JPH11140102A - Apple vinegar-derived antitumoral polysaccharide and preparation thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To extract and purify an antitumoral polysaccharide having such a new structure that has not been known until now, from an apple vinegar as a raw material. SOLUTION: This apple vinegar-derived antitumoral polysaccharide is a polysaccharide comprising the main component of a glucose which has in the mol. α-1,4-linkage and α-1,6-linkage and has a range of a mol.wt., in dextran equivalent, of 7,000 or more to 17,000 or less; it is extracted from an apple vinegar and purified, and also it can recover a malignant tumor by giving to a cancer patient.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an antitumor polysaccharide, and more particularly to a novel antitumor activity obtained by extracting and purifying apple vinegar brewed from apples specially produced in Aomori Prefecture. An object of the present invention is to provide an apple vinegar-derived antitumor polysaccharide having a simple structure.

[0002]

BACKGROUND OF THE INVENTION Vermont vinegar, a favorite of the people of the Vermont region of North America, is a drink prepared by adding a teaspoon of apple cider vinegar to a cup of honey and diluting with a glass of water. Is considered to be one of the reasons why people can maintain longevity, and it is said that it is very good for health, but information on the structural knowledge of bioactive substances showing the effect has been disclosed so far According to the above fact, it was one of the most interesting themes because of the unique region that boasts the best apple production in the whole country.

Various factors must be considered as conditions for achieving longevity in modern society. Among them, the rate of malignant tumors (cancer) is important in judging the conditions for longevity. It is already well known that this can be a factor.

[0004] On the other hand, it is a fact that many host-mediated antineoplastic polysaccharides have been found from various materials such as cereals and fruits. For example, β-cells extracted from mushrooms and the like have been found. -(1,3) (1,6) -glucan is very famous, and further examples of antitumor polysaccharides in which the glucosidic bond between each sugar residue has an alpha bond include, for example: As can be seen from the invention disclosed in Japanese Patent Publication No. 18722/1986, there is alpha- (1,6) -glucan using rice bran as a raw material, and alpha- ( 1,4) -Glucan is described as an invention in Japanese Patent Publication No. 88688/1999, and in Japanese Patent Publication No. 27929/1994.
In this publication, glycogen and alpha- (1,4) (1,6) -glucan using shellfish as a raw material are described as antitumor polysaccharides having a molecular weight of 100,000 or more.

[0005] As a result of analogy and consideration of these facts, apple vinegar, which is considered to be one of the causes of longevity in the people of Vermont, North America, may contain some kind of antitumor polysaccharide. According to the present invention, in order to find a novel antitumor polysaccharide component from apple vinegar, the present invention has been promptly and carefully analyzed and examined, and finally a novel polysaccharide component as described in detail below. An apple tumor vinegar-derived antitumor polysaccharide having a structure and a novel production method for extracting and purifying it have been successfully completed and put to practical use.

[0006]

The antitumor polysaccharide derived from apple cider vinegar of the present invention basically requires the following constitution. In other words, the main chain of alpha-1,4-linkage contains alpha-
The main component is glucose having a 1,6-bonded branched structure, and the molecular weight range is from 7000 to 1 in terms of dextran.
It is an antitumor polysaccharide derived from apple cider vinegar having an average molecular weight of about 10,000 or less.

The antitumor polysaccharide derived from apple cider vinegar of the present invention having the above-described structure as a basic structure has the following properties: (a) a molecular weight range of 7000 in terms of dextran;
Not less than 17000 and Sephacryl S-300
Has a main peak at an average molecular weight of 10,000 by gel filtration analysis according to the above. (b) Be a polysaccharide containing glucose as a main component. (c) having an alpha-1,4-linkage and an alpha-1,6-linkage in the molecule; As described above, the antitumor polysaccharide derived from apple cider vinegar is characterized by having at least three components.

[0008] More specifically, it is also possible to show the composition as an antitumor polysaccharide derived from apple cider vinegar, which has the following physical properties. That is, (a) a white or light brown powder that is easily soluble in water. (b) the molecular weight range is from 7000 to 17000 in dextran equivalent,
It has a main peak at an average molecular weight of 10,000 by gel filtration analysis using Sephacryl S-300.
(c) A polysaccharide containing glucose as a main component. (d)
Proton-NMR (Nuclear Magnetic)
(Resonance) spectrum, and a signal consisting only of sugar by NMR. (e) Do not pass through the dialysis membrane. (f) Positive in phenol sulfate reaction,
Show positive in iodine reaction. (g) Must not exhibit an ultraviolet absorption maximum in an aqueous solution. (h) having an alpha-1,4-linkage and an alpha-1,6-linkage in the molecule; Is an antitumor polysaccharide derived from apple cider vinegar comprising the following eight elements:

[0009]

[Related Invention] The present invention relates to the above-mentioned anti-tumor polysaccharide derived from apple cider vinegar and includes a method for producing an anti-tumor polysaccharide derived from apple cider vinegar having the following constitution. First, apple vinegar is concentrated under reduced pressure or dialyzed directly against distilled water (for example, Sanko Junyaku Co., Ltd., Dialysis membrane 36/3).
2, a molecular weight of 12000 to 14000) and freeze-drying the dialysis solution. Next, the freeze-dried product was subjected to gel permeation chromatography using Sephacryl S-300 gel, and it was 1000 to 400 in terms of dextran.
A step of obtaining a fraction of 00 or less and then desalting. Thereafter, weak anion exchange chromatography, such as DEA
E Sepharose Fast flow Anion exchange gel (trade name, manufactured by Pharmacia, etc.) was applied to column chromatography, eluted with a dilute neutral buffer at least twice the volume of the column carrier, desalted, and then lyophilized. Process. This is a method for producing an antitumor polysaccharide derived from apple vinegar, comprising the above steps.

The above dialysis, gel filtration chromatography,
In weak anion exchange chromatography or the like, it can be performed using another gel filtration carrier, an ion exchange resin, an ultrafiltration membrane or the like.

The properties of the antitumor polysaccharide extracted and purified by the method of the present invention are as follows from the results of chemical analysis. "Average molecular weight" From the effluent pattern by Sephacryl S-200 gel (trade name, manufactured by Pharmacia) filtration chromatography in FIG. 2, the semi-log plot of those molecular weights with respect to the effluent amount of the standard material shows the effluent amount of the antitumor polysaccharide. Was extrapolated to find that the molecular weight was estimated, and the average molecular weight of the antitumor polysaccharide derived from apple cider vinegar was 10,000.

"Proton NMR" In this chemical analysis, 2 milligrams of the dried material was dissolved in 99.8% D2O, shaken at room temperature for 12 hours, replaced with heavy water, and freeze-dried. The same operation was performed with 99.8% D2O and 99.95% D2O, and then dissolved in 99.93% D2O, and NMR was measured at 70 ° C. For NMR measurement, 270-MHZ
1H-NMR (JNM EX-270, manufactured by NEC) was used. The result is as shown in FIG.
3. a signal derived from α-1,4 bond at 73 ppm;
At 963 ppm, a signal derived from α-1,4,6 bond is shown, and at 3.4-4.4 ppm, a signal thought to be derived from polysaccharide is captured, and a signal consisting of only saccharide is shown.

"Chemical component analysis value" It is found that the tissue composition of sugar is composed only of glucose by the following analysis method. That is, a sample obtained by hydrolyzing with 2N hydrochloric acid for 2 hours is used as a sample, and a pyridyl amination apparatus (Takara Shuzo Co., Ltd.
Using a PALPAK Type A column (4.6 × 150 mm) obtained by apyridyl amination using ALSTATION model 4000), the solvent was 0.7 M
Potassium-borate buffer (pH 9.0) / acetonitrile (90/10, v / v), flow rate: 0.3 ml / min, column temperature: 65 ° C., detection: measurement of excitation wavelength 310 nm using a fluorescence detector HPLC at wavelength 380nm
(High performance Liquid Ch
As a result of the sugar composition analysis by romatography, as shown in FIG. 3, the separation pattern of the apple vinegar-derived anti-tumor polysaccharide by the pyridyl amination method of the apple vinegar-derived anti-tumor polysaccharide is shown as xylol, glucose, It was revealed that the polysaccharide was composed of mannose and galactose, most of which was composed of glucose (9.7%). Hereinafter, an example of the method for producing an antitumor polysaccharide and the method for administering an effective fraction in the present invention will be described in more detail.

[0014]

EXAMPLE 1 This example shows one example of a method for extracting an antitumor polysaccharide from apple cider vinegar.
After 0 ml was concentrated under reduced pressure to 100 ml, 70 ml of distilled water was added to make the total amount 170 ml, and the mixture was concentrated again under reduced pressure to give a total amount of approximately 100 ml, and dialyzed against distilled water. The dialysis solution (crude antitumor polysaccharide) was lyophilized and stored refrigerated. The dry weight of the dialysis solution is about 7% of the apple cider vinegar dry weight.
Met.

[0015]

EXAMPLE 2 Next, as an example of separation and purification, 20 mg of a crude antitumor polysaccharide was dissolved in an aqueous 0.1 M sodium chloride solution, and Sephacryl S-300 gel (trade name, manufactured by Pharmacia), column (2.3 × 118 cm), 0.1 M NaCl was used as the eluate, and the flow rate was
Purified at 0.5 ml / min. Neutral sugar was measured for each fraction (5 ml) by the phenol-sulfuric acid method, and the outflow pattern is shown in FIG. It was divided into three fractions F1, F2 and F3, desalted, and lyophilized. The yields were 1.6 mg (12.8%) for F1, 3.8 mg (30.4%) for F2, and 7.0 for F3.
Milligrams (56.8%).

Further, this F3 was fractionated and purified by weak anion exchange chromatography using DEAE Sepharose Fast Flow anion exchange gel (trade name, manufactured by Pharmacia). Separation was performed using 20 mmol / liter Tris-HCl buffer (pH 7.2) as an elution buffer (see FIG. 3). Neutral sugar was measured by the phenol sulfate method, divided into three fractions (abc), dialyzed, and lyophilized. The yield was 3.6 mg of a (5 mg).
6%), b is 1.7 milligrams (25%), and c
1.2 milligrams (19%). In the figure, a is apple cider vinegar-derived antitumor polysaccharide.

[0017]

Example 3 An example of an antitumor experiment is as follows. A 6-week-old female, BALB / C-CRJ mouse weighing an average of 20 grams was transplanted with 1 x 105 peritoneal peritoneal cancer cells Meth-A, which had been passaged intraperitoneally to syngeneic mice for 1 week. The test group (1 group) and the test group (1 group) were divided into two groups.

On the second, fourth, and sixth days after the transplantation of the cancer cells, the test group was prepared by dissolving 0.625 milligrams of the antitumor polysaccharide in 0.2 milliliter of physiological saline and intraperitoneally. Was administered. The control group was similarly administered only with physiological saline. Thereafter, the number of surviving days was observed up to 60 days, and the survival effect was calculated by the following equation. Survival rate (%) = (average surviving days of test group) / (average surviving days of control group) × 100 The antitumor effect of the antitumor polysaccharide derived from apple cider vinegar, which was tested by the above method, is as shown in the table below. Was.

[0019]

[Table 1]

[0020]

Embodiment 4 Next, a second example of an antitumor experiment will be described. 6 week old female, average weight 20g B
ALB / C-CRJ mice were intraperitoneally transplanted with 1 × 10 5 cancer cells Meth-A per mouse intraperitoneally for 1 week, and 5 control mice (1 group)
And a test group (1 group).

On the second, fourth, and sixth days after the transplantation of the cancer cells, the test group was prepared by dissolving 0.2 mg of the antitumor polysaccharide in 0.2 ml of physiological saline and intraperitoneally. Was administered. The control group was similarly administered only with physiological saline. Thereafter, the number of surviving days was observed up to 60 days, and the survival effect was calculated by the following equation. Survival rate (%) = (average survival days of test group) / (average survival days of control group) × 100 The antitumor effect of apple cider vinegar-derived antitumor polysaccharide tested by the above method is shown in the table below. Out.

[0022]

[Table 2]

[0023]

[Effects] As a treatment method for a tumor using an apple vinegar-derived anti-tumor polysaccharide, the apple vinegar-derived anti-tumor polysaccharide is dissolved in physiological saline or various salt solutions, and then administered directly to a lesion by direct injection. Intravenous administration is possible. In addition, because it is similar to substances that exist in the body, there is no toxicity,
Thus, the dosage can be controlled over a wide range depending on the condition.

In the present invention, its antitumor activity can be confirmed by the following method. That is, tumor cells could be cured by implanting tumor cells into the peritoneal cavity of a mouse, dissolving the apple vinegar-derived antitumor polysaccharide in physiological saline, and administering the solution. In this experiment, since no side effects were observed in the cured mice, it was considered that there was no acute toxicity, and it was confirmed that the mice were very promising substances for tumors.

Antitumor polysaccharides having such characteristics include β- (1,3) (1,1) extracted from mushrooms and the like.
6) -Glucan is famous, but the antitumor polysaccharide derived from apple cider vinegar in the present invention is a material which is completely different in that glycoside bonds between the sugar chain residues take reverse alpha bonds. In addition, it has been reported that there are several other polysaccharides exhibiting antineoplastic activity, and there are known alpha-glucans of these polysaccharides which have antineoplastic properties. The present invention relates to a product comprising only -1,4-linkage (polysaccharide obtained from the roots of ginseng 37) or a product comprising only alpha-1,6-linkage (polysaccharide derived from rice bran). Is significantly different from the apple vinegar-derived antitumor polysaccharide in its structure and molecular weight.

Glycogen having an alpha-1,4-linkage and an alpha-1,6-linkage (eg, a glycogen obtained from shellfish) is also known, but the antitumor polysaccharide derived from apple cider vinegar of the present invention is also known. Has a molecular weight of 20,000 or less, whereas those known are completely different substances having a molecular weight of 100,000 or more.

As described above, the present invention continues to develop and study the extraction of antitumor polysaccharide from normally eaten apple vinegar, and the antitumor activity of the obtained antitumor polysaccharide.
As a result of repeated trial and error, it was finally possible to confirm that the cider vinegar-derived antitumor polysaccharide of the present invention has strong antitumor properties. The absence of cytotoxicity in the anti-tumor polysaccharide derived from apple cider vinegar of the present invention activates the immune mechanism, that is, the self-defense mechanism that the living body should have, thereby increasing the resistance to tumor cells. As a result, the proliferation of tumor cells With this effective feature, apples that have become a specialty of the region can be used with greatly increased added value and contribute to the development of local industries, It is expected that a high reputation in human medicine will be obtained above all.

[Brief description of the drawings]

FIG. 1 is a proton-NMR spectrum of an antitumor polysaccharide derived from apple vinegar.

FIG. 2 is a gel filtration chromatography separation pattern of crude anti-tumor polysaccharide on Sephacryl S-300 gel.

FIG. 3 is a separation pattern obtained by analyzing a sugar composition of an antitumor polysaccharide derived from apple cider vinegar by a pyridyl amination method.

FIG. 4. Sephacryl S-, an antitumor polysaccharide derived from apple cider vinegar
200 is a gel filtration chromatography separation pattern using 200 gels.

FIG. 5. DEA of F3 fraction of gel filtration chromatography
E Sepharose Fast Flow It is an ion exchange chromatography separation pattern by a weak anion exchange resin.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Toshisada Kushibiki 15-23, Kurashimachi, Hirosaki City, Aomori Prefecture Inside Kanesho Co., Ltd. (72) Inventor Rikako Arai 9 Hirosaki Functional Food Development Cooperative

Claims (4)

[Claims] The present invention comprises glucose having a branched structure of alpha-1,6-linkage in the main chain of alpha-1,4-linkage as a main component, a molecular weight range of 7000 to 17000 in dextran equivalent, and an average molecular weight of about 10,000. Apple vinegar-derived antitumor polysaccharide. 2. An antitumor polysaccharide derived from apple cider vinegar, having the following physical properties. (a) The molecular weight range is 7000 or more in terms of dextran and 1
7000 or less and having a main peak at an average molecular weight of 10,000 by gel filtration analysis with Sephacryl S-300. (b) Be a polysaccharide containing glucose as a main component. (c) having an alpha-1,4-linkage and an alpha-1,6-linkage in the molecule; 3. An antitumor polysaccharide derived from apple cider vinegar, having the following physical properties. (a) White or light brown powder that is easily soluble in water. (b) The molecular weight range is 7000 or more in terms of dextran and 1
7000 or less and having a main peak at an average molecular weight of 10,000 by gel filtration analysis with Sephacryl S-300. (c) A polysaccharide containing glucose as a main component. (d) Proton-Nuclear Magnetic
4 shows a Resonance (NMR) spectrum, NM
R indicates a signal consisting only of sugar. (e) Do not pass through the dialysis membrane. (f) Positive in phenol sulfate reaction and iodine reaction respectively. (g) Must not exhibit an ultraviolet absorption maximum in an aqueous solution. (h) having an alpha-1,4-linkage and an alpha-1,6-linkage in the molecule; 4. Apple apple vinegar is concentrated under reduced pressure or dialyzed against distilled water (dialysis membrane 36/32, molecular weight 120).
00-14000) and freeze-drying the dialysis solution. The freeze-dried product is subjected to gel filtration chromatography using Sephacryl S-300 gel to obtain a dextran-equivalent fraction of 1,000 to 40,000 and desalted. And then subjected to weak anion exchange chromatography, for example column chromatography packed with DEAE Sepharose Fast Flow anion exchange gel, eluting with at least twice the volume of the column carrier in dilute neutral buffer, A method for producing an antitumor polysaccharide derived from apple cider vinegar, comprising the steps of desalting and freeze-drying.