostate cancer, lung cancer, kidney cancer, colon cancer and breast cancer. The method of claim 1,
The pharmaceutical composition is a pharmaceutical composition having anticancer and antioxidant activity, characterized in that it exhibits anticancer activity by inhibiting the expression of cyclinD ₁ against cancer cell lines. Anti-cancer and antioxidant effect enhancement health food comprising lutein isolated from soybeans as an active ingredient. The method of claim 1,
The lutein is a dietary supplement for enhancing cancer and antioxidant effects, characterized in that contained in the dietary supplement 0.01 to 50% by weight. Cosmetic composition having antioxidant activity comprising lutein isolated from soybeans as an active ingredient. 10. The method of claim 9,
The lutein is a cosmetic composition having an antioxidant activity, characterized in that contained in the cosmetic composition 0.01 to 40% by weight.

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