KR20010035299A - Raw-diet composition for anticancer - Google Patents

Abstract

PURPOSE: A cancer-preventive raw grain and vegetable composition is provided by mixing brown rice, Jobs-tear, millet, black beans, beans, pine needles, Angelica, purslane, kale, tangleweed, wild dropwort and perilla. CONSTITUTION: The raw grain and vegetable composition for preventing cancer contains brown rice 30.0-40.0wt.%, Jobs-tear 10.0-20.0wt.%, millet 10.0-20.0wt.%, black beans 3.0-5.0wt.%, pine needles 2.0-20.0wt.%, Angelica 1.0-6.0wt.%, purslane 1.0-6.0wt.%, kale 1.0-6.0wt.%, tangleweed 0.3-1.0wt.% and perilla 5.0-10.0wt.%. The composition has effects on anti-mutation, anti-cancer and cancer prevention.

Description

Raw-diet composition for anticancer

The present invention relates to a cancer prevention reproductive composition. More specifically, the present invention is a cancer prevention and anti-cancer composition consisting of cereals, such as brown rice, yulmu, sorghum, black beans, vegetables, such as pine needles, pine needles, bright leaf, purslane, kale, kelp, wild parsley and perilla It is about.

In modern society with advanced medicine, cancer mortality increases are a major problem. About 75 to 80% of cancers are reported to be caused by environmental factors, and about 30 to 40% of them are controlled by food. (Watson, RR, Leonard, TK, J Am Diet Assoc 86, 505-510 (1986)). Cancer occurs through three stages: initiation, promotion, and progression, which can occur only with a sufficient amount of carcinogen, but in reality a small amount of carcinogen in the environment or food It is known that cancer tissues begin to form when abnormal cell division occurs due to the stimulation of a tumor promotor as cell mutations (initiation cells) occur and the number of cells increases exponentially. Various ingredients in foods are carcinogens or promoters that are involved in carcinogenesis, but also have a function of inhibiting them.

On the other hand, the dietary habits of our country is in the trend of simplification and westernization, which is why the onset of circulatory diseases and malignant neoplasms is rapidly increasing [Kweon, Y.M. Ph.D. Pusan National University (1998), Soo-Ok Kim, Master's Thesis, Graduate School, Pusan National University (1999)]. In particular, studies have been actively conducted to search for bioactive factors such as mutation inhibition, antioxidant, and aging inhibitors related to anticancer effects due to the highest frequency of cancer deaths. There is a tendency to look for foods that have fewer side effects than pharmaceuticals and have preventive effects. In the case of anti-cancer, research is being conducted to search for active ingredients in foods that can suppress the initial stage of cancer (initiation) due to continuous intake rather than drugs that inhibit the promotion of potential tumor cells metastasis.

Currently, various cancer treatments have been developed and put into practice, but since there are many limitations, it should be emphasized rather than treatment. There are also passive ways to prevent cancer, such as quitting smoking, drinking alcohol, reducing the consumption of processed foods containing additives, or reducing the intake of irritating foods, but there is more to preventive cancer, such as chemoprevention. Much attention is being focused. The purpose of the chemoprevention of cancer is to stop the carcinogenic processes occurring in the body, normalize the cells that are already mutated to cancer cells, or convert the cancers from early cancer into invasive cancer. Or to prevent long-term use of drugs (vitamins, fiber, micronutrients, etc.).

The most likely clinical studies of nutrients for cancer prevention are known vitamins A, retinoids and carotinoids, which are known to reduce the frequency of cancers in epithelial cells. Okuyama, T., Takada, M., Planta Med., 57, 242-246 (1991)] Also anti-mutants in foods include porphyrin, fatty acids, polyphenols, hydroxides. Sulfur compounds, selenium, calcium, and dietary fiber are known to be active, and antimutagenic activity of grains such as rice and brown rice has been reported [Kim, ES, Kim, MK, J. Food Sci. ., 32 (4), 337-352 (1999)], Changes in Antimutagenic Activity of Rice Processing and Nitrite Scavenging Activity of Roasted Barley [Kang, TH, Park, YK, Ha, TY, Moon, KD, J Korean Soc.Food Nutr. 25 (3), 367-373 (1996)]. In addition, many studies have been conducted on soybeans, which are protease inhibitors, lecithin, and saponins (such as cholesterol-lowering, anticancer, intestinal and dementia-producing functions). saponin, isoflavone, β-sitosterol, etc. These ingredients are contained only in yellow beans and soybeans, but not in kidney beans, peas, red beans, etc. [Lee, YH, Choi , YS, Leem, SY, J. Korean Soc.Food Nutr. 25 (2), 188-192 (1996)] Trypsin inhibitors, isoflavones, phytic acid, etc. It is reported as a factor that prevents.

In addition, the more vegetables and fruits you eat, the more you can reduce the number of adult diseases and carcinogens. Because vegetables and fruits contain antioxidants such as vitamins C, E, β-carotene and flavonoids, This is because it can suppress the induced lipid peroxidation reaction and exhibits the effect of suppressing mutations. In particular, dithiothiones, thiocyanate, and iso- sorbents contained in broccoli, cabbage, and Brussel sprout. Thiocyanate and sulfoaphane components have particularly high antimutagenic effects.

Reproduction is simply the opposite of fire, and in ancient ancestors and reproductive villages, vegetarian foods centered on cereals and pine needles were called reproductive. However, in recent years, raw food refers to a powder freeze-dried a variety of organically grown cereals, soybeans, seeds, vegetables and seaweeds are sold as powder. When grains are ingested in the form of sugars and gelatinized starches, they are ingested in this case. In this case, the digestion and absorption rate is fast, and blood sugar rises rapidly, which is harmful to health. However, when ingesting raw grains, digestion and absorption gradually progress, and ingredients such as bitterness, astringent taste, and fishy taste of raw grains activate physiological functions and promote the secretion of digestive juices and saliva, so that the effect of health promotion is expected. Can be. In addition, vitamins, minerals, dietary fiber and other physiologically active substances in the endothelial and embryo of grains are greatly enhanced.

Cereals mainly used for reproduction include brown rice, yulmu, sorghum, glutinous rice, green tea, buckwheat, millet, barley, black rice, and whole wheat, and soybeans include black, white, frosted and red beans. , Perilla and sesame seeds. Other vegetables and seaweeds include pine needles, pumpkin, carrots, fresh vinegar, kale, persimmon leaves, mulberry leaves, jinjin mugwort, burdock, lotus root, comfrey, eoseongcho, buttercup, purslane, radish, laver, seaweed, seaweed, etc.

In view of the above, the present inventors screened cancer prevention and anticancer effects on various vegetables in order to develop reproduction having cancer prevention and anticancer effects, and based on this, a cancer prevention reproductive diet was prepared. The raw materials of the reproductive diet were vegetables that proved cancer prevention and anti-cancer effects through screening anti-cancer effects. Cereals, soybeans, seeds and seaweeds were previously studied [Kweon, Y.M., Ph.D. Pusan National University (1998)] was used brown rice, yulmu, sorghum, black soybean, perilla and kelp, respectively.

In this way, the genome of the present invention was compared with the cancer prevention activity and anticancer properties of several products sold in the reproductive system. > Cancer prevention and anticancer experiments were performed using Ames test and MTT assay in vitro, and the cancer prevention (anticancer) reproduction of the present invention was measured by measuring the effect of inhibiting solid cancer growth using micronucleus test and sarcoma-180 cell in vivo. Developed.

Accordingly, it is an object of the present invention to provide a reproductive composition having cancer prevention and anticancer effects.

Another object of the present invention to provide a method for ingesting the cancer preventive reproductive composition.

The above object of the present invention is brown rice 30.0-40.0% by weight, 10.0-20.0% by weight of radish, 10.0-20.0% by weight of sorghum, black beans 3.0-5.0% by weight, pine needles 2.0-20.0% by weight, light green leaf 1.0-6.0% by weight, purslane 1.0 A cancer preventive reproductive composition consisting of -6.0% by weight, 1.0-6.0% by weight of kale, 0.3-1.0% by weight of kelp, and 5.0-10.0% by weight of perilla was prepared, and antimutagenic, anticancer and cancer prevention effects were observed. Achieved by in vivo confirmation.

Hereinafter, the configuration of the present invention will be described in detail.

Figure 1 is a photograph of the hepatocellular tissue of rats fed with a pine needles diet was stained with H-E dye and observed under a microscope (× 400).

Figure 2 is a graph comparing the effect of the pine needles added to each step affecting the activity of spleen lymphocytes natural killer cells (NK).

3 is a graph comparing the effects of pineal leaf diets at different stages influencing the lipid peroxide content of Balb / c mice subcutaneously injected with sarcoma-180 tumor cells.

Figure 4 is a graph comparing the effects of pineal leaf diet at each stage affecting the liver glutathione content of Balb / c mice subcutaneously injected with sarcoma-180 tumor cells.

FIG. 5 is a graph comparing the effects of pineal leaf diets at different stages affecting hepatic glutathione-S-transferase activity in Balb / c mice subcutaneously injected with sarcoma-180 tumor cells.

6 is a graph comparing the micronucleus-induced inhibitory effect of the reproductive methanol extract of >> in vivo micronucleus test using ICR mice inoculated with mitomycin C, a nucleophile-inducing substance.

FIG. 7 is a graph comparing the effects of reproductive methanol extracts on hepatic glutathione-S-transferase activity in Balb / c mice subcutaneously injected with sarcoma-180 tumor cells.

FIG. 8 is a graph comparing the effects of reproductive methanol extracts on liver glutathione content in Balb / c mice subcutaneously injected with sarcoma-180 tumor cells.

The present invention, MNNG which is a toxic effect, direct mutant by conducting the Ames test and MTT assay in> in vitro on pine needles, Myeongil leaf, purslane, kale and wild parsley samples in order to find out vegetables excellent in cancer prevention and anti-cancer efficacy of raw materials. And testing the antimutagenic and anticancer effects on the indirect mutant AFB ₁ ; Preparing pine needle samples to examine the anticancer function and toxicity in vivo of pine needles found to be toxic in the experiment; The inhibitory effect of solid cancer growth, organ weight change, spleen spontaneous killer cells (NK) on Yac-1 cells in> in vivo anticancer test system using pine needle diet, sarcoma-180 tumor cells and Balb / c mice Immunopotentiating effects, changes in glutathione or peroxide content in mouse liver, changes in glutathione-S-transferase activity, aminotransferase (ALT, AST), an indicator of hepatotoxicity in serum, and blood urea nitrogen (BUN, an indicator of renal toxicity ) And creatinine concentration; After deciding the total energy requirement of 2500kcal per day, mix the appropriate amount of samples such as brown rice, yulmu, sorghum, black soybean, fresh day leaf, purslane, kale, kelp, wild parsley, and perilla, and the concentration of pine needles 2% by weight and 6% by weight. , 10% by weight, 20% by weight of the present invention to prepare a reproductive diet I-IV; In order to verify the cancer prevention and anticancer effect of each reproductive system of the present invention, extracting the reproductive powder with methanol and dissolving in DMSO to prepare an extract; Said present inventions I to IV and commercial reproduction of GMF Co., Ltd. Shirumigi reproduction (Diet A), Natural Reproductive Disease (Diet B) of Dr. Hwang Sung-Joo of Love Health Village Co., Ltd. (Diet C), Mom Love Co., Ltd. Ames test, micronucleus test using reticulocytes of mouse peripheral blood, to compare antimutagenicity, cancer prevention and anticancer effects in> in vitro such as Diet D, et al. Such as GMF Corporation's Shirumigi Reproduction (Diet A), Dr. Hwang Seong-Joo's Natural Reproduction (Diet B), Mother's Love Corporation's Diet (Diet C), Mother's Love Corporation's Diet (Diet D), etc. Conducting an MTT assay; To compare the effects of the present invention I, III and commercial reproductive system on the in vivo anti-cancer activity of dietary reproductive system (Diet A), natural reproduction (Diet B) of Dr. Hwang Sung-joo of the Healthy Village Co., Ltd. Performing a step of performing cytotoxicity test using sarcoma-180 tumor cells, measuring the effect of inhibiting solid growth growth, and measuring enzyme activity such as GST; As a result of the above experiments, the genus III having the most excellent anticancer and cancer prevention functions is developed into the genital vegetation of the present invention and subjected to the sensory evaluation.

As a strain used in the Ames test in this step,> Salmonella typhimurium TA100, a histidine nutrient-constituting strain of Salmonella typhimurium LT-2, was used as a B.N. Genetic traits such as histidine requirements, deep rough (> rfa) mutants,> uvrB mutants, and R factor were used when new frozen permanents were prepared or immediately before each use.

Aflatoxin B ₁ (AFB ₁ ) (USA, Sigma) dissolved in dimethylsulfoxide (DMSO) as an indirect mutant in the Ames test step, N-methyl-N'-nitro-N dissolved in sterile water -nitrosoguanidine (MNNG), USA, Aldrich) was used as the direct mutagenesis / carcinogen in the experiment.

(Microsomal) of liver granules for activating indirect mutants according to the method of Maron and Ames [Maron, DM, and Ames, BN, Mutat. Res., 113, 173-215 (1983)] in the Ames test step S9 mixture, which is an enzyme compound, was prepared and used. Aroclor 1254, a polychlorinated biphenyl (PCB) mixture, was diluted to a concentration of 200 mg per 1 mL of corn oil. Approximately 200 g of male Sprague-Dawley rats were injected once intraperitoneally (500 mg / kg) for the activity of liver enzymes. Liver was extracted at 4 ° C. aseptic state, washed several times with 0.15M KCl, and homogenized with a homogenizer (Hotogenizer, Potter-Elvehiem apparatus, USA) by adding 0.15M KCl solution of three times the liver weight. Centrifugation was performed at 9000 × g for 10 minutes to obtain the S9 fraction, the supernatant. Dispense 1-2 mL in a cryogenic tube, rapidly freeze on dry ice, and store in a deep freezer at -70 ℃. It was. The S9 fraction (10%) was mixed with MgCl-KCl salt (2%), 1M glucose-6-phosphate (0.5%), 1M NADP (4%), 0.2M phosphate buffer (pH 7.4) and sterile water to obtain S9. The mixture was prepared and used.

In this step, RPMI 1640, fetal bovine serum (FBS), 0.05% trypsin-0.02% EDTA and 100 units / ml penicillin-streptomycin were used as reagents for cell culture from GIBCO (USA). . Cell culture was performed using a CO ₂ incubator (Forma, model MCO96, USA).

In this step, the cell line used to confirm the> in vitro anticancer effect was obtained from AGS human gastric adenocarcinoma cells distributed from Korea Cell Line Bank (Seoul National University of Korea) at 100 units / ml of penicillin-streptomycin and 10% of FBS. Using RPMI 1640 containing 37 ℃, it was used incubated in a 5% CO ₂ incubator. The cultured cancer cells were refeeded 2-3 times a week, and after 6-7 days, washed with PBS to separate the cells attached with 0.05% trypsin-0.02% EDTA, centrifuged, and then put the medium into the integrated cancer cells and pipette them. The cancer cells were mixed well to distribute evenly and subcultured every 6 to 7 days. Each passage number was recorded during subculture, and when the number of cultures was 10 or more times, new cancer cells were taken out of the liquid nitrogen tank and used again.

Animals used in the present invention were male and female 6-week-old ICR-based mice (Korea Research Institute of Chemical Technology, Daejeon), and weighed about 35 g of body weight reared as standard feed. The animals were supplied with sufficient water and feed, and the animal laboratory maintained a temperature of 22 ± 1 ℃ and a humidity of 55 ± 5% and maintained a light-dark cycle every 12 hours.

Animals used in the present invention were male Balb / c mice (Korea Chemistry Research Institute, Daejeon), the body weight of about 25g was used, was bred as standard feed. When breeding, water and feed were supplied in sufficient quantities. The animal laboratory maintained a temperature of 22 ± 1 ℃ and a humidity of 55 ± 5% and maintained a light-dark cycle every 12 hours.

In this step, sarcoma-180 cells that were passaged and preserved at weekly intervals in the abdominal cavity of Balb / c mice were used as tumor cells. Sarcoma-180 cells cultured for one week in the abdominal cavity of mice were taken with ascites and centrifuged (1,200 rpm, 10 min.) With PBS (phosphate buffered saline) to separate tumor cells. The separated cells were suspended in PBS again, centrifuged again to remove the supernatant, and tumor suspensions were made to 1.0 × 10 ⁶ cells / ml.

In this step, casein, mineral mixture, vitamin mixture and cellulose were used as ICN (biomedical. Inc., Ohio., USA), and choline bitartarate was Sigma (Sigma). chemical company, St. Louis, USA).

Grain materials (Oryza stiva L., Brown rice), brown rice (Coix lacrymajobi L. var. Mayuen, Job's tears), and black soybeans (Phaseolus vulgaris L., Black soybean) are grown organically and organically in the high mountains of Gyeongbuk. Black sesame seeds (Sesamum indicum L., Black sesame) and sorghum (sorghum bicolor L. Moench, Sorghum) were grown and polluted in Boeun Hoenggol, Chungbuk, Korea, and were purchased from the Natural Health Research Institute (Dongdae Sin-dong, Busan). Purslane, wild parsley and kale were purchased from Busan Bujeon Market, pine needles, kelp, and perilla mother love Co., Ltd. (Kijang-gun, Busan). In addition, Myeongilyeop was purchased from Green and Human Corporation.

In the cancer preventive reproductive composition material of the present invention, saponin and phenolic compounds, such as coumarin flavonoids, and the like, are contained in various types of organic acids and unsaturated fatty acids. [Okuyama, T. and Takada, M., Planta Med., 57, 242-246 (1991)], and Ha Jung-ok's research reported high antimutagenic and anticancer effects. (1994)].

Purslane (> Portulaca oleracea) in the cancer prevention reproduction composition material of the present invention is used for edible, ornamental, and medicinal. According to a recent study, purslane ethanol extract was 40% for 4-NQO and 50% for MNNG in SOS chromotest. It showed antimutagenic inhibitory activity and strong antimutagenicity in Ames experimental system [Kangwon University Master's Thesis (1996)].

The wild parsley is a perennial herb belonging to the Apiaceae, which grows in watery places or streams. Buttercup has been used as a tasteful vegetable because it has a fresh color and distinctive aroma, and has many pharmacological effects such as long-leaf, jaundice, crayfish, appetite stimulation, fever, species, sheep blood, high blood pressure and neuralgia. [Kim Sang-sun; Scientific Review of Korean Traditional Foods, Sookmyung Women's University Press (1985)], antimutagenicity and anticancer activity are also known to be high [Lee, K.I., Ph.D. Pusan National University (1992).

Kale (> Brassica oleracea var. Acephala) is a type of cabbage of origin in Asia Minor, which contains vitamin C, vitamin E, beta-carotene, calcium, phosphorus, iron, potassium, and magnesium. Rich in vitamins and minerals, such as Hertog, MGL, Hollman, PCH, and Katan, MB J., Agric. Food Chem., 40, 2379 (1992). In addition, Lee's study showed that not only antimutagenic effects, but also high anti-cancer effects in> in vitro and> in vivo.

The pine needles (> Pinus densiflora Sieb. Et Zucc) of the cancer preventive reproductive composition material of the present invention include essential oil components such as α-oinene, β-pinene and camphene, flavonoids such as quercetin and kaempferol, resins, sugars and carotene ( carotene), known to contain vitamin C. In addition, it is known to be effective in the treatment of liver disease, gastrointestinal disease, nervous system disease, circulatory system disease, skin disease, etc., and studies on the function of pine needles affect the lipid metabolism [Kim, ES, Kim, MK, J. Food Sci. Nutr., 32 (4), 337-352 (1999)], Studies on Enzyme Activity and Liver Tissue of Rats on High Fat Diet [Kang, JD, Yoon, TH, Choei, M., Im, KJ, Moon , KD, J. Korean Soc. Food Nutr. 25 (3), 367-373 (1996)], cholesterol lowering effect [Lee, Y. H., Choi, Y. S., Lee, S. Y., J. Korean Soc. Food Nutr. 25 (2), 188-192 (1996)], and other antioxidant activities and antimutagenicity [Kang, Y. H., Park, Y. K., Sung, S. K., J. Korean Soc. Food Sci. Nutr. 28 (5), 984-989 (1999). Although these studies anticipate antimutagenicity and anticancer activity of pine needles, there is no direct research on the effects, so the present inventors compared the effects with other vegetables known to have antimutagenic and anticancer effects. In addition, the toxicity of overdose was not known, and the toxicity of liver and kidney was investigated by ingesting pine needles in vivo.

Cancer prevention and anti-cancer reproductive composition of the present invention in the above step is 30.0 ~ 40.0% by weight brown rice, 10.0 ~ 20.0% by weight, sorghum 10.0 ~ 20.0% by weight, black beans 3.0 ~ 5.0% by weight, pine needles 2.0 ~ 20.0% by weight, day It consisted of 6.0 weight%, 1.0-6.0 weight% of purslane, 1.0-6.0 weight% of kale, 0.3-1.0 weight% of kelp, and 5.0-10.0 weight% of perilla.

50 g of the cancer preventive reproductive composition of the present invention may be taken after soaked in soymilk or mixed with honey or mixed with yoghurt to reduce fishy smell.

In the grains, vegetables, seeds and soybeans of the present invention, other foods having similar conditions depending on calories or various nutrient contents can be replaced or added and used, and the distribution ratio can also be reduced.

Hereinafter, specific embodiments of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited thereto.

Example 1 Antimutagenicity and> in vitro Anticancer Effect of Vegetables

In order to identify vegetables with high cancer prevention (anticancer) properties in the reproductive material, antimutagenic and anticancer effects were compared in vitro for several vegetables known for cancer prevention and anticancer activity. As a result, pine needles showed very high cancer prevention (anticancer) effect, and Myeong Il-yeop showed good effect. The result is as follows.

The present invention Ames test to examine the presence of toxicity and mutagenicity was carried out by the following method. Dispense 2 ml of top agar into a sterile cap test tube, add 100 µl of Salmonella TA100 (> Salmonella typhimurium TA100) and 100 µl of the test sample, and stir lightly.In the case of toxicity test, nutrient agar plate In the case of mutagenicity experiments, the cells were aliquoted into a minimal agar plate, solidified and incubated at 37 ° C. for 24 to 48 hours, and then their toxicity and antimutagenicity were determined.

The preincubation test mainly carried out in the present invention is 0.5ml S9 mixture (when treated with indirect mutagen), Salmonella TA100 (> Salmonella typhimurium TA100) strain cultured overnight (1-2 × 10 ⁹ cells / ml) 0.1 ml, 50 μl of diluted test sample and 50 μl of mutagenesis were placed in a cap tube in an ice bath, and gently stirred and pre-incubated at 37 ° C. for 30 minutes. 2 ml of top agar at 45 ° C. was poured into each tube and stirred for 3 seconds to spread on minimal glucose agar plates, incubated at 37 ° C. for 48 hours, and the number of return mutations was counted. Meanwhile, the concentrations of the sample and the mutagen source used in the experiment were determined through preliminary experiments (dose response and toxicity experiment). Inhibition rate was calculated by the following equation.

Inhibition rate (%) = 100 × [(a-b) / (a-c)]

Where a is the number of back mutations induced by the mutagen, b is the number of back mutations when the sample is processed, and c is the number of natural back mutations in the absence of the mutagen and the sample.

First step. Ames test to measure the toxic effects of vegetables

As a result of testing the toxicity of vegetables by conducting the Ames test at the concentrations of 0.625 mg / plate, 1.25 mg / plate, and 2.5 mg / plate of the vegetable samples of the present invention, such as pine needles, wild leaf, purslane, kale and wild parsley, the other vegetable samples were tested. Was not toxic, but pine needle samples showed toxicity at the concentration of 2.5 mg / plate.

Second step. Ames test to measure antimutagenic effects

Antimutagenic effects on vegetable samples were measured. As a result of the first step, in the case of pine needles, antimutagenic experiments were performed at concentrations except for the concentration of 2.5 mg / plate, which is toxic.

For MNNG, a direct mutant, pine needles showed the highest mutation inhibition effect. At the concentration of 0.625 mg / plate, all vegetables showed a significantly higher effect than the control group (p <0.05), but there was no significant difference between the vegetables. The inhibitory rate of pine needles was high at 88% at 1.25mg / plate concentration, and 84% was higher at Myeongil leaf at 2.5mg / plate concentration (Table 1).

Inhibitory Effects of Vegetable Samples on Mutation of> Salmonella typhimurium TA100 by MNNG sample Revertants / plate 0.625mg / plate 1.25mg / plate 2.5mg / plate Natural mutation 98 ± 20 ¹⁾ 98 ± 20 98 ± 20 Control 2037 ± 151 ^a 2037 ± 151 ^a 2037 ± 151 ^a pine needles 823 ± 109 ^c (63) 324 ± 17 ^d (88) - Myeong Il-yeop 961 ± 17 ^c (56) 536 ± 161 ^cd (77) 401 ± 48 ^d (84) purslane 812 ± 130 ^c (63) 602 ± 98 ^cd (74) 645 ± 209 ^c (72) Kale 858 ± 62 ^c (61) 781 ± 110 ^bc (65) 698 ± 34 ^c (69) Parsley 1350 ± 135 ^b (35) 1541 ± 238 ^b (26) 1292 ± 102 ^b (38) NOTE ^{1) 1)} Mean means ± standard deviation. ^{a to} d mean significance (p <0.05) by Duncan's multiple range test. () Represents the inhibition rate (%).

Treatment with the indirect mutant aflatoxin B ₁ (AFB ₁ , aflatoxin B ₁ ) showed higher antimutagenic effects than the direct mutant MNNG (Table 2). Myeongil leaf showed a high inhibition rate of 91% at 2.5mg / plate concentration and pine needles showed a very high mutagenesis inhibitory effect at 96% at 1.25mg / plate concentration.

Inhibition of> Salmonella typhimurium TA100 Mutation by AFB ₁ in Vegetables sample Revertants / plate 0.625mg / plate 1.25mg / plate 2.5mg / plate Natural mutation 155 ± 31 ¹⁾ 155 ± 31 155 ± 31 Control 2636 ± 667 ^a 2636 ± 667 ^a 2636 ± 667 ^a pine needles 483 ± 65 ^c (87) 248 ± 30 ^c (96) - Myeong Il-yeop 771 ± 658 ^b (75) 496 ± 90 ^c (86) 377 ± 21 ^c (91) purslane 2443 ± 129 ^a (8) 2057 ± 293 ^ab (23) 2061 ± 316 ^ab (23) Kale 2458 ± 568 ^a (7) 2367 ± 390 ^ab (11) 1795 ± 594 ^b (34) Parsley 1593 ± 164 ^b (42) 1742 ± 49 ^b (36) 1717 ± 234 ^b (37) NOTE ^{1) 1)} means mean ± standard deviation. ^{a to} d mean significance (p <0.05) by Duncan's multiple range test. () Represents the inhibition rate (%).

Third step. MTT assay for the determination of anticancer effects in vitro

As a result of enzymatic action of viable cells, MTT [3- (4,5-dimethylthiazol-] is an experimental method for measuring the degree of suppression of death or proliferation of cancer cells by measuring the extent of MTT reduction and precipitation into formazan crystal. 2-yl) -2,5-diphenyl-tetrazolium bromide] assay was conducted to investigate the anticancer properties of vegetables against AGS human gastric cancer cells.

When AGS human stomach cancer cells were treated with 25 ㎍ / assay of vegetable samples, pine needles showed the highest inhibition rate (83%), purslane and wild parsley (37%). Was shown (Table 3). At 50㎍ / assay concentration, pine needles showed a very high inhibition rate (85%) and purslane also had a high inhibition rate (68%). Therefore, pine needles showed the highest inhibition rate when the vegetables showed> in vitro anticancer effects.

Results of MTT Analysis of Vegetable Samples on AGS Human Gastric Cancer Cells Vegetable Sample OD ₅₄₀ 25 µg / assay 50 µg / assay Control 0.323 ± 0.004 ^{a, 1)} 0.437 ± 0.015 ^a pine needles 0.054 ± 0.002 ^c (83) ²⁾ 0.066 ± 0.031 ^d (85) Myeong Il-yeop 0.227 ± 0.035 ^b (30) 0.232 ± 0.030 ^b (47) Kale 0.226 ± 0.026 ^b (30) 0.240 ± 0.015 ^b (45) purslane 0.202 ± 0.011 ^b (37) 0.138 ± 0.042 ^c (68) Parsley 0.202 ± 0.028 ^b (37) 0.231 ± 0.013 ^b (47) NOTES 1. ¹⁾ represents the mean ± SD _{.OD 540 of control - OD 540 of} sample2. ²⁾ Inhibition Rate (%) = --------- &#12316; --------- &#12316; --------- &#12316; ---- -× 100 OD ₅₄₀ of control 3. ^{a ~} d Mean significance by Duncan's multiple range test (p <0.05).

As shown in the above results, pine needles were found to be very high cancer prevention or anti-cancer activity in> in vitro, pine needles are expected to have a cancer prevention effect is contained flavonoids, carotene, vitamin C.

In general, excessive reproduction of pine needles is included. To determine whether excessive intake of pine needles is toxic to living organisms, the toxicity of pine needles to liver and kidney of mice in> in vivo was examined as in Example 2 below.

Example 2> in vivo anticancer functional and toxicity test of pine needles

In order to examine the anticancer function and toxicity of pine needles, the sarcoma-180 tumor cells were used in the in vivo anticancer test system to inhibit the growth of solid cancers in the mouse, and to change the weight of organs such as liver and spleen, changes in spleen immune function, Investigate changes in glutathione-S-transferase activity, glutathione content and lipid perxide content, and aminotransferase (ALT, AST) activity, an indicator of hepatotoxicity in serum. The concentrations of blood urea nitrogen (BUN) and creatinine (CRE) were measured.

First step. Preparation of Sample

Considering the general components of pine needles, pine needles were added to the AIN-76 diet by adding 2%, 10%, and 20% pine needles with the same energy level (Table 4). The standard diet AIN-76 diet was used as a control.

Scheme of Pine Needle Additives Control Group (AIN-76) pine needles 2 (% by weight) 10 (% by weight) 20 (% by weight) Casein 20.0 19.9 19.6 19.1 Methionine 0.3 0.3 0.3 0.3 Corn starch 15.0 15.0 15.0 15.0 Sucrose 50.0 49.6 48.0 46.1 Fibrin (cellulose) 5.0 4.7 3.7 2.3 Corn oil 5.0 4.9 4.6 4.2 Mineral mixture 3.5 3.5 3.5 3.5 Vitamin mixtures 1.0 1.0 1.0 1.0 Choline Bicarbonate 0.2 0.2 0.2 0.2 pine needles - 2.0 10.0 20.0

Second step. Measurement of the Effect of Pine Needle Growth on Solid Cancer Growth

The prepared pine needle added diet was ingested in Balb / c mice for 2 weeks before the experiment, and the intake was similar between the groups, and the left subcutaneous groin (groin) of the Balb / c mouse was examined to investigate the antitumor effect by pine needle ingestion. After 7 days old sarcoma-180 ascites cells were transplanted, each group was ingested and killed for 4 weeks. Mice were dissected to isolate tumors and weighed (Table 5).

Inhibitory Effects of Pine Needle Diet on Solid Cancer Growth in Balb / c Mice Infused with sarcoma-180 Cells group Tumor Weight (g) Inhibition Rate (%) S-180 + control 2.83 ± 0.62 ^{a, 1)} S-180 + Pine Needle 2% by weight 1.15 ± 0.22 ^bc 59 S-180 + Pine Needle 10% by weight 0.97 ± 0.12 ^c 65 S-180 + Pine Needle 20% by weight 1.77 ± 0.16 ^b 37 NOTE ¹ shows the mean ± standard deviation of five rats. ^{a to c} represent the significance level by Duncan's multiple range test (p <0.05).

As shown in Table 5, the weight of solid cancer in the control group was 2.83g, whereas the weight of solid cancer in the pine needle group was significantly reduced. In the pine needle added group with 2% pine needle added to the diet, the tumor weight was 1.15 g, showing 59% inhibition rate compared to the control group, and the 10% added group showed 0.97g inhibition rate (66%). Indicated. However, in the group added 20% by weight pine needles it was found that the solid rock was 1.77g, the inhibition rate was 37%, rather than the group added 10% by weight pine needles. Therefore,> 10 wt% of pine needle intake concentration was most suitable.

Third step. Measurement of Weight Changes in Organs of Pine Needle-Fed Mice

The weight of each organ was measured to examine the effect of pine needle diet and sarcoma-180 tumor cell transplantation on each organ. The weight ratio of the liver, which is responsible for the detoxification of various harmful substances during metabolism, was slightly increased in 10% and 20% of pine needles (PN diet group). The weight ratio was increased in the transplant group (S-180 + PN diet group). In addition, the spleen related to the immune system was increased in the S-180 + pine needle diet group as compared to the pine needle diet group. In other organs (heart, kidneys) there was no significant difference between groups (Table 6).

Changes in the Weight of Organs after Pine Needle Addition to Balb / c Mice Infused with Sarcoma-180 Cells sample Body weight (g) Spleen / Weight (wt%) Liver / Weight (% by weight) Heart / weight (% by weight) Height / weight (% by weight) Control 30.32 ± 1.46 ¹⁾ 0.41 ± 0.02 5.49 ± 0.09 0.56 ± 0.03 1.49 ± 0.02 Pine needle diet 2% 25.76 ± 1.80 0.41 ± 0.01 6.08 ± 0.14 0.59 ± 0.01 1.50 ± 0.06 Pine needle diet 10% 28.28 ± 1.80 0.36 ± 0.01 7.28 ± 0.18 0.65 ± 0.01 1.55 ± 0.04 Pine needle diet 20% 25.23 ± 2.19 0.39 ± 0.01 7.68 ± 0.19 0.63 ± 0.02 1.50 ± 0.06 S-180 + Control 30.43 ± 3.32 0.67 ± 0.04 5.93 ± 0.16 0.49 ± 0.02 1.53 ± 0.07 S-180 + pine leaf diet 2% 30.70 ± 1.47 0.62 ± 0.04 6.08 ± 0.14 0.55 ± 0.01 1.44 ± 0.05 10% of S-180 + pine needles diet 29.58 ± 1.65 0.61 ± 0.03 8.01 ± 0.29 0.54 ± 0.03 1.44 ± 0.05 20% of S-180 + pine needles diet 27.06 ± 1.71 0.65 ± 0.03 8.54 ± 0.22 0.54 ± 0.02 1.44 ± 0.02 NOTE ¹ represents the mean ± SD (standard deviation).

The histological changes of the hepatocytes of mice observed with a microscope (× 400) after staining with H-E dye are shown in FIG. 1.

Fourth step. Measurement of Immune Activity Involvement of Natural Killer Cells of Pine Needles

Natural killer cells (NK) are cytotoxic lymphocytes found mainly in the blood and spleen. They are involved in the nonspecific immune system and are known to kill microorganisms, viruses, and tumor cells. SC, Salome, MJ, Varghese, CD, Panikkar, B. and panikkar, KR Bio. Factors, 4 (1), 51 (1992).

The spleens of the mice were sterilely isolated to isolate splendid killer cells of the spleen and their cell disruption activity against Yac-1 cells was measured. Yac-1 cells were seeded at 5 × 10 ⁴ cells / ml as target cells, and effector cells were seeded at 500 × 10 ⁴ cells / ml (the ratio of effector cells to target cells was 100: 1). As a result, the pine needle added group significantly increased its activity compared to the control group (p <0.05), and among them, the cell-destructive activity of natural killer cells was very high in the group which ingested 10 wt% pine needles (Fig. 2). .

5th step. Changes in Lipid Peroxide Content in Mouse Liver with Pine Needle Diet

Lipid peroxide is a lipid that is peroxidated by the binding of oxygen to unsaturated fatty acid, a lipid component, especially the unsaturated fatty acids that make up phospholipid, which is the main component of cell membrane, with active oxygen. Mitochondria rich in unsaturated fatty acid and rich in phospholipid It is found in cell membranes such as mitochondria, granules, microsomes, erythrocytes, platelets, and the like. These lipid peroxides are produced by a series of chain reactions starting from the biosynthesis of superoxide radicals, which are active oxygen, and degradation products generated as a result of lipid peroxidation are known to interfere with normal functions in vivo. Bus, IS and Gibsin, JE, Elsevier Press, Review in biochemical toxicology, 125 (1979)]. Lipid peroxide content (malondialdehyde / g of tissue) of the pine needles and S-180 + pine needles group was measured and compared, the amount was increased in the S-180 + pine needles diet group (Fig. 3). In the S-180 + pine needle diet group, the control group who consumed AIN-76 semipurified diet had 11.2 lipid peroxide, whereas the control group that consumed 2%, 10% and 20% pine needles added 4.4, 4.9, 4.0, respectively. The amount of lipid peroxide was significantly reduced. However, no statistical significance was found between the groups that consumed pine needles (p <0.05).

Sixth step. Changes in Glutathione Content in Mouse Liver with Pine Needle Diet

The phase II stage of the metabolic enzyme system in the liver contains the endogenous or exogenous toxic substances or converts them into water-soluble substances and then removes them. Protective System Glutathione has sulfur hydroxide radicals and is involved in detoxification and reduction of lipid peroxides in the metabolism of electrophilic substances and free radicals and lipid peroxides. Glutathione is conjugated with activators such as toxins and peroxides to excrete urine by the action of enzymes such as glutathione-> S-transferase (GST). By removing toxins from the body [Burk, RF, Trumble, MJ, and Lawerence, RA] Biochem. Biophts. Acta. 618, 35-41 (1980). The glutathione content in the mouse liver was measured.

Glutathione content was slightly lower in the S-180 + pine needle diet group than in the pine needle diet group. Glutathione content (umol / g of tissue) in the pine needle diet group intake 10% by weight of the pine needles group was 0.29, which was slightly increased compared to the control group 0.17. In addition, in the S-180 + pine needle diet group, the control group was 0.08, whereas the group ingesting 2% by weight, 10% by weight, and 20% by weight of pine needles significantly increased to 0.15, 0.25, and 0.20, respectively. It was found that the most increased in the group containing the diet containing 10% by weight (p <0.05) (Fig. 4).

Step 7. Changes in Glutathione-> S-Transferase (GST) Activity in Mouse Liver with Pine Needle Diet

Glutathione-S-transferase activity in mouse liver following pine needle diet was measured.

The GST activity (nmol / mg protein / min) of the control group was 302.6, whereas the pine needle diet group supplemented with 2%, 10%, and 20% of pine needles increased 378.5, 366.8, and 405.9, respectively. (p <0.05). In addition, GST activity of the S-180 + pine needles diet group was increased compared to the control group, and GST activity was increased in proportion to the concentration of pine needles (Fig. 5).

Step 8. Liver Toxicity Test in Mouse Serum According to Pine Needle Diet

Aminotransferase is an enzyme present in the hepatocyte membrane which is used as an indicator of hepatotoxicity due to relatively high blood concentration, which is released into the blood when hepatocytes are damaged by toxic substances. Aminotransferases include aspartate aminotransferase (AST) and alanine aminotransferase (ALT), which are known to catalyze amino group transfer reactions between amino acids and α-keto acids. AST and ALT are present in different organases (isoenzymes) depending on their intracellular location and are present in all organs. AST is particularly present in the heart, liver, and skeletal muscle, and ALT is present in the liver and its specificity is recognized. AST and ALT, which are indicators of liver failure, increase their activity as alcohol metabolism is inhibited by alcohols, organic solvents and other toxins, and liver parenchymal cells are impaired and their release into the blood is enhanced. As shown in Table 7, the control group ingested only with AIN-76 diet had 132.4 AST (Karmen unit / mL of serum), whereas the groups ingested with 2% by weight, 10% by weight, and 20% by weight of pine needles were 121.0, 156.3 and 172.8 showed that excessive intake of pine needles increased AST. In addition, the AST of the control group ingesting the AIN-76 basic diet and transplanting the tumor was 136.7, whereas the AST of the group in which the pine leaf supplemented with 2%, 10%, and 20% by weight of pine needles was implanted was 125.4, respectively. , 125.6, and 156.7, which increased only in the group fed with 20% pine needles, but not in the other groups (p <0.05).

Karmen unit / ml of serum (ALT) showed a similar trend to AST. The ALT of the control group fed the AIN-76 diet was 28.3, while the 20 wt% pine needles group increased to 42.8. In addition, the ALT of the S-180 + pine needle diet group was slightly increased compared to the pine needle diet group, and there was no significant difference between the groups of the S-180 + pine needle diet group (Table 7).

Effect of Pine Needle Supplementation on AST and ALT in Sarb-c Injected Balb / c Mice process AST ALT Karmen unit / mL of serum Control 132.4 ± 25.2 ^bc 28.3 ± 2.6 ^ab Pine needle dietary weight 2% by weight 121.0 ± 22.7 ^c 18.4 ± 17.4 ^b 10% by weight of pine needles 156.3 ± 13.3 ^ab 42.7 ± 6.7 ^a 20% by weight of pine needles 172.8 ± 15.7 ^a 42.8 ± 6.4 ^a S-180 + Control 136.7 ± 9.8 ^bc 23.8 ± 2.1 ^b S-180 + pine needles dietary 2% by weight 125.4 ± 30.5 ^bc 29.1 ± 16.4 ^ab S-180 + pine needle dietary weight 10% by weight 125.6 ± 4.3 ^bc 19.4 ± 1.5 ^b 20% by weight of S-180 + pine needles diet 156.7 ± 5.5 ^ab 30.6 ± 3.9 ^ab NOTE 1. a to ^c mean significance by Duncan's multiple range test (p <0.05).

Step 9. Kidney Toxicity Test in Mouse Serum

Blood urea nitrogen (BUN) increases with the incidence of uremia due to high protein intake, tissue disruption due to fasting, excretory disorders or renal dysfunction. BUN values in normal rats are known to range from 15 to 21 mg / dl [Baker]. , HJ, Lindsey, JR, and Weisbroth, SH The laboratory rat. Academic Press, Inc., New York, Vol. II, 123 (1984)]. We examined the renal toxicity of pine needle added group.

The BUN level of the control group ingested only with AIN-76 diet was 12.6 mg / dl, whereas the BUN levels of 2, 10, and 20 wt% pine needles were 11.9, 19.1, and 18.8 mg / dl, respectively. As the amount of was increased, the S-180 + pine needle diet group showed similar trend. The group ingested with 10% and 20% pine needles increased somewhat compared to the group not ingested, but they were all within the normal range (Table 8).

Effect of Pine Needle Supplementation on BUM and CRE in Balb / c Mice Infused with Sarcoma-180 process BUM CRE mg / dL Control 12.6 ± 4.5 ^ab 1.2 ± 0.8 ^a Pine needle dietary weight 2% by weight 11.9 ± 4.3 ^ab 1.4 ± 0.6 ^a 10% by weight of pine needles 19.1 ± 3.5 ^ab 1.3 ± 0.4 20% by weight of pine needles 18.8 ± 1.2 ^a 1.2 ± 0.3 ^a S-180 + Control 15.2 ± 4.1 ^ab 1.2 ± 0.3 ^a S-180 + pine needles dietary 2% by weight 8.5 ± 0.5 ^b 1.3 ± 0.4 ^a S-180 + pine needle dietary weight 10% by weight 14.1 ± 6.8 ^ab 1.1 ± 0.4 ^a 20% by weight of S-180 + pine needles diet 18.5 ± 7.9 ^ab 1.3 ± 0.4 ^a [Note] 1. ^a~b shows the significance of the Duncan's multiple range test.

Creatinine clearance (the number of creatinine excreted in the plasma after taking 5 g of creatinine) is used as a measure of renal excretion capacity.The amount of creatinine in the blood increases in glomerulonephritis and the amount of creatinine in normal rats is 0.4-1.5 mg / Known as [[Baker, HJ, Lindsey, JR, Weisbroth, SH The laboratory rat. Academic Press, Inc., New York, Vol. II, 123 (1984)]. The control group intake of the AIN-76 diet was 1.21 mg / dl, whereas the serum creatinine levels in the pine needle-added diet were all within the range of 1.1 to 1.4 mg / dl, showing little difference from the normal group. (Table 8).

As shown in the above experiments, the toxic effects after ingesting 2%, 10%, and 20% by weight of pine needles were examined, and as a result, the aminotransferase activity, which is an indicator of liver damage, was increased during excessive intake. You can give a bunch. On the other hand, the kidney indicators BUN and CRE slightly increased, but within the normal range, it was not toxic to the kidneys. Accordingly, a genital diet centered on pine needles, which was the most effective in vegetable screening and did not find significant toxicity even when ingested up to 20% by weight, was prepared. It was examined in vivo.

Example 3 Preparation of Cancer Prevention Reproductive System of the Invention

First step. Preparation of the genital diet of the present invention

The genital meal of the present invention was prepared using a sample of cereals, beans, seeds, seaweeds, and vegetables found to have an anticancer effect as a result of the screening of the vegetables. The method of reproductive diet was selected by standard food exchange method for 20-49 year old males, and the total daily energy requirement was 2500㎉. [Korean Nutrition Society, 6th Amendment of Korean Nutrition Recommendation, Launcher (1995) ]. The dietary composition was prepared based on the protein (15%): fat (20%): carbohydrate (65%) of Koreans, and the vegetable screening test showed that the concentration of pine needles, which had the highest anticancer effect, was 2% by weight and 6% by weight. Four diets were prepared, varying by%, 10% by weight, and 20% by weight (Diet I, II, III, IV).

Nutritional composition and food exchange table for cancer prevention reproduction Ⅰ Exchange group food Weight per unit (g) Protein (g) Fat (g) Carbs (g) Calories (Kcal) Cereals Brown rice 300.0 (38.4 wt%) 33.0 7.5 230.4 1104.0 Rule 100.0 (12.8 wt%) 10.4 3.7 61.1 373.0 Sorghum 100.0 (12.8 wt%) 12.0 4.7 69.5 336.0 Beans Black Beans 35.0 (4.5% by weight) 4.5 6.2 6.6 144.5 Vegetables pine needles 21.0 (2.7% by weight) 0.9 0.8 4.1 33.8 Myeong Il-yeop 42.0 (5.4% by weight) 1.1 0.1 3.0 16.4 purslane 42.0 (5.4% by weight) 1.2 0.2 3.1 17.6 Kale 20.0 (2.6% by weight) 0.9 0.2 0.4 6.0 Kelp 5.0 (0.6% by weight) 0.3 0.1 1.9 10.1 Parsley 45.5 (5.8% by weight) 1.0 0.6 1.5 15.9 Seeds Perilla 70.0 (9.0% by weight) 11.2 27.7 14.1 378.0 Total 780.5 (100.0% by weight) 84.5 51.7 395.8 2435.4 Protein: fat: carbohydrate = 14.1: 19.5: 66.3

Nutritional composition and food exchange table for cancer prevention reproduction Ⅱ Exchange group food Weight per unit (g) Protein (g) Fat (g) Carbohydrate (g) Calories (Kcal) Cereals Brown rice 270.0 (30.0% by weight) 19.4 6.8 207.4 993.6 Rule 100.0 (11.1 wt%) 21.3 3.7 61.1 373.0 Sorghum 100.0 (11.1 wt%) 10.3 4.7 69.5 336.0 Beans Black Beans 50.5 (5.6% by weight) 15.0 6.4 6.8 148.7 Vegetables pine needles 60.0 (6.6% by weight) 2.7 2.3 11.8 96.6 Myeong Il-yeop 60.0 (6.6% by weight) 1.5 0.2 4.3 23.4 purslane 60.0 (6.6% by weight) 1.7 0.2 4.4 25.2 Kale 60.0 (6.6% by weight) 2.8 0.5 1.3 18.0 Kelp 5.0 (0.6% by weight) 0.3 0.1 1.9 10.1 Parsley 65.0 (7.2% by weight) 1.4 0.8 2.1 22.8 Seeds Perilla 72.0 (8.0% by weight) 11.5 28.4 14.5 388.8 Total 902.5 (100.0% by weight) 88.1 54.1 385.1 2436.1 Protein: fat: carbohydrate = 14.8: 20.5: 64.7

Nutritional composition and food exchange table for cancer prevention reproduction Ⅲ Exchange group food Weight per unit (g) Protein (g) Fat (g) Carbohydrate (g) Calories (Kcal) Cereals Brown rice 270.0 (36.3% by weight) 19.4 6.8 207.4 993.6 Rule 120.0 (16.2 wt%) 25.6 4.4 73.3 447.6 Sorghum 120.0 (16.2 wt%) 12.4 5.6 83.4 403.2 Beans Black Beans 30.0 (4.0% by weight) 12.5 5.3 5.6 123.9 Vegetables pine needles 75.0 (10.1% by weight) 3.4 2.9 14.7 120.8 Myeong Il-yeop 15.0 (2.0% by weight) 0.4 0.0 1.1 5.9 purslane 15.0 (2.0% by weight) 0.4 0.1 1.1 6.3 Kale 15.0 (2.0% by weight) 0.7 0.1 0.3 4.5 Kelp 5.0 (0.7% by weight) 0.3 0.1 1.9 10.1 Parsley 15.0 (2.0% by weight) 0.3 0.2 0.5 5.3 Seeds Perilla 63.0 (8.5% by weight) 10.1 24.9 12.7 340.2 Total 743.0 (100.0% by weight) 85.5 50.5 240.5 2461.3 Protein: Fat: Carbohydrate = 14.2: 18.9: 66.9

Nutritional composition and food exchange table for cancer prevention reproduction IV Exchange group food Weight per unit (g) Protein (g) Fat (g) Carbohydrate (g) Calories (Kcal) Cereals Brown rice 270.0 (30.0% by weight) 19.4 6.8 207.4 993.6 Rule 100.0 (11.6 wt%) 21.3 3.7 61.1 373.0 Sorghum 100.0 (11.6 wt%) 10.3 4.7 69.5 336.0 Beans Black Beans 35.0 (4.0% by weight) 14.6 6.2 6.6 144.6 Vegetables pine needles 180.0 (20.7 wt%) 8.1 7.0 35.3 289.8 Myeong Il-yeop 35.0 (4.0% by weight) 0.9 0.1 2.5 13.7 purslane 30.0 (3.6% by weight) 0.9 0.1 2.2 12.6 Kale 35.0 (4.0% by weight) 1.6 0.3 0.8 10.5 Kelp 5.0 (0.6% by weight) 0.3 0.1 1.9 10.1 Parsley 30.0 (3.6% by weight) 0.7 0.4 1.0 10.5 Seeds Perilla 55.0 (6.3% by weight) 8.8 21.7 11.1 297.0 Total 875.0 (100.0% by weight) 86.9 51.1 399.3 2491.3 Protein: fat: carbohydrate = 14.4: 19.1: 66.4

Commercially available reproduction for comparing the effects of cancer prevention reproduction of the present invention, such as Shirumigi reproduction (Diet A) of GMF Corporation, Natural Reproduction (Diet B) of Dr. Hwang Sung-Joo of the Healthy Village Co., Ltd. (Diet C) ), Mother's Love Co., Ltd. (Diet D) was used.

Second step. Preparation of Extract

Each genus was extracted three times with 10 times methanol. Concentrated using a rotary vacuum concentrator (Buchi Oll & 461, Switzerland) and then dissolved in DMSO (dimethyl sulfoxide) was used in the following experiment.

Experimental Example 1: Ames test for antimutagenic and cancer prevention functional test of the reproductive system of the present invention

The anti-mutagenic effect of the reproductive system of the present invention was measured by performing the Ames test used in Example 1.

When treated with MNNG, which is a direct mutagen, the antimutagenicity of the genomes I, II, III and IV of the present invention prepared in consideration of cancer prevention is higher than that of commercial diets A, B, C and D at the concentration of 1.25 mg / plate. It showed an effect. Cancer prevention reproduction I, II, III, and IV showed a tendency to increase antimutagenicity with increasing pine needle concentration. In addition, commercially available diets of mother love (Diet C) showed a high rate of inhibition of 48%, GMF Shirumi reproduction (Diet A) of 43% showed a high effect. In addition, except for Diet C, the antimutagenic effect of the reproductive system of the present invention was superior to that of the commercial reproductive system at the concentration of 2.5 mg / plate. In the present invention, pine needles contained 10 wt% concentration, and diet III, which had slightly increased grains, had the best antimutagenic effect with 87% inhibition rate. Among commercial reproduction, mother's love reproduction (Diet C) showed a significantly higher effect (Table 13).

Antimutagenic Effects of Reproductive Samples of the Present Invention on Direct Mutation Treated with MNNG Revertants / plate 1.25mg / plate 2.5mg / plate Natural mutation 125 ± 29 ⁹⁾ 125 ± 29 Control 1056 ± 100 ^a 1056 ± 100 ^a Diet Ⅰ ¹⁾ 507 ± 67 ^de (59) ¹⁰⁾ 334 ± 8 ^de (78) Diet Ⅱ ²⁾ 578 ± 105 ^cd (51) 353 ± 22 ^d (76) Diet Ⅲ ³⁾ 453 ± 18 ^e (65) 242 ± 15 ^e (87) Diet Ⅳ ⁴⁾ 398 ± 64 ^e (70) 468 ± 18 ^c (63) Diet A ⁵⁾ 657 ± 32 ^de (43) 517 ± 18 ^c (58) Diet B ⁶⁾ 808 ± 28 ^b (27) 666 ± 38 ^b (42) Diet C ⁷⁾ 607 ± 13 ^cd (48) 284 ± 38 ^de (83) Diet D ⁸⁾ 694 ± 12 ^bc (39) 643 ± 12 ^b (44) ¹⁾ Diet I: Cancer prevention reproduction with ² % by weight of pine needles ²⁾ Diet II: Cancer prevention reproduction with 6% by weight of pine needles ³⁾ Diet III: Cancer prevention with 10% by weight of pine needles Reproduction ⁴⁾ Diet Ⅳ: Cancer prevention with 20% by weight of pine needles ⁵⁾ Diet A: GMF Shirumi reproduction ⁶⁾ Diet B: Healthy village of Dr. Hwang Sung-joo ⁷⁾ Diet C: Mother's love ⁸⁾ Diet D: Maternal Love Co., Ltd. ⁹⁾ Mean ± standard deviation (SD). ¹⁰⁾ represents the inhibition rate. ^{a to e} represent significance (p <0.05) by Duncan's multiple range test.

When the indirect mutant AFB ₁ was treated, the tendency was similar to the results of experiments with MNNG, and the commercial expression of the present invention was more effective at all concentrations. Although there was no significant difference between the reproductions of the present invention, diet III showed the highest inhibition rate of 94%. In the commercial diet, sirumi reproductive (Diet A) showed high effect with 67% inhibition rate (Table 14).

Antimutagenic Effects of Reproductive Samples of the Invention on Indirect Mutations Treated with Aflatoxin B ₁ (AFB ₁ ) Revertants / plate 1.25mg / plate 2.5mg / plate Natural mutation 133 ± 21 ⁹⁾ 125 ± 29 Control 2254 ± 317 ^b 1056 ± 100 ^a Diet Ⅰ ¹⁾ 499 ± 24 ^e (83) ¹⁰⁾ 375 ± 7 ^d (89) Diet Ⅱ ²⁾ 347 ± 7 ^e (90) 305 ± 24 ^d (92) Diet Ⅲ ³⁾ 420 ± 36 ^e (87) 260 ± 35 ^d (94) Diet Ⅳ ⁴⁾ 377 ± 15 ^e (89) 279 ± 35 ^d (93) Diet A ⁵⁾ 837 ± 116 ^d (67) 446 ± 18 ^d (85) Diet B ⁶⁾ 3481 ± 291 ^a (-) 2683 ± 100 ^a (-) Diet C ⁷⁾ 1156 ± 140 ^c (52) 709 ± 89 ^c (73) Diet D ⁸⁾ 1060 ± 55 ^cd (56) 893 ± 149 ^c (64) ¹⁾ Diet I: Cancer prevention reproduction with ² % by weight of pine needles ²⁾ Diet II: Cancer prevention reproduction with 6% by weight of pine needles ³⁾ Diet III: Cancer prevention with 10% by weight of pine needles Reproduction ⁴⁾ Diet Ⅳ: Cancer prevention with 20% by weight of pine needles ⁵⁾ Diet A: GMF Shirumi reproduction ⁶⁾ Diet B: Healthy village of Dr. Hwang Sung-joo ⁷⁾ Diet C: Mother's love ⁸⁾ Diet D: Maternal Love Co., Ltd. ⁹⁾ Mean ± standard deviation (SD). ¹⁰⁾ represents the inhibition rate. ^{a to e} represent significance (p <0.05) by Duncan's multiple range test.

Experimental Example 2> in vivo micronucleus test using reticulocytes of peripheral blood

In order to examine whether the reproduction of the present invention exhibits cancer prevention effects even in> in vivo, micronucleus experiments were conducted using the methanol sample of the reproduction of the present invention. Micronucleus experiments targeting rodent erythrocytes are cytogenic assays for the induction of micronuclei, an abnormal chromosomal component formed during the differentiation of red blood cells. Micronuclei are chromosomal fragments or chromosomes that are spontaneously formed during the differentiation of unnuclear mature erythrocytes from nucleated erythrocyte blasts or by clastogens at the end of cell division. Formed by failure. Acridine orange fluorescence stains with the micronucleus, a DNA component, to emit yellow-green fluorescence, and when combined with RNA, it fluoresces red. Micronucleus experiments using peripheral blood counted reticulocytes of type I, II, and III, and the frequency of micronucleated reticulocytes containing micronucleus was counted to evaluate the significance by statistical treatment by Cochran Armitage method. Singh, PN, Stephens, RE and Schneider, EL J. Radiat. Biol., 66, 23-28 (1994)].

A positive control mitomycin C (mitomycin C; MMC, 0.1mg / ml, USA Sigma) was dissolved in physiological saline and injected intraperitoneally in ICR mice to a body weight of 0.1mL / 10g. Samples were prepared using sterile distilled water and 500 or 1000 mg / kg mice were administered orally.

10 μl of acridine orange distilled water mixture of 1 mg / ml concentration was dropped in the center of the pre-heated slide glass at 70 ° C., then uniformly smeared with a glass rod, dried, and kept at room temperature until sealed and used. Acridine orange-coated slides were made.

48 hours after MMC administration, 5 μl of blood was taken from the tail blood vessels of the mice, dropped onto an acridine orange-smear slide, covered with a cover glass, and left at 4 ° C. for 2 hours to sufficiently react the cells with the acridine orange.

The slides were observed at 40 × 10 times by fluorescence microscope (Olympus, model U-ULH, Japan), and the number of reticulocytes counted from type I to type III, and the number of reticulocytes with micronucleus was counted. Was determined.

48 hours after the tail vein of mice induced micronucleus by injecting micronucleus-inducing mitomycin C (MMC) 6 hours after oral administration at 1,000 mg / kg and 500 mg / kg concentrations in each test group After the blood was collected, the result of counting the incidence of micronuclei is shown in FIG. 6. All groups orally administered the present invention showed significantly higher nucleation-inducing ability than the control group. As a result, the present invention showed superior anti-nuclear proliferation ability than commercially available reproduction A and B because of its excellent cancer prevention function, and showed the highest micronucleus prophylaxis activity in the diet III in which pine needles were added 10% and grains slightly increased.

Experimental Example 3. MTT assay for measuring anticancer enhancement effect in> in vitro for testing anticancer effect of reproduction

Human gastric cancer cell (AGS) single cell suspension was prepared and samples were administered. In the case of cancer cells attached to the culture flask, add 0.05 ml trypsin-EDTA to 2-3 ml, remove 5 ml of RPMI 1640 medium, centrifuge at 1000 × g for 5 minutes, and add a moderate amount of medium to the tube. Single cell suspensions were made. Prior to this experiment, the appropriate inoculation cell number for each well to be used for MTT assay was determined for each cell line. The cell number in the present invention say the optimal number of cells inoculated in cell proliferation and OD ₅₄₀ can fully exhibit a high value actively used in the range of 1~2 × 10 ³ cell / well. After making preliminary experiments to make the appropriate number of cells inoculated according to the cell type, add 100 µl to the 96well plate, add 80 µl of the culture medium, and dilute the sample with PBS (diluted with 0.02% DMSO). After diluting 10 times, 20 μl was added to each well, and only 20 μl of PBS was added to one column as a control. The plate was again incubated for 4 days at 37 ° C., 5% CO ₂ incubator.

Four days later, 0.1 mg (50 μl of 2 mg / mL) of MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide) was added to each well of the plate to determine viable cell count. Was added and incubated for another 4 hours at 37 ° C. to reduce the MTT. After the incubation, formazan (formazan) crystals formed were allowed to settle, and all of the medium was extracted with only 30 μl of the medium remaining. 150 μl of DMSO was added to each well from which the medium was removed, followed by gently shaking for about 5 minutes until the formazan crystal was dissolved. The absorbance was immediately measured at 540 nm with an ELISA reader. This absorbance represents the amount of MTT reduced by the cells and is therefore proportional to the number of viable cells present in each well.

Survival was calculated based on the measured cell number. The mean value of one column from each well was calculated to yield a percentage of the mean value of the control group (100% surviving group). This percentage is a value corresponding to the cell viability of the compared experimental group, the cell survival rate was calculated by the following equation.

Cell viability (%) = T (absorbance of treated cells) / C (absorbance of control cells) × 100

The results are shown in Table 15.

MTT assay results of the inventive reproductive extracts of AGS human gastric cancer cells sample OD ₅₄₀ 25 µg / assay 50 µg / assay Control 0.692 ± 0.079 ^a 0.766 ± 0.028 ^{a 9)} Diet Ⅰ ¹⁾ 0.449 ± 0.027 ^de (35) 0.361 ± 0.063 ^d (53) ¹⁰⁾ Diet Ⅱ ²⁾ 0.536 ± 0.026 ^bc (23) 0.662 ± 0.036 ^b (14) Diet Ⅲ ³⁾ 0.406 ± 0.077 ^ef (41) 0.349 ± 0.036 ^d (54) Diet Ⅳ ⁴⁾ 0.338 ± 0.031 ^f (51) 0.166 ± 0.012 ^e (78) Diet A ⁵⁾ 0.450 ± 0.009 ^de (35) 0.585 ± 0.122 ^bc (24) Diet B ⁶⁾ 0.595 ± 0.010 ^b (14) 0.617 ± 0.009 ^bc (19) Diet C ⁷⁾ 0.367 ± 0.047 ^ef (47) 0.357 ± 0.081 ^d (53) Diet D ⁸⁾ 0.507 ± 0.054 ^cd (27) 0.537 ± 0.027 ^c (30) ¹⁾ Diet I: Cancer prevention reproduction with ² % by weight of pine needles ²⁾ Diet II: Cancer prevention reproduction with 6% by weight of pine needles ³⁾ Diet III: Cancer prevention with 10% by weight of pine needles Reproduction ⁴⁾ Diet Ⅳ: Cancer prevention with 20% by weight of pine needles ⁵⁾ Diet A: GMF Shirumi reproduction ⁶⁾ Diet B: Healthy village of Dr. Hwang Sung-joo ⁷⁾ Diet C: Mother's love ⁸⁾ Diet D: Maternal Love Co., Ltd. ⁹⁾ Mean ± standard deviation (SD). ¹⁰⁾ represents the inhibition rate. ^{a to e} represent significance (p <0.05) by Duncan's multiple range test.

Reproductive I, II, III, IV of the present invention prepared by adding 2% by weight, 6% by weight, 10% by weight, and 20% by weight of pine needles, which were most effective in vegetable screening, and the reproduction A, B, C, D currently on the market After preparing the methanol extract of AGS human gastric cancer cells, the results of the above experiments examined> in vitro anticancer activity, the inhibition rate of the present invention produced higher cancer prevention function than the commercial reproduction was high. In particular, the reproductive diet of the present invention, diet III and diet IV had a high inhibition rate.

Experimental Example 4. Measurement of anticancer enhancement effect using a mouse

First step. Cytotoxicity test

Cytotoxicity is a nonspecific defense mechanism that not only directly damages cancer cells, but has been reported to enhance its cytotoxic effects by stimulating effector cells that have cytotoxic effects on target cells, such as lymphocytes and macrophages in vivo. . To see the effect of direct cell killing on tumor cells, sarcoma-180 tumor cells were seeded, and various concentrations of samples were dispensed into the medium, and the survival rate of the tumor cells was observed after 24 hours.

In order to examine the direct cytotoxicity of the anti-cancer samples, cytotoxicity test was performed in> in vitro using dye exclusion method. 1 ml of sarcoma-180 tumor cell suspension (2.0 × 10 ⁵ cells) and 1 ml RPMI medium of 20% HFCS (heat inactivated fetal calf serum: Gibco Lab., USA) were added to a disposable 24 well plate. 100 μl of pine needle sample was added thereto and incubated in 37 ° C. and 5% CO ₂ incubator for 24 hours. After incubation, 50 µl of the cells were mixed well with 50 µl of 0.2% trypan blue solution, and the total number of cells, non-viable cells and non-stained cells using a hemocytometer. The number of s was measured and the survival rate was calculated by comparison with the control cell group without the sample. The calculation is as follows.

Viable cells (%) = number of viable cells per mL of sample / total cells per mL of sample × 100

The cytotoxicity test results are shown in Table 16.

Cell viability of sarcoma-180 cells in medium containing reproductive samples sample Final conc. (mg / mL) Total cell count (× 10 ⁴ ) Cell survival rate (%) Control 71.2 ± 7.1 ⁵⁾ 95.8 S-180 + Diet Ⅰ ¹⁾ 0.1 62.7 ± 2.1 94.1 0.25 50.8 ± 1.2 93.3 0.5 46.4 ± 2.7 90.1 0.75 34.6 ± 2.4 82.7 1.0 45.2 ± 2.3 83.6 S-180 + Diet III ²⁾ 0.1 56.5 ± 0.7 94.7 0.25 27.3 ± 1.2 93.9 0.5 46.4 ± 0.9 92.2 0.75 39.6 ± 2.1 83.3 1.0 36.0 ± 2.3 78.9 S-180 + Diet A ³⁾ 0.1 113.3 ± 1.0 96.7 0.25 75.2 ± 2.9 94.9 0.5 64.2 ± 1.1 94.7 0.75 63.4 ± 2.2 93.7 1.0 62.6 ± 3.3 91.7 S-180 + Diet B ⁴⁾ 0.1 99.3 ± 0.6 96.3 0.25 81.8 ± 1.1 95.4 0.5 88.2 ± 1.2 95.5 0.75 72.8 ± 1.7 93.9 1.0 75.8 ± 1.6 94.9 ¹⁾ Diet I: Cancer preventive reproduction with ² % by weight of pine needles ²⁾ Diet III: Cancer prevention reproduction with 10% by weight of pine needles ³⁾ Diet A: Shirumigi reproduction of GMF Corporation. ⁴⁾ Diet B: Natural reproduction ⁵⁾ of Dr. Hwang Sung-joo of Healthy Healthy Village Co., Ltd. shows the mean ± standard deviation (SD) calculated three times. ^{a to e} represent significance (p <0.05) by Duncan's multiple range test.

Overall survival was decreased at concentrations of 0.5 mg / ml and later, and the concentrations were selected and the following solid cancer growth inhibition effect experiments were performed within a range that does not significantly affect sarcoma-180 tumor cells.

Second step. Solid rock growth inhibition effect

24 hours after subcutaneous transplantation of 0.2 ml (6.0 × 10 ⁶ cells / mouse) of sarcoma-180 tumor cell suspension, which is passaged in the laboratory at weekly intervals, into the left groin of 6 selected animals. Sample solutions were injected intraperitoneally once daily. On the 26th day of tumor cell transplantation, the solid tumors produced by lethality were extracted and weighed, and then the tumor growth inhibition ratio (IR:%) was calculated according to the following equation. Where Cw is mean tumor weight of control group and Tw is mean tumor weight of treatment group.

I.R. (%) = (Cw-Tw) / Cw x 100

Antitumor Activity of Reproductive Extracts in Mice Injected with Sarcoma-180 Tumor Cells sample Tumor Weight (g) Inhibition Rate (%) S-180 + PBS 3.80 ± 0.73 ^{a, 5)} S-180 + Diet Ⅰ 2.42 ± 1.24 ^bc 36 ⁶⁾ S-180 + Diet Ⅲ 1.84 ± 0.70 ^c 52 S-180 + Diet A 3.36 ± 0.19 ^b 12 S-180 + Diet B 3.65 ± 0.45 ^b 4 ¹⁾ Diet I: Cancer preventive reproduction with ² % by weight of pine needles ²⁾ Diet III: Cancer prevention reproduction with 10% by weight of pine needles ³⁾ Diet A: Shirumigi reproduction of GMF Corporation. ⁴⁾ Diet B: Natural reproductive health of Dr. Hwang Sung-joo, Ph.D., of Love Village, Ltd. ( ⁵⁾ shows the mean ± standard deviation (SD) calculated three times. ⁶⁾ represents the inhibition rate. ^{a to e} represent significance (p <0.05) by Duncan's multiple range test.

In the PBS-only group, the solid cancer weight was 3.79 g, whereas the reproductive diet I of the present invention with 2% by weight of pine needles was 2.42 g, and the diet III with 10% by weight of pine needles was 1.84 g with inhibition rate of 36, respectively. %, 52%. On the contrary, GMF Corporation's Shirumi Reproductive System (Diet A) had a solid cancer weight of 3.36g, an inhibition rate of 12%, and Dr. Hwang Sung-ju's natural reproduction (Diet B) of 3.65g of solid cancer. As a result, a low inhibition rate of 4% was seen. > In vivo, the anti-cancer properties of the present invention (Diet I, III) prepared in consideration of cancer prevention are higher than those of commercially available reproduction (Diet A, B).

In addition, in order to measure the effect of the sample on other organs, the tumor cell group was injected with PBS and the sample treatment group was administered by injection for 20 days after tumor cell transplantation with the same amount of methanol extract as the control group. As a result, the weight of the liver, which is responsible for the detoxification of various harmful substances generated during metabolism, was different from that of the control group, dietary group of GMF Co., Ltd. (Diet A) and natural health (Diet B) of Dr. Hwang Sung-joo of Love Health Village Co., Ltd. The same result was observed in the case of spleen. Organs such as heart and kidney did not differ significantly between groups (Table 18).

Changes in Weight of Mouse Organs with Reproductive Samples sample Body weight (g) Spleen / Weight (wt%) Liver / Weight (% by weight) Heart / weight (% by weight) Height / weight (% by weight) Control 23.25 ± 1.69 ⁵⁾ 0.42 ± 0.00 4.78 ± 0.10 0.56 ± 0.00 2.35 ± 0.34 s-180 + PBS 26.10 ± 1.31 0.96 ± 0.03 6.77 ± 0.39 0.53 ± 0.01 1.05 ± 0.16 s-180 + Diet I ¹⁾ 27.08 ± 1.28 0.72 ± 0.02 5.88 ± 0.11 0.58 ± 0.02 1.55 ± 0.02 s-180 + Diet II ²⁾ 26.94 ± 1.58 0.69 ± 0.02 6.66 ± 0.22 0.59 ± 0.03 1.73 ± 0.05 s-180 + diet A ³⁾ 26.24 ± 1.34 0.85 ± 0.01 7.32 ± 0.45 0.55 ± 0.03 1.62 ± 0.05 s-180 + diet B ⁴⁾ 25.95 ± 1.96 0.91 ± 0.04 7.09 ± 0.29 0.50 ± 0.03 1.48 ± 0.04 ¹⁾ Diet I: Cancer preventive reproduction with ² % by weight of pine needles ²⁾ Diet III: Cancer prevention reproduction with 10% by weight of pine needles ³⁾ Diet A: Shirumigi reproduction of GMF Corporation. ⁴⁾ Diet B: Natural reproduction of Dr. Hwang Sung-joo of Love Health Village Co., Ltd. ⁵⁾ shows the mean ± standard deviation (SD).

Third step. Enzyme Activity Measurement

An enzyme source for measuring enzyme activity was prepared.

After killing the rats, the liver was perfused with saline of 4 ° C. or lower to remove blood remaining in the liver, and the liver was extracted. 0.1 M potassium phosphate buffer (pH 7.4) was added per gram of liver tissue and ground with a glass teflon homogenizer under ice cooling. This grinding solution was used as a homogenate fraction, which was centrifuged at 13,000 rpm for 10 minutes to remove the nucleus and unbranched cell sections, and the supernatant obtained by ultracentrifugation at 105,000 × g for 1 hour was used as the cytosol fraction. . The homogeneous fraction thus obtained was used to measure glutathione content and cytosol fraction to measure glutathione-S-transferase activity.

The activity of glutathione-S-transferase was measured. Habig et al., Habig, W.H., Pabist, M.J and jakoby, W.B. J. Biol. Chem., 249, 7130-7139 (1974), add 75 µl of 0.04 M reduced glutathione and 0.1 ml of enzyme solution in 0.1 M potassium phosphate buffer (pH 6.5), and add 20% trichloroacetic acid in blank. acid) 0.5 ml was added to inactivate the enzyme and allowed to react at 25 ° C. for 5 minutes. 25 µl of 0.12M 1-chloro-2,4-dinitrobenzene was added to the blank and the sample, and the mixture was reacted at 25 ° C. for 2 minutes. After the reaction was completed by adding 20% trichloroacetic acid to the sample, the supernatant obtained by centrifugation was absorbed at 340 nm. The activity was calculated using the mole extinction coefficient of 9.6mM-1cm-1 of 1-chloro-2,4-dinitrobenzene. The unit of enzyme activity was expressed as nmole number of 2,4-dinitrobenzene-glutathione produced by 1 mg of protein for 1 minute.

GST activity in the liver (nmol / mg protein / min) was found to be 296.22 in the normal group, whereas the activity was increased to 300.69 in the control group injected with sarcoam-180. And the reproductive diet I and diet III of the present invention the activity was higher than the control group, diet A and B showed a somewhat lower result. The results are shown in FIG.

The content of glutathione in the tissue was measured. Ellamn's Method [Ellamn, G.L. Arch.Biochem.Biophys., 193, 265 (1951)], disulfide reagent (0.1 M 2.7 ml of 5,5'-dithiobis (2-nitrobenzoic acid) was dissolved in sodium phosphate buffer pH8.0), and the resulting blue color was absorbed at 412 nm and calculated according to the standard curve.The unit is glutathione μmole per gram of tissue. Marked as.

In the normal group, the glutathione content was as high as 0.288, but in the control group in which the tumor was transplanted, it was as low as 0.215. However, the content was higher in the group administered the raw methanol extract sample. The glutathione content of the genus I and III of the present invention was higher than that of the commercially available types A and B, and the diet III showed the highest content of 0.303 (Fig. 8).

Based on the above experimental results, the genital genus of the present invention, which has been developed to increase cancer prevention function, has a higher cancer prevention and anticancer effect than the existing commercially available reproductive organs, and in particular, 10% added pine needles and slightly increased grains in the diet. Phosphorus diet Ⅲ showed the highest cancer prevention and anticancer effect.

Example 4 Development and Sensory Evaluation of Cancer Preventive Reproduction of the Present Invention

First step. Diet composition of the present invention and method of taking

Through the above examples and experimental examples, 10% by weight of pine needles are added, grains slightly increased, the reproductive diet III excellent in cancer prevention and anticancer effect was developed as cancer prevention reproduction of the present invention. The diet composition is shown in Table 19.

Cancer Prevention and Anticancer Reproductive Diet Formula food Weight per unit (g) Protein (g) Fat (g) Carbohydrate (g) Calories (kcal) Brown rice 270.0 (36.3% by weight) 19.4 6.8 207.4 993.6 Rule 120.0 (16.2 wt%) 25.6 4.4 73.3 447.6 Sorghum 120.0 (16.2 wt%) 12.4 5.6 83.4 403.2 Black Beans 30.0 (4.0% by weight) 12.5 5.3 5.6 123.9 pine needles 75.0 (10.1% by weight) 3.4 2. 14.7 120.8 Myeong Il-yeop 15.0 (2.0% by weight) 0.4 0.0 1.1 5.9 purslane 15.0 (2.0% by weight) 0.4 0.1 1.1 6.3 Kale 15.0 (2.0% by weight) 0.7 0.1 0.3 4.5 Kelp 5.0 (0.7% by weight) 0.3 0.1 1.9 10.1 Parsley 15.0 (2.0% by weight) 0.3 0.2 0.5 5.3 Perilla 63.0 (8.5% by weight) 10.1 24.9 12.7 340.2 gun 743.0 (100.0% by weight) 85.5 50.5 402 2461.3

The amount of the intake of the present invention reproductive diet was set to 50g per package, the amount of calories per package (50g) is 165kcal, protein 5.8g, fat 3.4g, carbohydrate 16.2g is included.

Since this diet is used without ripening cereals and soybeans, the use of soy milk can reduce fishy odor.

Second step. Sensory Evaluation of Cancer Prevention Reproduction of the Present Invention

Among the reproductions made by enhancing cancer prevention function, the cancer prevention reproduction of the present invention having high cancer prevention and anticancer effect and no risk of toxicity was compared with the natural reproduction and preference of commercial reproduction mother love.

The two reproductions were presented by putting 50 g of a bag in 200 mL of soymilk. Eight sensory personnel evaluated two samples and expressed them in 9-point conversion. The description items were color, odor, taste, and comprehensive evaluation. The closer to 1, the better.

As a result, the organoleptic scores of the reproductive system of the present invention prepared in consideration of cancer prevention function as a whole were high. In particular, higher scores were obtained for odor and taste (Table 20).

In other opinions, there was an opinion that mother's love was too bitter to eat raw food, and the raw food of the present invention had a lot of fishy taste. There was also an opinion that it would be nice to eat it with honey because of its lack of sweetness. Also, are you willing to buy the above reproductive system? The answer was that if the functional reproduction would accept. In addition, the question 'Is reproductive sachets (reproduction 165kcal + soymilk 120kcal = 285kcal) suitable as a substitute for one meal?' Came out similarly to the opinion that it was insufficient.

Sensory Evaluation of Reproductive and Mother-Love Reproduction of the Invention color smell flavor Comprehensive evaluation Mom love reproduction 5.8 ± 0.7 4.9 ± 0.8 4.5 ± 1.3 5.4 ± 1.1 Invention reproductive 6.3 ± 1.6 6.4 ± 1.1 6.0 ± 1.9 6.1 ± 1.1 [Note] We display by nine points conversion method

As described above, the present invention, as described through the above Examples and Experimental Examples, the present invention is 30.0 to 40.0% by weight brown rice, 10.0 to 20.0% by weight, sorghum 10.0 to 20.0% by weight, black beans 3.0 to 5.0% by weight, pine needles 2.0 to 20.0 weight %, 1.0-6.0% by weight, 1.0-6.0% by weight of purslane, 1.0-6.0% by weight of kale, 0.3-1.0% by weight of kelp, 5.0-10.0% by weight of perilla, to prepare a cancer preventive reproductive composition and antimutagenicity of the reproduction The effects of anti-cancer and anti-cancer effects on> in vitro and> in vivo showed that it has a superior effect of providing cancer prevention reproductive compositions with excellent solid cancer growth retardation, anticancer, antimutagenic and immune enhancing effects. It is a very useful invention for industrial and national health promotion.

Claims (2)

Brown rice 30.0 ~ 40.0%, yulmu 10.0 ~ 20.0%, sorghum 10.0 ~ 20.0%, black soybeans 3.0 ~ 5.0%, pine needles 2.0 ~ 20.0%, light green leaf 1.0 ~ 6.0%, purslane 1.0 ~ 6.0%, kale A cancer prevention reproduction composition comprising 1.0 to 6.0% by weight, kelp 0.3 to 1.0% by weight, and perilla perilla 5.0 to 10.0% by weight. A cancer prevention food prepared by adding 45 to 55 g of the cancer prevention reproduction composition according to claim 1 to 200 mL of soy milk and eating it as a meal substitute.