WO2006129901A1

WO2006129901A1 - Anticancer agent, food and manufacturing method of anticancer agent and food

Info

Publication number: WO2006129901A1
Application number: PCT/KR2005/002523
Authority: WO
Inventors: Tae-Sook Kim; Ok-Ja Kim
Original assignee: Blc Bio Tech Inc.
Priority date: 2005-06-01
Filing date: 2005-08-03
Publication date: 2006-12-07
Also published as: KR20060125075A

Abstract

The present invention relates to anticancer agents, and health functional foods and other functional foods with anticancer function, which are obtained 5 from Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai, as well as preparation methods thereof. More particularly, the present invention relates to preparing an extract or powder from a mixture of Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai so as to make anticancer agents, and feeding livestock with a dilution of the extract in order to be able to obtain anticancer functional foods, such as meat and milk, and also using the extract or concentrated powder to prepare anticancer healthy functional foods, such as tablets and capsules.

Description

ANTICANCER AGENT, FOOD AND MANUFACTURING METHOD OF ANTICANCER AGENT AND FOOD

Technical Field The present invention relates to anticancer agents, and health functional food and other functional foods with anticancer function, which are obtained from Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai, as well as preparation methods thereof. More particularly, the present invention relates to preparing an extract or powder from a mixture of Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai so as to make anticancer agents, and feeding livestock with the diluted extract in order to be able to obtain anticancer functional foods, such as meat and milk, and also using the extract or concentrated powder to prepare anticancer healthy functional foods, such as tablets or capsules.

Background Art Sargassum fulvellum, which is a brown alga widely distributed in the sea around Korea, Japan and the like, is 1-3 m in height and has a disk shape at the lower portion. Its branches are prismatic or triangular in shape and twisted. Its leaves are curved downward in a spoon or oval shape and have a weak midrib up to their middle. Its upper leaves resemble a needle, have a saw-tooth portion without a midrib, and are yellowish brown in color. Also, it has several air vesicles which are different in shape according to the position and larger in the base of the branches than in the upper portion. Its generative organs are long cylindrical in shape, have a short stem, and are arranged in an overall shape. In addition, it grows on rocks at the low-water line, is used as food and feed and distributed in Korea, Japan and so on.

Test results for cervical cancer cells showed that the survival rate of the cancer cells was 0.6% when treated with 1,600 μg/ml of a Sargassum fulvellum extract, indicating that the extract had a carcinostatic activity of

99.4% (Korean patent application No. 10-2003-0002053).

Meanwhile, Gleditsia japonica var. koraiensis Nakai has entire leaflets, small thorns and turned pods, and are distributed in the whole area of Korea, Japan, China and so on. One with no thorn is called "Gleditsia japonica for. inarmata"^', one with fruits slightly crooked without twisting is called "Gleditsia japonica var. stenocarpa", one with thick thorns round in cross section and untwisted fruits is called "Gleditsia sinensis", and one with small leaves of 15-35 mm, thorns round in cross section similarly to Gleditsia sinensis and untwisted pods similarly to Gleditsia japonica var. stenocarpa is called "Gleditsia japonica" . This plant belongs to Gleditsia Linn. However, there is still no suitable method of using Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai, which are frequently distributed in Korea.

Disclosure of Invention Accordingly, it is an object of the present invention to use Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai to prepare anticancer agents, health functional foods and other functional foods, which have excellent anticancer function. To achieve the above object, the present invention provides anticancer agents, health functional foods and other functional foods with anticancer function, which comprise a mixture of Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai, as well as preparation methods thereof. A method for making a mixture of Sargassum fulvellum with Gleditsia japonica var. koraiensis Nakai in the anticancer agents comprises: finely cutting Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai; making the amounts of portions of each of the plants (in the case of Gleditsia japonica var. koraiensis Nakai, the term "portions of each of the plants" means leaf, fruit and thorn portions and has the same meaning below) ; mixing 5- 15% by weight of the Gleditsia japonica var. koraiensis Nakai powder with the Sargassum fulvellum powder in an amount of 5-15% by weight based on the total weight of the Sargassum fulvellum powder; and subjecting the mixture to hot water extraction with distilled water. Another method comprises: finely cutting Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai; making the amounts of portions of each of the plants; drying the Sargassum fulvellum pieces to a water content of 15-25%; crushing each of the cut plants to a size of 50-150 mesh to form powder, and mixing the Gleditsia japonica var. koraiensis Nakai powder with the Sargassum fulvellum powder in an amount of 5-15% by weight based on the total weight of the Sargassum fulvellum powder.

Also, the present invention provides a method for preparing health functional foods with anticancer function, such as tablets, capsules, pills and granules, the method comprising: feeding hens with the above powder or extract; removing the shell of eggs laid by the hens, crushing the eggs to form powder, and using the powder to prepare health functional foods.

Also, the present invention provides a method for preparing anticancer functional foods, the method comprising: allowing livestock either to eat feed containing the powder or to drink a dilution of the extract, and obtaining meat from the livestock; or allowing milking cows either to eat feed containing the powder or to drink a dilution of the extract, and obtaining milk from the milking cows. In addition, the present invention provides functional foods with anticancer function, which contain the powder or extract added to food powder.

Hereinafter, the anticancer properties of concentrated BLC powder and concentrated BLC egg powder according to the present invention will be described.

Concentrated BLC egg powder produced by laying hens which have taken a BLC extract for 30 days without dilution resulted in a 30% reduction in the number of breast cancer cells at a concentration of 400 μg/ml in cell tests, and a 71.4% reduction in the weight of breast cancer tumors at a dose of 1000 mg/kg/day in animal tests (the term "kg" means the body weight and has the same meanings below) .

In order to examine a mechanism where BLC and BLC/HEN eggs inhibit the growth and proliferation of human breast cancer cells, the cells were treated with BLC and BLC/HEN eggs at a concentration of 100 μg/ml for 48 hours followed by Hoechst 33258 staining, and the morphology of the cells was analyzed. As a result, as shown in FIG. 1, the cells treated with BLC and BLC/HEN eggs showed a reduction in their proliferation as compared to a control group (untreated cells) . Also, in apoptosis-induced cells, it could be observed that nucleic acid fragmentation and concentration, which are the characteristic phenomena of apoptosis, were induced.

Also, in order to examine if the apoptosis of human breast cancer cells is induced when exposed to BLC and BLC/HEN eggs, DNA laddering was performed in which MCF-7 cells were treated with BLC and BLC/HEN eggs at a concentration of 100 μg/ml for 48 hours. As a result, as shown in FIG. 2, a ladder was formed where characteristic DNA fragmentation could be observed.

The apoptosis mechanism of breast cancer cells by such BLC eggs was considered to occur by the cleavage of PARP, a DNA repair-related death substrate, by increasing the expression of a Bax protein promoting cell death and at the same time, reducing the expression of a Bcl-2 protein inhibiting cell death.

Also, it was found that the BLC eggs inhibited the proliferation of human breast cancer cells by increasing the expression of a p53 protein, a typical cancer inhibitory gene of the human body.

Also, the concentrated BLC egg powder was added to EtOH to make a stock, diluted in media at varying concentrations, and analyzed for an inhibitory effect on the growth of human breast cancer cells for 72 hours. As a result, as shown in FIG. 5, the proliferation of breast cancer cells was reduced in a concentration-dependent manner, and particularly, the number of human breast cancer cells treated with a concentrated powder of BLC eggs was shown to be 30% of the control group (untreated cells) at a concentration of 400 μg/ml.

And, nude mice were allowed to freely eat feed obtained by blending an AIN-76A diet with 4,000 ppm of a concentrated powder of BLC eggs and allowed to freely drink tap water sterilized with vapor under high pressure. During 28 days before implanting MCF-7 cells into the mice, the mice were allowed to eat the feed containing the concentrated BLC egg powder, and at day 29, the mice were implanted with MCF-7 cells, human breast cancer cells. And, during 28 days (day 29 to day 56) after the cell implantation, the mice were allowed to eat the same amount of the feed containing the concentrated BLC egg powder, subjected to autopsy and measured for the weight of tumors by autopsy. As a result, as shown in FIGS. 3 and 4, the weight of tumors in the animals treated with the BLC egg powder was 71.4% reduced as compared to a control group untreated with the BLC egg powder.

And, the concentrated BLC powder was added to EtOH to make a stock, diluted in media at varying concentrations, and analyzed for an inhibitory effect on human breast cancer cells for 72 hours. As a result, as shown in FIG. 5, the proliferation of breast cancer cells was reduced in a concentration-dependent manner. Particularly, at a concentration of 400 μg/ml, the number of breast cancer cells in the animals treated with the concentrated BLC powder was shown to be 62% of a control group untreated with the concentrated BLC powder.

And, nude mice were allowed to freely eat feed obtained by blending an AIN-76A diet with 2,000 ppm of the concentrated BLC powder and allowed to freely drink tap water sterilized with vapor under high pressure. During 28 days before implanting MCF-7 cells into the mice, the mice were allowed to eat the feed containing the concentrated BLC powder, and at day 29, the mice were implanted with MCF-7 cells, human breast cancer cells. And, during 28 days (day 29 to day 56) after the cell implantation, the mice were allowed to take the same amount of the feed containing the concentrated BLC egg powder, subjected to autopsy and measured for the weight of tumors by autopsy. As a result, as shown in FIGS. 3 and 4, the weight of tumors in the animals treated with the concentrated BLC powder was 42.1 reduced as compared to a control group untreated with the concentrated BLC powder.

The above tests for BLC extracts and BLC/HEN eggs were simultaneously performed in College of Veterinary Medicine, Seoul National University.

Also, Sargassum fulvellum contains Ig protein, policonoid, viotinogen, asphaltic acid, alaninogen, nitrominoid, linanogen, geroniolein, rivoflavinoid and the like.

Among them, Ig protein was verified to inhibit the growth of prostate cancer cells (Koch S; 2000), and policonoid was proven to have inhibitory activity against the metastasis and proliferation of adult female cancer cells (Adamek H E; 1999) and activities for the production of human blood cells, the development of enzymes, the activation of cerebral cells, the inhibition of production of the main enzyme of cervical cancer cells (HeLa cells) , and the inhibition of cancer development in the human neural tube (Cus kelly G; 1999) .

And, biotinogen was proven to be effective in treating human pancreatic cancer (Murat A; 2000), and avidin-biotin-peroxidase was demonstrated to function to inhibit human esophagus cancer (Raida M; 1999) .

Asphaltic acid was verified to function to grow human antitumor erythrocytes (Gigoux V, Univ. Rang. France; 1999), alaninogen was found to have activity to inhibit the growth of cancer cells in a cerebral nerve (Wu S N;2000), and nitroxninoid was proven to inhibit the proliferation of lymphoma leukemic cells (gall cancer cells) (J Tokida; 1959) , and have the effect of treating a cancer tumor cell line (White I N; 1989) .

Linanogen was proven to have the effect of inhibiting the growth of cancer cells in the cell membrane of a human nervous system (Elisabetsky E; 1999) , geroniolein was proven to have activity to inhibit the growth of human pulmonary cancer cells and skin cancer cells (Yu S G, Wisconsin Univ.; 1995), and rivoflavinoid was found to have the effect of treating human oral cancer and pharyngeal cancer (Negri E; 2000) .

Particularly, Gleditsia japonica var. koraiensis Nakai contains nine components consisting of Gleditsia- saponin, ceryl alcohol, nonacosan, stigmasterol, sitosterol, fisetin, gleditsin, fustin, and triacantine. Among them, Gleditsia-saponin has anticancer, antibacterial, wound drainage, antiphlogistic, and phlegm discharge effects, and G. sapogenin is divided into triterpenoid and steroid components, which are non- carbohydrates produced by the decomposition of G. saponin. Gledigenin and gledinin have the effects of G. sapogenin.

Olenolic acid has the same kind of effects as triterpenoid, and ethynocystic acid has the effect of promoting the respiration of normal cells, and prosapogenin, which is the precursor glycoside of sapogenin, has the effects of G. sapogenin.

Pentose consists of aldose and ketose, and arabinose is a kind of altopentose and has the precursor function of ethynocystic acid.

Thus, according to the present invention, it is possible to obtain anticancer agents, and health functional foods and other functional foods with anticancer properties, using Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai, which are easily obtainable in Korea.

Brief Description of Drawings FIG. 1 is a photograph showing apoptosis by BLC and BLC/Hen eggs according to the present invention.

FIG. 2 shows DNA ladders by BLC and BLC/HEN eggs according to the present invention.

FIG. 3 is a graphic diagram showing changes in the size of tumors by BLC and BLC/HEN eggs according to the present invention.

FIG. 4 is a photograph showing changes in the size of tumors by BLC and BLC/HEN eggs according to the present invention . FIG. 5 is a graphic diagram showing the effect of the inventive BLC and BLC/HEN eggs on the inhibition of growth of breast cancer cells.

Best Mode for Carrying Out the Invention Hereinafter, preferred examples of the present invention will be described in more detail. 1. Preparation of BLC extract

Sargassum fulvellum and Sargassum fulvellum Gleditsia japonica var. koraensis Nakai are collected, selected, estimated for their origin, and identified. Then, the collected plants are finely cut, and the amounts of portions of each of the cut plants are made equal to each other, and 5000 g of the Sargassum fulvellum pieces and 500 g of the Gleditsia japonica var. koraensis Nakai pieces are taken. Then, the mixed plant is placed in a round bottom flask double extractor to which 15-20 liters (18 liters in Example of the present invention) of distilled water is added. Then, the mixed plant is subjected to sterile extraction by boiling at 150-200 ⁰C (160 ⁰C in Example of the present invention) , followed by filtration, thus preparing about 9 liters of an extract (hereinafter, referred to as a "BLC extract") .

2. Preparation of BLC powder

Sargassum fulvellum and Sargassum fulvellum Gleditsia japonica var. koraensis Nakai are collected, selected, estimated for their origin, and identified. Then, the collected plants are finely cut, and the amounts of portions of each of the cut plants are made equal to each other. The Sargassum fulvellum powder is dried to a water content of 15-25%. Then, 5000 g of the Sargassum fulvellum pieces and 500 g of the Gleditsia japonica var. koraensis Nakai pieces are taken, crushed into a size of 100 mesh, and mixed with each other, thus preparing powder (hereinafter, referred to as "BLC powder") .

3. Preparation of concentrated BLC powder

The BLC extract is freeze-dried at a temperature of - 40 ⁰C to -50 ⁰C and a vacuum pressure of 1 x 10^"1 mb for 20- 40 minutes (30 minutes in Example of the present invention), thus preparing powder (hereinafter, referred to as "concentrated BLC powder") .

4. Preparation of anticancer agents with concentrated BLC powder Using the concentrated BLC powder as a main raw material, agents for the prevention and treatment of breast cancer are prepared in the form of tablet, capsule, pill, granule, powder and liquid formulations. The tablets are made by compressing the concentrated BLC powder into a given shape, the capsules are made by filling the concentrated BLC powder in capsules or encapsulating the concentrated BLC powder with a capsule base, the granules are made by forming the concentrated BLC powder into granules, the powders are prepared by making the particle size of the concentrated powder smaller than that of granules, and the liquid formulation is made of the concentrated BLC extract.

5. Preparation of BLC eggs The BLC extract is fed to laying hens without dilution for 20-40 days (30 days in Example of the present invention) . The daily drinking amount of laying hens in laying week 2 is about 190 ml at a suitable henhouse temperature of 21 ⁰C. Alternatively, feed is prepared by adding 30-50% by weight (45.5% by weight of Sargassum fulvellum and 4.5% by weight of Gleditsia japonica var. koraensis Nakai in Example of the present invention) of the above-prepared powder to general feed for laying hens. The feed containing the mixed powder of Sargassum fulvellum and Gleditsia japonica var. koraensis Nakai powder was fed to laying hens for 20-40 days (30 days in Example of the present invention) . The daily amount of feed taken by a laying hen weighing 1.8 kg is about 120 g. The characteristics of BLC eggs laid by hens raised as described above are as follows. General eggs have an ovum which is yellow in color and smells a little bloody after the removal of the shell, whereas the inventive anticancer eggs are light reddish in color and have a good odor and taste without a bloody smell. 6. Preparation of concentrated BLC egg powder

The BLC eggs of which shell has been removed are freeze-dried at a temperature of -40 ⁰C to -50 ⁰C and a vacuum pressure of 1 x 10^"1 mb for 30 minutes and crushed to form powder. 7. Preparation of health functional foods with anticancer property

Functional foods are prepared by adding the BLC extract to each food at a concentration of 25-100 μg/ml, or adding the concentrated BLC powder to each food at a concentration of 500-1,000 ppm, or adding the BLC powder to each food at a concentration of 5-20% by weight based on the total weight of the food, or adding the concentrated BLC egg powder to each food at a concentration of 2,000- 4,000 ppm. Examples of the foods include gruel, soup, tea, noodle, bread, wheat flour, drinks, milk products, ice creams, gums, and the like.

Also, it is possible to prepare highly efficient health functional foods, such as tablets, capsules, pills, granule and powders, which contain the concentrated BLC powder in an amount of 50-80% by weight based on the total weight of powders (e.g., various cereal powders, such as unhulled rice powders, and herbal powders, such as Angelica gigas and Back-Bo-Ryoung) .

8. Preparation of functional milk with anticancer properties The BLC extract is diluted in distilled water at a ratio of 1 (BLC extract): 40-60 (distilled water) (1:55 in Example of the present invention) , and the dilution is fed to milking cows for 90-120 days (90 days in Example of the present invention) . Milk is obtained from the milking cows. A milking cow weighing 300 kg drinks about 270 ml of the dilution.

Alternatively, feed is prepared by adding the BLC powder to general feed for milking cows in an amount of 5- 15% by weight (10% by weight in Example of the present invention) based on the total weight of the general feed. The prepared feed is fed to milking cows, and milk is obtained from the milking cows.

9. Preparation of functional foods with anticancer properties

In addition, the BLC extract is diluted in distilled water at a ratio of 1 (extract) : 40-60 (distilled water) (1:55 in Example of the present invention). Alternatively, the BLC powder is added to general feed for each livestock in an amount of 5-15% by weight (10% by weight in Example of the present invention) based on the total weight of the general feed. The prepared dilution or feed is fed to cows and pigs for 90-120 days (90 days in Example of the present invention) or fed to chicken and ducks for 30-40 days (30 days in Example of the present invention) . From the raised animals, functional beef, pork, chicken and duck meat with anticancer properties are obtained.

Industrial Applicability Although anticancer tests in Example of the present invention have been conducted for breast cancer, various components contained in Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai show anticancer effects against various cancers in addition to breast cancer. Thus, the present invention will be applicable against various cancers including breast cancer.

Claims

What Is Claimed Is:

1. An anticancer agent prepared by mixing Gleditsia japonica var. koraiensis Nakai with Sargassum fulvellum in an amount of 5-15% by weight based' on the total weight of Sargassum fulvellum.

2. A method for preparing an anticancer agent, comprising the steps of: finely cutting Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai, making the amounts of portions of each of the cut plants equal to each other, adding the Gleditsia japonica var. koraiensis Nakai pieces to the Sargassum fulvellum pieces in an amount of 5-15% by weight based on the total weight of' the Sargassum fulvellum pieces, adding 1,100 g of the mixture to 3-4 liters of distilled water, subjecting the plant solution to hot water extraction at a temperature of 150-200 ⁰C for 3-5 hours, followed by filtration, thus preparing an extract; freeze-drying the extract at a temperature of -40 ⁰C to -50 ⁰C and a vacuum pressure of 1 x 10^""1 mb for 20-40 minutes, thus preparing concentrated powder; and using the concentrated powder as a main raw material to prepare any one formulation selected from the group consisting of tablet, capsule, pill, granule, powder and liquid formulations.

3. A method for preparing health functional foods with anticancer properties, the method comprising: adding an extract obtained in the extraction step of Claim 2 to each of foods, including gruel, soup, tea, noodle, bread, wheat flour, drinks, milk products, ice creams, and gums, at a concentration of 25-100 μg/ml, thus preparing health functional foods; or adding the concentrated BLC powder obtained in the concentrated powder preparation step of Claim 2 to each of the foods at a concentration of 500-1,000 ppm, thus preparing health functional foods; or adding the concentrated powder to food powder in an weight of 50-80% by weight based on the total weight of the food powder, thus preparing highly efficient health functional foods in the form of any one of tablet, capsule, pill, granule and powder formulations.

4. A method for preparing health functional foods with anticancer properties, the method comprising the steps of: finely cutting Sargassum fulvellum and Gleditsia japonica var. koraiensis Nakai, making the amounts of portions of each of the cut plants equal to each other, drying the Sargassum fulvellum pieces to a water content of 15-25%, crushing each of the cut plants into a size of 50-150 mesh to form powder, adding the Gleditsia japonica var. koraiensis Nakai powder to the Sargassum fulvellum powder in an amount of 5- 15% by weight based on the total weight of the Sargassum fulvellum powder, and adding the mixed powder as a food additive to each of foods, including gruel, soup, tea, noodle, bread, wheat flour, drinks, milk products, ice creams, and gums, at a concentration of 5-20% by weight based on the total weight of the food.

5. A method for preparing health functional foods, comprising the steps of: removing the shell of eggs obtained by allowing laying eggs either to eat feed containing general feed and

30-50% by weight, based on the total weight of the feed, of a mixed powder of Claim 4, for 20-40 days, or to drink an extract of Claim 2; freeze-drying the eggs at a temperature of -40 ⁰C to -50 °C and a vacuum pressure of 1 x 10^'1 mb for 20-40 minutes, thus preparing concentrated egg powder; and adding the concentrated egg powder as a food additive to each of foods, including gruel, soup, tea, noodle, bread, wheat flour, drinks, milk products, ice creams, and gums, at a concentration of 2,000-4,000 ppm.

6. Health functional foods with anticancer properties, prepared by a method as set forth in Claim 4 or 5.

7. A method for preparing functional foods with anticancer properties, the method comprising the steps of: diluting a BLC extract of Claim 2 in distilled water at a ratio of 1 (BLC extract) : 40-60 (distilled water) or adding a powder of Claim 4 to general feed for each livestock in an amount of 5-15% by weight based on the total weight of the feed; feeding the dilution or feed to milking cows, cattle or pigs for 90-120 days or to chicken or ducks for 30-40 days; and obtaining functional milk and meat from the animals.

8. Health functional foods with anticancer properties, prepared by a method as set forth in Claim 7.