JP3026435B1 - Leukemia cell growth inhibitor and method for producing the same - Google Patents

Abstract

An object of the present invention is to obtain a leukemia cell proliferation inhibitor that is effective in treating leukemia by effectively inhibiting the proliferation of leukemia cells from easily available raw materials. A leukemia cell growth inhibitor comprising non-cellulosic dietary fiber derived from woody tissue of a plant as a main component,
The plant woody tissue powder is brought into contact with pressurized hot water at 100 to 140 ° C. to extract and remove soluble components.
Decomposition and extraction are performed by contacting hot pressurized water at 40 to 220 ° C. to separate and produce non-cellulosic dietary fiber.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel leukemia cell growth inhibitor, and more particularly, to a leukemia cell growth inhibitor containing dietary fiber as a main component extracted from plant tissue under specific conditions, and to its use. It relates to a manufacturing method.

[0002]

2. Description of the Related Art Leukemia is a disease in which white blood cells in the bone marrow autonomously proliferate, so-called cancers of the blood, which cause anemia, leukocytosis, thrombocytopenia, etc.
It is known to create secondary invasive foci (metastasis) or secondary hematopoietic foci in other organs such as lymph nodes.

[0003] As such a therapeutic agent for leukemia, various synthetic substances, antibiotics, naturally occurring substances, interferon, interleukin, TNF (tumor necrosis factor), CSF (colony formation stimulating factor) inhibitor and the like have hitherto been used. Biotechnology products are being developed. These act on leukemia cells to inhibit their growth,
It causes necrosis or necrosis to treat the disease.

[0004] However, these drugs for treating leukemia are:
In addition to difficulties in obtaining raw materials, it is unavoidable that the production method is complicated and costly. On the other hand, a cell growth inhibitor is known to induce cell growth inhibition in specific cells by inducing cytotoxicity. However, the mechanism of this cytotoxicity expression is very complicated. It is completely unpredictable to what cell the substance causes cytotoxicity, and it has been very difficult to obtain an effective cell growth inhibitor for leukemia cells.

[0005]

SUMMARY OF THE INVENTION The object of the present invention is to provide a leukemia cell growth inhibitor which effectively inhibits the growth of leukemia cells and is effective for the treatment of leukemia from readily available raw materials. is there.

[0006]

Means for Solving the Problems The present inventors have conducted various studies to develop a substance that inhibits the growth of leukemia cells. The present inventors have found that dietary fiber has a good growth inhibitory effect on T-cell leukemia cells, and have accomplished the present invention based on this finding.

That is, the present invention relates to a leukemia cell growth inhibitor comprising a non-cellulosic dietary fiber derived from plant wood tissue as a main component and a plant wood tissue powder,
After extracting soluble components by contacting with hot pressurized water at a temperature of 140 ° C., 140 ° C. to 220 ° C. is contacted with hot water at a temperature of 140 to 220 ° C. to cause decomposition and extraction to separate non-cellulosic dietary fiber. And a method for producing a leukemia cell growth inhibitor.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The leukemia cell growth inhibitor of the present invention is obtained by using plant wood tissue as a raw material, treating it under specific conditions, and extracting the wood tissue. There is no limitation, and for example, those belonging to any of bamboos, hardwoods, conifers and the like may be used. Examples of such woody materials include cycads, ginkgo, pine, yew, magnolia, walnut, bayberry, beech, camellia, mallow, willow, azalea, plum, oyster, rose, cypress, cedar, palm,
Woody parts such as elm can be mentioned. For use as the leukemia cell growth inhibitor of the present invention, those containing a relatively large amount of lignin or hemicellulose are preferred.

According to the method of the present invention, the woody tissue of these plants is used as a raw material, and is treated and extracted with hot pressurized water in two stages. That is, first, 50 to 500
Raw material powder having a particle size of μm is charged into, for example, a pressurized hot water flow type reaction apparatus and treated with pressurized hot water at 100 to 140 ° C., whereby components contained in cells such as pigments and tannin are easily extracted. You. When this treatment is continued for about 20 to 60 minutes, the above-mentioned components are almost completely removed.
When the wood part is continuously contacted with pressurized hot water at 220 ° C., it is hydrolyzed by the pressurized hot water and eluted. The eluate is collected and freeze-dried to obtain the leukemia cell growth inhibitor of the present invention as a pale brown powder. The leukemia cell growth inhibitor thus obtained contains, as a main component, a hemicellulose-derived monosaccharide, a water-soluble oligosaccharide, a water-soluble lignin and a sugar-lignin conjugate as a result of sugar analysis and ultraviolet absorption spectrum analysis. I understood that.

[0010] The cell growth inhibitor of the present invention can be administered parenterally. The dose varies depending on the degree of the disease to be treated, but is usually 0.01 to 10 per day.
It is selected within the range of mg / kg. Since this compound exhibits a cell growth inhibitory effect due to cytotoxicity, a sufficient effect can be obtained by administering a smaller amount than other cell growth inhibitors. It is formulated as an aqueous solution having a concentration of 0.1 to 10%, and may be used as an injection, such as a solubilizing agent, a buffer, an isotonic agent, a stabilizer, a preservative, or a soothing agent, if desired. Commonly used additives can be used in combination.

On the other hand, this decomposed extract is used as a leukemia cell growth inhibitor in a small amount, but contains a large amount of dietary fiber, so that it can be used as a functional food by adding it to general foods.

[0012]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Bamboo powder adjusted to a particle size of 177 to 250 μm (composition, alcohol-benzene extract: 7% by weight, lignin: 21)
% By weight, hemicellulose: 25% by weight, cellulose: 4
1 g of a stainless steel fixed-bed reactor (φ10.2 mm, length 46.7 mm, 3.6 m) capped at both ends with a sintered metal filter (pore diameter 5 μm).
1 volume), and pressurized hot water of 135 ° C., which is pressurized to 100 atm so as not to evaporate, is passed at a flow rate of 10 ml / min.
For about 15 minutes to remove soluble components. Then 200
Decomposition products were collected by continuously passing hot water of 15 ° C. for 15 minutes. At this time, in order to suppress the secondary decomposition of the components decomposed from the reactor outlet and solubilized in hot water and flowed out, the components were immediately brought into contact with cooling water at a flow rate of 2.5 ml / min. The obtained extract was freeze-dried and the recovery rate per charged amount was determined. The yield of the first soluble component (group I) was 12%.
% By weight, and the recovery of the decomposition product (Group II) was 43% by weight.

As a result of the analysis of the sugars and the ultraviolet absorption spectrum of the groups I and II obtained by the two-step heating of the pressurized hot water as described above, the sugar compositions were different from each other. Was found to contain mainly arabinose, xylose and xylooligosaccharide derived from hemicellulose. This is 205 nm and 28
Since it has an aromatic absorption wavelength of 0 nm and its strong absorption, it is considered that water-soluble lignin and a sugar-lignin conjugate coexist.

From the above, when the temperature of the pressurized hot water is raised in two stages, the intracellular components (group I) which are easily extracted by the relatively low-temperature pressurized hot water can be extracted with the 135 ° C. pressurized hot water. The hydrothermal decomposition product containing the separated hemicellulose-derived monosaccharide, oligosaccharide, water-soluble lignin and sugar-lignin conjugate was 200
It was found to be separated by hot pressurized water at ℃.

Example 2 Using the freeze-dried product of Group II obtained in Example 1,
The water-soluble fraction was fractionated by the following procedure. After solubilizing 200 mg of the lyophilized product in 1.5 ml of distilled water, 0.1%
The filtrate obtained by filtering excess solid content with a 2 μm filter was used as a water-soluble decomposition extract A. Similarly, 200 mg of the lyophilized product was used to remove fat-soluble components with 5 ml of ethanol, and the solid residue after ethanol extraction was solubilized in 1.5 ml of distilled water, and the filtrate filtered with a 0.2 μm filter was used. Each extract was used as a water-soluble decomposition extract B and subjected to a leukemia cell growth inhibition test. In the leukemia cell proliferation inhibition test, the decomposed extracts of A and B described above were used as representative leukemia cells of three types of T-cell leukemia cells (RPMI8402, MOLT4,
Jurkat) and B-cell leukemia cells (NALM6)
For all four species.

The method of measuring leukemia cell inhibition is determined in advance by 3
Leukemia cells (90 μl, 2.22 × 10 ⁵ cells / ml) cultured in RPMI-1640 medium at 7 ° C. were dispensed into 96 wells, and further incubated at 37 ° C. in 5% CO ₂ humid air under atmospheric pressure. After culturing at 24 ° C. for 24 hours, 100 μg / ml, 50 μg / ml, 25 μg / ml,
12.5 μg / ml, 6.25 μg / ml, 3.125
[mu] g / ml phosphate buffered saline to a concentration of those prepared in (pH 7.4, Ca ions and Mg ions is not included), after each 10μl added, under atmospheric pressure, 5% CO ₂ The cells were cultured in humid air at 37 ° C. for 20 hours. In addition, the same treatment was carried out for a control obtained by adding 10 μl of a phosphate buffered saline to 90 μl of a cell suspension as a control. Culture solution 1 cultured for 24 hours
Celltiter for 00 μl
96 (manufactured by Promega), and the cell growth inhibitory activity of the decomposed extract on the cell line was examined using the activity of mitochondrial dehydrogenase as an index. At this time, the cell growth inhibitory activity was calculated as cell growth inhibition rate (%) = 100− (absorbance at 590 nm of culture solution cultured for 24 hours after addition of the decomposed extract / absorbance at 590 nm of control) ×
Represented by 100. The results are shown as graphs in FIGS.

FIG. 1 shows the T cell leukemia cell RPMI840.
24 when various concentrations of the decomposed extract were added to
It shows the leukemia cell growth inhibition rate after time. The results for RPMI8402 show that B
Only at a concentration of μg / ml, about 20% of growth inhibition was observed, and at concentrations lower than that, no effect of cell growth inhibition was observed. FIG. 2 shows the cell growth inhibitory effect on T cell leukemia cells MOLT4. From this figure,
A cell growth inhibitory effect of about 10% was shown with respect to a concentration of 50 μg / ml of B, and at a concentration of 100 μg / ml, A
It can be seen that cell growth inhibitory effects of about 10% and about 80% are shown for B and B, respectively. FIG. 3 is a graph showing the results of cell growth inhibition on T cell leukemia cells Jurkat. From these results, it was found that A and B
at a concentration of 100 μg / ml A and B at a concentration of 100 μg / ml
It can be seen that each of them exhibits a cell growth inhibitory effect of about 50% and about 80%. FIG. 4 is a graph showing the inhibition rate after 24 hours when various concentrations of the decomposed extract were added to B cell leukemia cell NALM6. As can be seen from this figure, no cell growth inhibitory effect was observed on any of the degraded extracts A and B.

From the above results, from the lyophilized product of Group II decomposed and extracted with hot pressurized water at 140 to 220 ° C.,
The component (A) obtained by extraction with distilled water, and the lyophilized powder obtained by removing the fat-soluble component using ethanol and then the component (B) extracted with distilled water, both inhibit the growth of leukemia cells. You can see the effect. In particular, it was found that the growth inhibitory effect on T-cell leukemia cells was effective, and that the effect of the extracted component B was large. From this, it was confirmed that the decomposition extract of Group II obtained by pressurized hot water had an effect of inhibiting the growth of leukemia cells.

[0020]

According to the present invention, the plant wood tissue powder is
After contacting pressurized hot water heated to 40 ° C. or lower to remove soluble components such as components contained in cells, 140 to 2
The decomposed extract obtained by contacting hot pressurized water at 20 ° C. has an effect of inhibiting the growth of leukemia cells. The decomposed extract is used as a leukemia cell growth inhibitor in a small amount, but contains a large amount of dietary fiber, and thus is useful as a material as a functional food when added to general foods.
Also, the production method is a safe and simple method because it can be easily obtained only with water without using a catalyst such as an acid or an alkali.

[Brief description of the drawings]

FIG. 1 is a graph showing the cell growth inhibition rate after 24 hours when various concentrations of a decomposed extract were added to T cell leukemia cells RPMI8402.

FIG. 2 is a graph showing the cell growth inhibition rate after 24 hours when various concentrations of a decomposed extract were added to T cell leukemia cells MOLT4.

FIG. 3 is a graph showing the cell growth inhibition rate after 24 hours when various concentrations of a decomposed extract were added to T cell leukemia cells Jurkat.

FIG. 4 is a graph showing the cell growth inhibition rate after 24 hours when various concentrations of a decomposed extract were added to B cell leukemia cells NALM6.

──────────────────────────────────────────────────の Continuing on the front page (72) Inventor Hiroki Ando 1445-1 Oda, Hayato-cho, Aira-gun, Kagoshima Prefecture Inside the Kagoshima Industrial Technology Center (72) Ikuko Furukawa 1-445-1, Oda, Hayato-cho, Aira-gun, Kagoshima Kagoshima Kogyo Inside the Technology Center (72) Inventor Tetsuro Kunio 1445-1 Oda, Hayato-cho, Aira-gun, Kagoshima Prefecture Inside the Kagoshima Prefectural Industrial Technology Center (56) References JP-A-3-49662 (JP, A) JP-A-3-205402 , A) JP-A-4-71466 (JP, A) JP-A-4-218501 (JP, A) US Patent 4,978,817 (US, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) 35/78 A61K 31/715 BIOSIS (DIALOG) CA (STN) MEDLINE (STN)

Claims (2)

(57) [Claims] 1. A leukemia cell proliferation inhibitor comprising a non-cellulosic dietary fiber derived from a woody tissue of a plant as a main component. 2. A method for preparing a plant wood tissue powder at 100 to 140 ° C.
After extracting and removing soluble components by contacting hot pressurized water of
A method for producing a leukemia cell growth inhibitor, comprising separating non-cellulosic dietary fiber by contacting the residue with hot pressurized water at 140 to 220 ° C. to cause decomposition and extraction.