JPS5936889B2 - Method for producing antitumor substances - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for extracting antitumor substances from the surface layer of grains, beans, and other seeds by effective means.

Conventionally, anti-tumor substances have been made from parts of bamboo, bamboo grass, and other specific plant species, such as the roots, bark, seeds, and poppy seeds, which are then immediately treated with a solvent at 100°C or below. There are methods for extracting anticancer substances, but because the raw materials are special and limited, mass production is not possible, and it is also uneconomical and impractical (G).

This also applies to the use of conventionally known microorganisms such as lichens, basidiomycetes, yeasts, bacteria, and molds, which often require special cultivation and are not necessarily inexpensive. However, there are some drawbacks that make it difficult to say that it is a suitable raw material.

In view of this conventional viewpoint, the present inventor focused on the seed coat, which is produced in large quantities on a daily basis and is easy to handle, and as a result of conducting research, succeeded in obtaining a substance with extremely strong antitumor properties using special means. The method for producing an antitumor substance of the present invention involves preparing the surface layer of seeds selected from rice grains, barley grains, millet grains, legumes, and oil-smelling grains, either while still attached to the grains or in a state in which they are separated from the grains. The method is characterized in that it is subjected to a wet pressure and heat treatment, and then an antitumor substance is collected from the treated material.

Next, embodiments of the present invention will be described in detail.

Any “seeds” such as grains, beans, etc. that are the raw materials of the present invention
Rice grains, barley, barley, wheat, oats, rye and other wheat grains, buckwheat, millet, millet, corn, millet,
Millet grains, soybeans, adzuki beans, mung beans, arm beans, fava beans,
Common name for beans such as peanuts, oil-smelling fruits such as cottonseed, and rapeseed.

Typical seeds generally used as raw materials are the above-mentioned grains,
Sesame seeds, cottonseeds, rapeseeds, sunflower seeds, etc., which can be grown by hand.

The present invention was based on the discovery that the surface layer of these seeds contains an anti-pulmonary substance, and the surface layer is defined as the bran layer on the surface of brown rice in rice grains, the nuclear layer on the surface of wheat grains, and the surface layer of grains on grains. The term refers to the wrinkles or skin layer of the surface layer of seeds, the epidermis layer of beans and seeds, and the epidermis layer of the seeds of oilseeds.

According to the present invention, the surface layer of the seed meal of each of the above-mentioned seed plants is targeted, but in actual production, although it is possible to use each of the seeds themselves as a raw material, it is uneconomical and inconvenient. Although it is efficient and it is possible to specifically produce only the seed coat and use it as a raw material, it is not practical.

Therefore, the most practical, large-scale, and economical method is to use various seed meal that is produced as a by-product or waste, especially in the food processing industry. It can open up new ways to develop materials that are only used as fertilizers.

For example, bran produced by milling rice, wrinkles produced by milling or milling wheat husks, beer lees produced as a by-product during the brewing process of beer, corn starch lees after starch is collected, starch extraction lees such as milo lees, etc. , soybean meal after oil and fat collection, corn oil meal, rapeseed meal, oil extraction meal such as defatted rice bran, protein extraction meal such as okara after protein collection, and other products after collecting the desired product from seeds. Various industrial by-products and waste materials are used, and these materials can be easily obtained in large quantities as raw materials for the present invention, and the antitumor substance can be produced in large quantities and economically at low cost. It will be done.

The present invention is based on the discovery that an effective antitumor substance can be obtained from the surface layer of such seeds by subjecting the surface layer of the seed to a high-pressure heat treatment before extraction, without subjecting the surface layer to normal pressure extraction or treatment. .

In other words, as described below, even if the surface layer of the same seed is used as raw material,
This can be processed and extracted using conventional methods, that is, extracted with water or any other solvent at room temperature, or extracted at 100°C.
Although the desired substantially effective antitumor substance could not be obtained even after heat treatment at a temperature of up to 100 °C and extraction treatment, extraction was performed after heat treatment at a temperature exceeding 100 °C. It was discovered that a powerful antitumor substance could be obtained by this method.

The pressure and heat treatment is approximately 0.2 to 15 kg/cyi, approximately 1
It is normal to carry out at 03 to 200°C for about 10 to 0.1 hours, especially about 0.5 to 3 kg/d, about 110 to
It has been found that it is practically preferable to carry out the reaction at 140° C. for about 3 to 0.5 hours.

The pressure and heat treatment is preferably wet pressure and heat treatment of the raw material.

That is, this is wet heating in which the raw material is heated with heated steam such as heated water vapor, heated liquid such as hot water, or a mixture of these steams or liquids, and this enables high-temperature treatment without burning the raw material.

The steam or liquid for pressurization and heating in this case may be any one that also has an extraction effect on the raw material, such as water, aqueous liquid, hydrophilic or hydrophobic organic solvent, and these are appropriately selected and used.

In particular, pressurized heat treatment involves steam heating the raw material using pressurized steam, or adding water or an aqueous solvent to the raw material so that the raw material is moistened or immersed, and then steam is blown into the raw material or external heat is applied. This is generally carried out by heating, which is preferable from a work standpoint.

After the pressure and heat treatment as described above, the antitumor substance is collected from the solid or filtrate of the treated material.

This collection method includes extraction with aqueous solvents such as water and alkali;
Antitumor substances are usually produced in the form of white to light brown powder by combining two or more of the following methods, such as concentration, centrifugation, salting out, dialysis, adsorption, decolorization, filtration, and freeze-drying. get.

The degree of purification may be as long as it is industrially or practically necessary.

The toxicity of the purified product is extremely low (particularly when extracted with alkali) compared to when extracted with other substances.

It was found that the higher the degree of purification, the greater the antitumor effect.

According to elemental analysis, this extracted material consists of C-H.0,
Alternatively, it may sometimes contain a trace amount of N.

In addition, this substance was negative in protein coloring reactions such as ninhydrin reaction and Biuret reaction, and positive in water extraction by Molisch reaction and Anthrone reaction.

Furthermore, column chromatography, infrared absorption spectrum,
Combining the results of ultraviolet absorption spectra, paper chromatography of acid decomposition products, elemental analysis, and other results, we found that this substance is a non-protein polymeric substance, that water extraction is a polysaccharide or its near-green substance, and alkaline extraction It is assumed that it is a higher fatty acid or its near-green substance.

In addition, this substance has particularly high thermal stability, for example, at 150°C, 3
It has been found that the antitumor effect does not change at all even after being heated at high temperatures for hours.

In animal tests using mice, this substance showed a strong antitumor effect even when administered in extremely small amounts, and a particularly notable feature was that it exhibited extremely high antitumor effects not only when administered intraperitoneally but also when administered orally.

The optimal dose for intraperitoneal administration is 0.2-5~/k (j1 day x
10 days, and it is noteworthy from a practical point of view that the optimal dose for oral administration is different from that for intraperitoneal administration, and that the effect does not decrease with large doses.

Regarding the toxicity of this substance, the alkali-extracted one was administered intraperitoneally to mice. ? /kg, 20g/kg by oral administration
However, it has no adverse effects, and as a result of seven experiments to the extent possible, no acute toxicity has been observed, and no pathological side effects appear at all, indicating that it is an extremely safe substance.

In the case of water-extracted substances, the substance of Example 2 has an LD of 56150 ml when administered intraperitoneally. /kg was observed.

The main antitumor effect was confirmed as follows.

That is, approximately 4 million Sarcoma 180 cells were transplanted into the lower thigh skin of 20 ICR-JCL mouse mice, and 24 hours after the transplantation, the administration group was injected with various concentrations of this substance obtained in each of the following Examples. A suspension prepared in distilled water was administered intraperitoneally once a day for 10 days, and the tumor area was calculated every week to observe changes in the antitumor effect.

The inhibition rate % was calculated from the average total tumor area of the control group and that of the administration group.

The results are shown in Figure 1. As shown in the figure, the effect over time for 4 weeks is significant.

Furthermore, after 4 weeks, the tumor was excised and weighed, the tumor growth inhibition rate of this substance was determined as a percentage, and the disappearance rate was calculated for the administration group.

As a result of the above test, a strong antitumor effect was observed at a dosage of 0.1 to 10 m9/kg/day x 10 days.

One example is shown in the table below. Further, there is an optimum amount for each of the example products, and the range is generally about 0.
2~7171J? /kg/day xio day.

Next, a similarly excellent antitumor inhibitory effect was obtained by oral administration, but no decrease in the inhibitory effect was observed even when the drug was administered in a much larger amount, for example, in grams, compared to the intraperitoneal dose.

A similar test was conducted on a product in which 4 million Ehrlich ascites carcinoma cells were transplanted into the lower skin of the thigh, and the same excellent effects as described above were obtained.

As a control, the same raw materials used in Examples 1 to 11 were extracted with any solvent such as water at room temperature, alkali aqueous solution, or an organic solvent, or after heat treatment with steam at 100'C for a long time. Extract with any solvent, or mix raw materials with water,
After heat treatment with an alkaline aqueous solution or any other organic solvent at a temperature of 100°C or lower, extraction treatment is performed from this,
After that, similar to Kempo, we conducted animal tests on each powder obtained by removing impurities such as protein removal, starch removal, defatting, and low molecular weight substances, and found that the best rejection rate was about 30. %, and the disappearance rate was about 1/1o, so it was found that there was no substantial effect and the yield of the target substance was significantly reduced, making it impractical.

Example 1 200g of rice bran in an autoclave at fkg/i, 12
After heating under pressure at 0°C for 1 hour, this was treated with hot water to remove impurities such as soluble protein and starch, and then 500ml of 4% caustic soda was added thereto and heated at room temperature for 48 hours or about 80 minutes.
Extract with stirring while heating at ~100°C for 1 hour, then filter through 2'500ml of water, neutralize the resulting extract with hydrochloric acid to a slightly acidic pH of 5. 500m
1 under reduced pressure.

Add 750 ml of absolute ethanol of 1.5 times the volume to this concentrated solution, centrifuge for 15 minutes at 0°C, 10,000 r, p, m, and add 500 ml of water for further purification of the resulting precipitate. °C, 15000r, p,
Repeat the washing operation of centrifuging for 20 minutes at 100 m, then redissolve the precipitate in 2% caustic soda 2007711, add the same amount of absolute ethanol, and incubate at 0 °C and 1000 °C.
Centrifugation was performed at 0r, p, m for 15 minutes to remove ethanol-insoluble components (fibers, etc.) in the sediment, and the resulting supernatant was dialyzed in running water using a cellophane membrane for 50 hours to remove the dialyzed fluid. The precipitate precipitated by concentration under reduced pressure was freeze-dried to obtain 1.4 g of white powder.

This substance is insoluble in acetone and methanol.

The elemental analysis value of this substance is C70.81%, HI3.07%
, NO, 56%.

In addition, the average molecular weight is determined by column chromatography.
- It was estimated to be around 200,000, as it ranks higher than globulin.

FIGS. 2 and 3 show its infrared absorption spectrum and ultraviolet absorption spectrum, respectively.

Example 2 After treating 200 g of rice bran in an autoclave under the same pressure and heating conditions as in Example 1, 1500 genus water was added thereto and heat treated at 100°C for 6 to 8 hours to obtain P obtained by E-filtration.
The extract of the liquid was concentrated under reduced pressure to 450 ml, and an equal amount of ethanol was added to this concentrated liquid to remove the resulting precipitates (starch, etc.), and the supernatant was dealcoholized and concentrated to 270 ml. Amount of anhydrous ethanol 10,108O was added to form a precipitate, and this was heated at 0°C, 10,000 r, p, m,
After centrifugation for 15 minutes, the resulting precipitate was dissolved in water and left in a cold room for 48 hours. The resulting precipitate was washed twice with water and then lyophilized to obtain 0.4I as an off-white powder. Ta.

The infrared absorption spectrum and ultraviolet spectrum of this material are shown in FIGS. 4 and 5, respectively.

Example 3 200g of dried beer lees was boiled in a high-pressure pot by blowing high-pressure steam into 1
After pressurizing and heating at 8 kg/cflL1130°C for 30 minutes, 2000 ml of a 25% caustic soda aqueous solution was added and the mixture was extracted with stirring all day and night.

Next, this was centrifuged at 0°C, 10,000 r, p, m for 15 minutes to remove insoluble components of the precipitate, and after neutralizing the supernatant with hydrochloric acid, twice the amount of absolute ethanol was added to remove the precipitate. °C, 1
The mixture was separated and collected by centrifugation at 0000 r, p, m for 10 minutes.

This precipitate was dissolved in 1000 Tll of water and incubated at -5°C for 15
After repeating the washing operation twice to collect water-insoluble components by centrifugation at 000 r, p, m for 20 minutes, the mixture was freeze-dried to obtain 2.2 g of a gray spongy substance.

Example 4 200g of dried beer lees was added with 500ml of water in a high-pressure pot and heated with a burner from the outside to produce 1kg/ff.
After pressurizing and heating at 120°C for 50 minutes, 2000 ml of a 4% caustic soda aqueous solution was added and extracted with stirring for 10 hours. After removing the residue by filtration, the P solution was neutralized with hydrochloric acid.
Concentrate under reduced pressure to 500 TL, add twice the amount of absolute ethanol (1000 TL), and incubate the resulting precipitate at -5°C at 100 TL.
The supernatant was removed by centrifugation at 00r, p, m for 15 minutes, and the supernatant was collected and dealcoholized, and the resulting precipitate was heated at -5°C.
13000r, p, m.

After removing by centrifugation for 15 minutes, 4 times the volume of ethanol was added again, and the resulting precipitate was incubated at 0°C for 1ooo.

The supernatant was removed by centrifugation at R, P, M for 15 minutes, and the supernatant was dealcoholized again. The resulting precipitate was left in a cold room overnight, collected by centrifugation, and freeze-dried to obtain a brown powder. .8g was obtained.

Example 5 200g of wrinkled wheat was moistened with 507rL of water and heated under pressure in an autoclave at 1.4 kgi = 1125°C for 50 minutes, then 2000ml of water was added and the mixture was heated at 100°C for 8 hours.
After heat treatment, the supernatant was removed, 1000 TL of a 2% caustic soda aqueous solution was added to the residue, and the mixture was stirred and extracted at room temperature overnight at -5°C, 10,000 r, p, m.

Centrifuge for 20 minutes to remove the precipitate, add an equal volume of absolute ethanol to the supernatant, redissolve the resulting white precipitate in 5% caustic soda aqueous solution 10,100O, add the same volume of 70% ethanol, and incubate at 0° C100OOr, p, m, 10
After centrifuging for a minute to remove the precipitate and neutralizing the resulting supernatant with hydrochloric acid, it was concentrated to 150mA! The mixture was closed and left overnight in a cold room to remove the precipitate, and the resulting supernatant was freeze-dried to obtain 2.9I in the form of a light brown powder.

Example 6 200 g of dried corn gluten feed, which is commercially available as feed, was washed with cold water, drained, placed in a high-pressure cooker, and blown with heated steam to produce 3 kg/art at 140°C.
After heat treatment under pressure for 0 minutes, 10100O water was added and kept at 100°C for 3 hours, followed by solid-liquid separation.The residue was purified in the same manner as in Example 1 to obtain a white powdery substance 1.
2g was obtained.

Example 7 A commercially available Okara 500I housed in a high-pressure cooker was charged with 0.
Add 05N caustic soda aqueous solution and blow in high pressure steam.
0.5kg/cI? After heating under pressure at 110° C. for 30 minutes, the P solution was purified in the same manner as in Example 1 to obtain 2.4 g of off-white powder.

Example 8 200g of cottonseed meal was heated to 1.8kg/11 in an autoclave.
After pressurizing and heating at 30° C. for 25 minutes, the mixture was treated in the same manner as in Example 1 to obtain 2.8 g of grayish brown powder.

Example 9 Add 300 ml of water to 200 g of dried milo cake, blow high-pressure steam into it in an autoclave, and heat to lky/ci 120°C.
After pressurizing and heating the mixture for 50 minutes, the mixture was treated in the same manner as in Example 1 to obtain 2.4 g of a light brown powdery substance.

Example 10 200g of rapeseed meal in an autoclave at 2kg/d13
After pressurizing and heating at 2°C for 30 minutes, the mixture was treated in the same manner as in Example 4 to obtain 2.1 g of light brown powder.

Example 11 After crushing 200g of barnyard fish, 1k was crushed in an autoclave.
g/=, after heating under pressure at 120°C for 60 minutes, it was then furnace-separated with a sawcloth in running water, and the remaining solids were extracted with a caustic soda aqueous solution in the same manner as in Example 3, and treated in the same manner. An off-white powder of 0.39% was obtained.

The pressure and temperature for pressurization and heating according to the present invention are preferably within the ranges of the above embodiments, but may exceed these ranges.

As described above, the present invention focuses on the surface layer of the above-mentioned specific seeds, and extracts the oil after applying a wet pressure and heat treatment to the surface layer of the seeds, thereby producing an extremely effective anti-tumor substance in high yield. It is possible to develop a new field of use of materials that can be collected, especially those that are by-products or discarded in the food industry, and in particular, it is possible to use materials that have traditionally been used in large quantities as feed fertilizer in an easy and economical way. It has the effect of leading to mass production of anti-tumor substances.

[Brief explanation of drawings]

Figure 1 is a diagram showing the change over time in the inhibitory effect of the antitumor substance obtained by this method, and Figures 2 and 3 are the infrared absorption spectra and graphs of one example of the same substance obtained by this method. 4 and 5 show infrared absorption spectra and ultraviolet absorption spectra in other regions, respectively.

Claims (1)

[Scope of Claims] 1. The surface layer of seeds selected from rice grains, barley grains, millet grains, legumes, and oil-smelling grains is subjected to a wet pressure heat treatment while attached to the grains or in a state separated from the grains, A method for producing an antitumor substance, which comprises: collecting the antitumor substance from the object to be treated. 2. The production method as set forth in claim 1, wherein the surface layer is the bran layer of rice grains, the core layer of wheat grains, the wrinkles or cortex of coarse grains, the cortex of legumes, and the cortex of oil-smelling seeds. . 3. In the food industry, the surface layer is made of rice, rice bran from wheat, wrinkles, starch such as beer lees, corn starch lees, and milo lees, which are produced as by-products after separating edible components such as starch, oil, fat, and protein from the seeds. Various by-products or wastes after collecting the desired product from any of the seeds, such as extracted lees, soybean lees, corn oil lees, rape lees, oil extraction lees such as defatted bran, and protein extraction lees such as okara. A manufacturing method according to claim 1. 4. Approximately 0.2 to 15 kg of the above raw materials are placed in a pressurized container.
cr7t1 about 103-200℃, about 10-0.1 hours,
The manufacturing method according to any one of claims 1 to 3, characterized in that a wet pressure and heat treatment is performed.