JP2001031585A - Anticancer seaweed extract - Google Patents

Abstract

PROBLEM TO BE SOLVED: To find out a component exhibiting high anticancer effect and safety from daily foods and develop a method of efficiently extracting it. SOLUTION: By extracting a seaweed, particularly a brown algae, with an organic solvent, e.g. methanol, ethanol, propanol, acetone or hexane, or the hydrous solvent containing a suitable concentration of water, the substance of inducing cell apoptosis or a mixture between the substance and an immunopotentiator is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a novel cancer inhibitor comprising seaweed extract which induces apoptosis in cancer cells, a method for extracting the same, and a component which induces immunostimulation together with a component which induces apoptosis. How to do it.

[0002]

2. Description of the Related Art In 1981, cancer surpassed cerebrovascular disease and became the leading cause of death in Japan in 1981, and the number of deaths from cancer has continued to increase. In order to reduce the number of deaths caused by this cancer, various synthetic chemical drugs have been clinically used as anticancer drugs, but these drugs cannot avoid serious side effects due to their properties. .

[0003] Under such circumstances, attention has been focused on food components that are effective in suppressing cancer (Food Phytochemical in Cancer Prevention Ian).
d II, ACS, Washinton (1994)).

Seaweed, which has been used as food for a long time in Japan and Korea, absorbs nutrients in the sea, which is the source of life, and is rich in vitamins, minerals, dietary fiber, etc. It has been popular as a food material, but recently its function of preventing lifestyle-related diseases has also attracted attention (J. Nutr. 129, 146-151, 1999).

[0005] Regarding seaweed components, many anticancer activities have been reported by polysaccharides, such as fucoidan and alginic acid, which are water-soluble components.
269 (1983): Kitasato Arch. Of Exp. Med., 60, 105
(1987): Journal of the Fisheries Society of Japan, 55, 1265 (1989): Antican
cer Res., 13, 2045 (1993): Carbhydr. Res., 224, 34
3 (1993): Anticancer Res., 13, 2011 (1989))
There are few reports on the anticancer activity of fat-soluble ingredients. Noda et al. Reported that the fat-soluble constituents of seaweed have anticancer activity, but the test showed that the route of administration was intraperitoneal and did not mention the mechanism of action of anticancer activity (Japan Journal of the Society of Fisheries Science, 55, 1265 (1989)).

[0006] It has been reported that polysaccharides such as fucoidan and alginic acid, which are water-soluble components of seaweed, have an anticancer activity due to the enhancement of the immune system. , 51, 741 (1991)) and water (Pro
c. 10th Int. Seawood Symp. vol 12, 109 (1982)), and no extraction with organic solvents such as alcohols has been reported.

[0007]

SUMMARY OF THE INVENTION It is an object of the present invention to find a component having a high cancer-suppressing effect and a high safety from foods taken daily, and to develop an efficient extraction method therefor. It is.

[0008]

Means for Solving the Problems In view of the above-mentioned problems, the present inventors have conducted intensive studies on food materials having a cancer-suppressing effect, and as a result, have found that a fat-soluble component of seaweed has a strong cancer-suppressing effect. The present invention has been completed.

In the past, it was a general idea that cell death due to an anticancer drug occurs passively as a result of dysfunction of cells that have accumulated damage due to the drug. However, with the advance of cell biology research, it has become clear that much of the physiological cell death progresses through a genetically programmed mechanism called apoptosis. Apoptosis is characterized by nuclear chromatin pseudo-degeneration and DNA fragmentation. Today, it has been found that the process of apoptosis proceeds even with cell death by an anticancer drug (Japanese Patent Publication No. 5-65491). Therefore, a food component that induces apoptosis in cancer cells and exhibits a cancer-suppressing effect can be a useful anticancer agent from the viewpoint of safety.

[0010] The present invention searches for a food ingredient that exhibits a cancer-suppressing effect of inducing apoptosis, finds that the fat-soluble ingredient of seaweed has a remarkable effect, and furthermore finds that the specific ingredient such as 50% by volume aqueous ethanol is used. By using a water-containing hydrophilic solvent having a water content as an extraction solvent, water-soluble polysaccharides such as fucoidan and alginic acid are simultaneously extracted together with a fat-soluble cancer-suppressing component, and the cancer-suppressing effect is more effectively expressed. It was found that.

[0011] Seaweed is a biological resource distributed worldwide, and it is difficult to estimate the current available amount.
It is certain that there is a considerable amount of unused resources, and edible seaweed such as wakame, kelp and mozuku have been cultivated using aquaculture techniques, and there is no problem in securing resources and there is an advantage in terms of raw materials. It is.

[0012]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Sea algae include brown algae, red algae, green algae and diatoms. Among the sea algae used in the present invention, brown algae are particularly useful, for example, wakame, wakame mekabu, mozuku, Alame, kelp, hijiki, hondawara, akamoku, hibamata and the like can be mentioned. In the extraction of the active ingredient of the present invention from these seaweeds, the state of the seaweed, which is the raw material, may be raw or dried, but in the present invention, the raw material may be in any state, such as raw or dried, Although the shape is not limited, it is preferable to use it in the form of a slurry by powdering or thundering to increase the efficiency of extraction.

The extraction of the fat-soluble cancer-suppressing substance of the present invention is performed using an organic solvent. Examples of the organic solvent include hexane, acetone, methanol, ethanol, propanol, isopropanol, butanol, ethyl acetate, toluene, propylene glycol, and the like.Hexane, acetone, methanol, ethanol, propanol, isopropanol, and butanol are more preferable. It is possible to use one or more of these as a mixture. Further, among them, ethanol used for food is particularly preferable.

In the extraction of the cancer-suppressing substance of the present invention using these solvents, the presence of water is allowable up to a water content of 65% by volume, including the water content derived from the raw materials. Furthermore, by containing water, a polysaccharide having a water-soluble immunity-enhancing effect is simultaneously extracted together with a cancer-inhibiting substance having a fat-soluble apoptosis-inducing effect by containing water, whereby a composition that further enhances the cancer-inhibiting effect can be obtained. . As the ratio of the organic solvent increases, the amount of the apoptosis-inducing substance increases, and as the ratio of water increases, the amount of the immune-enhancing substance increases, but without significantly decreasing the amount of the apoptosis-inducing substance.
The water content at which both these actives are extracted simultaneously is preferably in the range of 25 to 50% by volume.

The extraction can be carried out under heating or under low temperature. However, the lower the temperature, the more time is required for the extraction, and it may take several hours to several days. Is efficient and preferred.

When ethanol is used, the extract can be used as it is or after removing ethanol by distillation. When another solvent is used, it can be used as a liquid product obtained by removing the solvent by distillation, further as a concentrated liquid, or as a dried product obtained by drying them. Further, it is possible to use an auxiliary substance necessary for preventing decay of the liquid material obtained by distilling off the solvent or for maintaining the shape.

The extract thus obtained is prepared as a liquid, powder, granule, tablet, capsule or the like by itself or by mixing with an appropriate excipient, and is taken orally. It is possible. Alternatively, they can be mixed and added to foods such as rice, soup, candy, jelly, tablet confectionery, beverages, noodles, rice crackers, Japanese confections, frozen desserts and the like.

[0018]

Hereinafter, the present invention will be described with reference to Examples.
The present invention is not limited to this embodiment.

[0019]Preparation of seaweed extract of Examples and Comparative Examples  After cutting 1 kg of dried mekabu and desalting with cold running water for 10 minutes,
Add 12 liters of 85% by volume ethanol (about 75% by volume
The mixture was heated under reflux for 2 hours. this
, And the filtrate was concentrated under reduced pressure to obtain 136 g of a concentrate.
(Example 3: sample). The lipid in this extract is 20.3
%, Total sugar (phenol-sulfuric acid method) 9.8%, sulfate group content 1
%, Salt content (Mohr method) was 20.8%.

Similarly, test samples of Examples and Comparative Examples shown in Table 1 were prepared.

[0021]

[Table 1]

[0022]Test-1: Apoptosis evaluation  Using HL60 cells (human leukemia cells) as a cancer tissue model
Used.

5 × 10 ⁴ HL60 cells are seeded on a 6-well plate, and cultured at 37 ° C. for about 18 hours in a 5% CO ₂ incubator. Thereafter, the number of cells in each well was calculated using a hemocytometer, and the test samples of Examples and Comparative Examples were diluted with phosphate-buffered saline (PBS) and added to a final concentration of 1 mg / ml each. I do. After adding the test sample, the culture was continued for 24 hours at 37 ° C. in a 5% CO ₂ incubator, and the cells were collected, and the number of cells and viability were measured by trypan blue staining and DNA fragmentation was detected. . The result is shown in FIG.

Cont. Indicates a control to which no test sample was added, and a phosphate buffered saline (PB) to which no test sample was added.
S) and BFA is Breferringin A and a positive control. BFA is known to induce apoptosis, demonstrating the validity of this evaluation.

Samples according to the present invention, ~ <11> and <12> show increased number of dead cells by staining with trypan blue. In contrast, the sample of Comparative Example and <12> had an insufficient apoptotic effect.

For representative samples, DNA was extracted from cells 4 hours after co-culture, and DNA fragmentation was detected by agarose electrophoresis. The result is shown in FIG.
Shown in place of the drawing.

M is a DNA molecular weight marker, NC is phosphate buffered saline, SF is bereferidin A
Is shown. Example 1 (sample: electrophoresis photograph N
o.149), Example 3 (sample: electrophoresis photograph No. 14)
4), Example 4 (sample: electrophoresis photograph No. 150),
In Example 5 (sample: electrophoresis photograph No. 147) and Example 8 (sample: electrophoresis photograph No. 151), a remarkable ladder was observed in DNA extracted from cells, and apoptosis was definitely induced. It was shown that.

On the other hand, no clear ladder was observed in Comparative Example 1 (sample: electrophoresis photograph No. 146) and Comparative Example 2 (sample: electrophoresis photograph No. 145).

[0029]Test-2: Evaluation of macrophage activity (N
O production ability and poor eating ability)  5 × 10^FiveRaw264.7 (mouse-derived macrophage-like cells
Cells) were seeded on a 24-well plate and incubated at 37 ° C, 5% CO_TwoInn
50 U / ml INF-γ sample (final concentration)
(1 mg / ml) for 24 hours. After culturing, culture supernatant
And recover NO in the supernatant._Two ^-Depending on the grease reagent system
Quantified. The result is shown in FIG.

LPS is a lipopolysaccharide, which is a positive control for NO production, indicating that this evaluation system is valid.

Example 3 (sample), Example 4 (sample), and Example 5 (sample) showed that macrophages were activated (NO production ability was improved).

Further, for Example 3 (sample) and Example 4 (sample), Raw264.7 cells were collected, 1 × 10 ⁶ was made into a cell suspension with a medium, and transferred to a 15 ml centrifuge tube. Latex beads to be deprived and a sample are added to a final concentration of 1 mg / ml, and shaken in a 37 ° C. water bath for 6 hours.

Thereafter, the cell suspension was crushed by ultrasonic waves, the prepared cell lysate was transferred to a 96-well plate (100 μl), and the fluorescence intensity (ex 485 nm:
em 530 nm).

FIG. 3 shows the results.

Example 3 (sample) and Example 4
(Sample) was found to actually improve the phagocytic ability of macrophages.

[0036]

The seaweed extract produced by the method of the present invention has an effect of inducing apoptosis in cancer cells, and at the same time as inducing apoptosis, inducing immunostimulation and suppressing cancer.

The seaweed used in the present invention has been used as a foodstuff since ancient times. The product of the present invention is safe and can be added to various foods or easily taken in the form of capsules or tablets. .

[Brief description of the drawings]

FIG. 1 is a chart showing the effect of seaweed extracts of Examples 1 to 12 of the present invention to induce apoptosis in cancer cells, which is shown in comparison with a control product and a comparative product.

FIG. 2 is a chart showing the NO-producing ability of the seaweed extracts of Examples 1, 3, 4, 5, and 6 of the present invention, which are shown in comparison with a control product and a comparative product.

FIG. 3 shows the phagocytic ability of the seaweed extracts of Examples 3 and 4 of the present invention in comparison with a control product and a comparative product.

FIG. 4 shows D extracted after co-culture of Examples 1, 3, 4, 5, 6, 7, 8 of the present invention, a control product and a comparative product with cancer cells.
4 is a drawing-substituting photograph showing the result of detection of NA fragmentation by agarose electrophoresis.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Takahisa Nakano 3-32-7 Higashi Komatsugawa, Edogawa-ku, Tokyo (72) Inventor Shinya Tamura 10-9, Nihonbashi-Kofuna-cho, Chuo-ku, Tokyo Inside Nagase Industries Co., Ltd. (72 Inventor Naoko Hidano 10-9, Nibashi-Kofuna-cho, Chuo-ku, Tokyo F-term (reference) 4C088 AA12 AA13 AA14 AA15 AC15 BA08 BA10 NA14 ZB26 ZC41

Claims (3)

[Claims] 1. Induction of apoptosis obtained by extracting from seaweed using one or a mixture of two or more selected from methanol, ethanol, propanol, isopropanol, butanol, acetone and hexane. Seaweed extract having a cancer-suppressing effect due to 2. It is obtained by extracting from seaweed using one or a mixture of two or more of methanol, ethanol, propanol, isopropanol, or butanol in the coexistence of 65% by volume or less of water. A seaweed extract having a cancer-suppressing effect by inducing apoptosis and improving immunity. 3. The seaweed extract having a cancer-suppressing effect according to claim 1, wherein the seaweed is a brown algae.