JPH07258100A - Glycolipid contained in sweet potato and exhibiting anticancer action by suppressing proliferation and promoting differentiation of cancer cell and purification process therefor - Google Patents

Abstract

PURPOSE:To produce a raw material for a carcinostatic or foods and drinks for the prevention of cancer by collecting a glycolipid suppressing the proliferation and promoting the differentiation of cancer cell from the whole parts of sweet potato, i.e., the leaf, runner, stalk and rhizome and purifying the glycolipid. CONSTITUTION:The leaves, runners and stalks of sweet potato are dried, pulverized and extracted by boiling in water, homogenizing in a mixture of methanol, chloroform and water or extracting under irradiation with ultrasonic wave. A glycolipid is separated by a binary phase partition method and purified by silica gel column chromatography to obtain a purified carcinostatic substance consisting of two fractions active to suppress the proliferation of cancer cell and promote the differentiation of cancer cell. The edible part is used as it is, a starch waste left after the extraction of starch is extracted with water or a starch-washing liquid is attached as it is to an ion exchange resin, etc., and purified by a purification method. As an alternative, sweet potato is sliced into thin pieces, dried in the sun, pulverized, extracted with steam, etc., and purified to obtain a carcinostatic food or obtain a raw material for a carcinostatic food.

Description

Detailed Description of the Invention

[0001]

INDUSTRIAL APPLICABILITY The present invention can be provided as foods and drinks having an anticancer agent and a cancer preventive effect by extracting glycolipids having a medicinal effect from the edible part of sweet potato and all parts such as leaves, vines and stems It is a thing.

[0002]

[Prior Art] Sweet potato rhizomes are dug out, dried and incinerated, and the above-ground parts are laid on cultivated land and rotted to be used as compost, or they are rotted by the side of cultivated land and returned to the ground. ing. In addition, one part is used as livestock feed.

In the edible portion of sweet potato, about 75% of the production amount is used for various fruits and vegetables, and about 25% or about 300,000 tons is used as a raw material for starch production. In this case, the solid component is starch meal, 1 part is used as an oxalic acid raw material, 1 part is used as livestock feed, but most are discarded as industrial waste. Further, the soluble component is made to flow as a waste liquid together with the washing water for starch purification.

[0004]

[Problems to be Solved by the Invention] In sweet potato, it is confirmed that the anticancer glycolipid is distributed not only in the edible part but also in the leaves and the stem.
Reported by one of the inventors at the Japanese Cancer Society in October 1993. On the other hand, if sweet potatoes are eaten as fruits and vegetables, when the human body is estimated to be a culture system, it will reach an effective amount by continuously eating 3 kilograms every day for about one and a half months. However, ingestion of this amount of sweet potato cannot bring about various nutritional disorders including protein deficiency.

On the other hand, the leaves, stems and vines of sweet potatoes have little industrial and industrial value, and the amount of sweet potatoes as a raw material for starch, which reaches 300,000 tons per year, is several times as much as the amount of raw materials used. Is being discharged. The leaves, stems, starch meal and starch waste liquid that are mainly disposed of contain anticancer glycolipids as well as the edible portion. If anti-cancer glycolipids can be extracted from these discarded parts, it is considered to be significant from the aspect of environmental protection as well as industrial and medical value.

[0006]

[Means for Solving the Problems] Leaves, stems, vines and starch meal are washed with water and then dried or boiled in that state and extracted into water. Alternatively, extraction is carried out by ultrasonic irradiation or homogenization with an organic mixed liquid such as methanol-chloroform-distilled water having relatively strong hydrophilicity. The latter can extract only glycolipids depending on the mixing ratio of organic solvent and water.

The waste liquid from the starch production is filtered through the cation-exchange resin to remove the cation component along with the filtration of the suspended matter, and the effluent is subsequently passed through the anion-exchange resin to adsorb the anion component. The adsorbed component is eluted with a small amount of 1M vinegar ammonium solution. The boiling extract of leaves, stems, vines and starch meal is also cooled and then concentrated using an ion exchange resin.

The concentrated effluent obtained from the anion exchange resin is desalted, and then the ganglioside is adjusted by the Bligh-Dyer partitioning method or the like.

The crude ganglioside fraction obtained was dissolved in chloroform-methanol-distilled water (30: 60: 8), applied to a DEAE-Sephadex A-25 column, washed with water, and then eluted with a 0.1% ammonium acetate solution. Fractions are collected and designated as fraction I. Further, after washing with a 0.3% ammonium acetate solution, a fraction flowing out with a 0.5% ammonium acetate solution is collected to obtain a fraction II.

Both of these fractions were identified by thin-layer chromatography. Fraction I was found to be effective in the fraction having mobility between G _M3 and G _M4 , and fraction II had G _T1b and G _T1b.
A medicinal effect is observed in the fraction having mobility between _D1b and _D1b .
However, after the fraction I was ultrasonically extracted from the thin layer, fraction I
It is considered that the agent showing the same mobility as that of I and lacking a partial lipid in fraction I is the agent of fraction II.

The concentration at which the growth of cancer cells is almost completely suppressed in this fraction is 27 μg / ml. Even if it is assumed that there is no affinity of the active substance for cancer cells, the body weight is 60 kg.
In the case of cancer patients, it is considered that the administration of 1.62 g suppresses the growth of cancer and can supply an ideal anticancer drug.

[0012]

EFFECT OF THE INVENTION Starch meal, a waste solution thereof and wastes such as leaves, stems and vines, which have not been used as so-called fruits and vegetables of sweet potatoes or as a raw material for starch and an oxalic acid raw material, are currently disposed of. The supply of various anti-cancer drugs will help cancer treatment. It is also a desirable solution in terms of waste treatment.

The anti-cancer effect of fractions I and II was tested using human myeloid leukemia HL-60 cells, human cervical cancer HeLa cells and mouse melanoma B-16 cells.

A. Anti-cancer effect on human myeloid leukemia HL-60: HL-60 cells were adjusted to a cell number of 1 × 10 ⁵ / ml in RPMI-1640 medium containing 10% fetal bovine serum, and each was added to a 25 ml culture bottle. Dispense 5 ml and culture in a carbon dioxide incubator. The next day, at the time when each cultured cell started to grow, 25 μg of each of fractions I and II, 5
0 μg, 75 μg, 100 μg, 125 μg and 15
It was adjusted to 0 μg / 500 μl distilled water, sterilized by filtration, and 500 μl of each was added to the cultured cells to observe the growth. In addition, the growth inhibition rate was examined by measuring the number of each viable cell every day and using the growth of cultured cells to which 500 μl of sterile distilled water was added as a control. As a result, in fraction II, 25 μ
Sufficient growth inhibition was observed even with l, but with fraction I, the inhibition rate varied depending on the dose, indicating a clear dose-action relationship. Furthermore, the degree of dead cells was not found at each concentration of addition of Fraction II. It was suggested that it was not killed by cytotoxin.

B. Anti-cancer effect on human cervical cancer HeLa cells: Proliferated HeLa cells were treated with trypsin to separate the cells, and then Eagl containing 10% fetal calf serum
Adjust the e-MEM medium so that the cell number 4 × ¹⁰ 4 / 5ml, were cultured dispensed each 25ml culture flask 5 ml. For the treatments of fractions I, II and the control, the same concentration as that of HL-60 was added on day 3 when the cells adhered to and grew on the wall of the culture bottle. Photographs 1, 2 and 3 show micrographs on the 4th day after the treatment. Photo 1 is a control to which 500 μl of sterilized distilled water was added. 150 μg / 500 of fraction I of Photo 2
μl treatment and 25 μg / 500μ of Fraction II in Photo 3
The cells are smaller than the cells treated with 1 and have a globular shape, and the morphology peculiar to cancer cells such as the layering of cells is observed. Photo 2
In Nos. 3 and 3, morphological changes accompanied by prominent cell enlargement and differentiation into squamous epithelial cells are observed.

C. Mouse skin cancer (melanoma) B-1
Anti-cancer effect on 6 cells: The anti-cancer effect on B-16 cells is determined by the test of the effect on HeLa cells and the number of cells,
Exactly the same test was performed on the medium and the treatment method. 8 Photo 4 were trypsinized after incubation for 7 days after treatment, but was taken on day 4 subcultured by adjusting the number of cells in 4 × 10 ^4/5 ml in each treatment group. Photo 4 shows the deposition of melanin pigment by a spherical small layer peculiar to cancer cells on the 4th day of passage after addition of 500 μl of sterile distilled water. Photos 5 and 6 are 150 μg / 5 of fraction I, respectively.
On day 4 of passage of the cells to which 00 μl and 25 μg / 500 μl of the fraction II were added, a spindle-shaped enlargement of the cells was observed in both of them. Its action is 150 μg / like HeLa cells
500 μl treatment is strong. Photo 7 also suggests weak morphological changes and cell differentiation depending on the dose-action reaction even at 25 μg / 500 μl treatment, which was not prominent in HL-60 cells.

The difference in melanin deposition due to the overlay of B-16 cells is visually observed as shown in Photo 8.

The action of fractions I and II is considered to be derived from the promotion of differentiation of undifferentiated cancer cells as observed in HeLa and B-16 cells, and the acquired traits are maintained even after passage. It was saved without being lost. Further, _{GM3 and the} like are known as anti-cancer gangliosides, but these are considered to be effective only in serum-free medium and inhibit their uptake into cancer cells by binding with serum proteins.
In this respect, the action fraction distributed in sweet potato is effective in a medium containing 10% fetal bovine serum, and can be expected as an ideal anticancer agent.

Fraction I is HPTLC-Fretigpla
ten kieselge 160F ₂₅₄ (MERC
At the time of development with K), a pale blue fluorescent light is emitted by UV lamp irradiation, but sorbic acid sulfate reagent does not stain sialic acid. The extract substance irradiated with ultrasonic waves from the thin layer reacts with sorbinate sulfuric acid to be colored, and at the same time, the mobility is the same as that of fraction II. In addition, the working concentration of both fractions was about 6 times as strong as that of Fraction I, and the mobility was much higher than that of Fraction I in Fraction I in a strongly organic developing solution. Fraction I
Long fatty chains protect the ganglioside sialic acid, suggesting that it was cleaved upon extraction from thin layers. This is considered to mean active absorption in the small intestine as lipid droplets.

[0020]

[Example 1] purchasing stems and leaves from a sweet potato cultivator,
After washing with a tea leaf washer, cut to about 1 cm and dry or ferment in the same manner as tea to give 0.1 g per bag of tea pack.
Is added with the tea leaves and supplied as tea with a cancer prevention effect. In this case, 1 g corresponds to an amount corresponding to 25 l of the above-mentioned administration concentration of fractions I and II. If you drink 10 bags a day, it will be 62.5μg / 500μ for a 60kg person.
1 corresponds to the administration of fraction I. This can be expected to have a sufficient preventive effect on the latent cancer promoter.

[0021]

[Practical example 2] A washing solution for producing starch from sweet potato is passed through a cation exchange resin and then an anion exchange resin,
Fraction I is eluted from the anion exchange resin with a 0.1 M ammonium acetate solution and fraction II is eluted with a 0.3 M ammonium acetate solution for desalting and crystallization, and then supplied as a food additive or added to a beverage or the like.

[0022]

INDUSTRIAL APPLICABILITY In the sweet potato starch manufacturing industry, a huge amount of starch meal and most of the waste liquid at the time of manufacture are discarded as industrial waste and waste water. It is the current situation that such purification requires not only enormous volume of treatment ponds, treatment plants and electric power consumption but also causes environmental problems such as bad odor and water pollution.

INDUSTRIAL APPLICABILITY The present invention is used as an ideal anti-cancer agent and cancer-preventive food and drink from discarded materials in sweet potato cultivation and starch production, and can add value to troublesome waste. Its social, environmental and economic effects are enormous.

[0024]

[Brief description of the photo]

[Photo 1] HeLa cells derived from human cervical cancer (10%
Micrograph of Eagele-EME medium containing fetal bovine serum) added with 10% distilled water of the medium and cultured for 4 days

[Photo 2] Micrograph of cultured HeLa cells to which 10% sweet potato fraction I (150 μg dissolved in 500 μl distilled water) of the medium was added and cultured for 4 days

[Photo 3] Micrograph of cultured HeLa cells to which 10% sweet potato fraction II (25 μg dissolved in 500 μl distilled water) of the medium was added and cultured for 4 days

[Photo 4] B-16 cells derived from mouse melanoma
Micrograph of La cells cultivated in the same medium, 10% distilled water of the medium was added and cultivated for 7 days, and then subcultured for 4 days with the number of cells being equalized.

[Photo 5] Cultured B-16 cells containing 10% sweet potato fraction I
(150 μg / 500 μl) Treatment micrograph on day 4 after passage

Photo 6: Cultured B-16 cells containing 10% sweet potato fraction I
Micrograph of I (25 μg / 500 μl) treated 4 days after passage

[Photo 7] Cultured B-16 cells containing 10% sweet potato fraction I
(25 μg / 500 μl) Treatment micrograph on day 4 after passage

[Photo 8] 10% sweet potato fraction I from cultured B-16 cells
(25 μg / μl) Reduction of melanin pigmentation on day 4 after passage of treatment (compared to the same distilled water treatment)

Claims (2)

[Claims] 1. Purification of glycolipids by extracting sweet potato leaves, vines, stems and subterranean stems containing edible parts, either by drying or extracting raw water with hot water and steam, or ultrasonically or homogenizing with water or an organic solvent. And column chromatography / thin layer chromatography to isolate / purify the active substance 2. An anticancer glycolipid extracted and purified by the method according to claim 1.