JPS59184129A - Polysaccharide t25g i and its preparation - Google Patents

Abstract

NEW MATERIAL:The polysaccharide T25G I . Appearance, white powder; solubility, soluble in water and insoluble in organic solvents such as alcohol, benzene, etc.; sugar composition, composed mainly of glycose and contains arabinose and galactose; molecular weight, 61,000; specific rotation, [alpha]<22>D=+54.9 deg. (C= 0.5, H2O). USE:An antitumor agent exhibiting strong suppressing effect to sarcoma 180 solid carcinoma and having low toxicity. PREPARATION:The whole grass of Tetragonia expansa is extracted e.g. with hot water of >=80 deg.C, and the extract liquid is purified e.g. by alcohol precipitation, etc. and molecular sieve treatment. In the above process, the molecular sieve treatment is carried out first by using a molecular sieve having a fractionation molecular weight of between 1,000-50,000 and 1,000-100,000, and the first eluted saccharide fraction is treated with a molecular sieve having a fractionation molecular weight of between 1,000-100,000 and 1,000-200,000. The objective polysaccharide T25G I is collected from the first sugar fraction of the two eluted fractions of polysaccharide.

Description

Detailed Description of the Invention (1) Background of the Invention 1 Seed Spot I The present invention relates to a novel polysaccharide T25G I and a method for producing the same.

More specifically, the present invention relates to polysaccharide T25G I isolated and purified from a hot water extract of the whole plant of Thurna, and a method for producing the same. The polysaccharide of the present invention is useful as a low toxicity antitumor agent.

Le tachisara zuru tsuruna is a Japanese folk medicine, also known as hamajisha, and is said to be good for stomach cancer and stomach ulcers. However, its ingredients and pharmacological effects are unknown, and books on medicinal plants published in recent years state that it is ``effective against stomach cancer, but it is doubtful.'' Showa 55
Published on April 1, 2015, see pages 21-22].

The present inventors first extracted the whole plant of Tuluna with hot water and purified the obtained extract by alcohol precipitation method or dialysis membrane method to obtain Trunna extract with antitumor activity (Japanese Patent Application Laid-open No. 5
7-193416). As a result of further purifying this extract using a molecular sieve, the present inventors succeeded in isolating a polysaccharide having an antitumor effect, thereby completing the present invention.

II. OBJECTS OF THE INVENTION Accordingly, an object of the present invention is to provide a novel polysaccharide having antitumor properties. As described below, the polysaccharide of the present invention exhibits a strong inhibitory effect on Sarcoma 180 solid cancer and has low toxicity.

A further object of the present invention is to provide a method for producing the above polysaccharide. According to the method of the present invention, the above-mentioned polysaccharide is produced by treating a hot water extract of the whole plant of Tulna with molecular sieve treatment, particularly gel filtration, and collecting a compound having a molecular weight of about 81,000.

(1) Detailed Description of the Invention The present invention consists first of all of the polysaccharide T25G I obtained from the whole plant of Tulna and having the following properties.

(b) Color and shape white powder (b) Solubility Soluble in water, alcohol, benzene, vinegar Insoluble in organic solvents such as ethyl chloride (c) Sugar composition Consists primarily of glucose, and Contains arabinose and galactose.

(This) Molecular Weight A single peak is obtained by high performance liquid chromatography with a molecular weight cutoff of 10,000 to 100,000, and the molecular weight is approximately 61,000.

(e) Specific rotation (f) Infrared absorption spectrum As shown in FIG.

Enter yna 2920, 1660 (g) Ultraviolet absorption spectrum Does not show absorption maximum when measured in aqueous solution, Shows terminal absorption.

The second aspect of the present invention comprises a method for producing the polysaccharide T25G I, which comprises subjecting a hot water extract of the whole plant of Tulna to molecular sieving to collect a compound having a molecular weight of about 81,000.

Thirdly, the present invention provides that the molecular sieve treatment is performed so that the molecular weight cut-off is i, ooo ~ 50,000 to 1,000 to 100.0.
Of the two components of the resulting polysaccharide, the first eluted sugar fraction is further sieved with a molecular weight cut-off of 1,000 to 100,000 to 1.0.
The above polysaccharide T25G I is obtained by performing molecular sieving treatment using a molecular sieving agent in the range of 00 to 200.000, and collecting the polysaccharide from the first eluted sugar fraction of the two fractions of the resulting polysaccharide. Consists of manufacturing method.

Fourthly, the present invention comprises a method for producing the polysaccharide T25GI, wherein the molecular sieve treatment is gel filtration, and the molecular sieve agent is a gel filtration agent.

Fifthly, the present invention provides that the gel filtration agent is dextran gel,
It consists of a method for producing the above-mentioned polysaccharide T25GI, which is a polyacrylamide gel, a woody polyvinyl gel, or a porous glass bead.

When producing the polysaccharide of the present invention, a hot water extract of the whole plant of Thurna is subjected to molecular sieve treatment, particularly gel filtration, and the molecular weight is about 81.
000 is taken.

Trunna belongs to the family Trunaceae (Pomegranate, Aizodan7), and its scientific name is Tetra onia tetra onoi.
des(PALL.

) O, Kuntze. In the present invention, the whole plant extract is used as a raw material. There are no particular restrictions on the season for collecting trunna. The operation of extracting the whole trunna plant with hot water is carried out by adding hot water of 80°C or higher to the whole trunna plant, or by adding water to the whole trunna plant and heating the mixture to boiling. . Heating can be carried out in a boiling water bath or over an open fire. The extraction time is appropriately determined depending on the quality of the raw materials, etc., but is usually 1 to 48 hours. After the extraction process is completed, an extract is obtained by filtering the extraction mixture. Since the extract thus obtained contains a large amount of impurities, it is desirable to purify the extract by alcohol precipitation or dialysis before being subjected to the molecular sieve treatment step of the present invention. For example, when purifying by alcohol precipitation, an alcohol such as methanol or ethanol is added to the above extract, and the resulting precipitate is collected by a conventional method, for example, by centrifugation, to obtain the desired extract.

In the case of purification by the dialysis membrane method, the extract is placed in a dialysis tube, dialyzed by immersion in water, and the dialyzed solution is concentrated to dryness or freeze-dried as desired to obtain the desired extract. As a dialysis membrane, regenerated cellulose, such as Spectra O Pore (
Spectrum Medical Industries, Inc. product)
Alternatively, Visking tube (Union Carbide product) is used. The extract obtained by purification by an alcohol precipitation method or a dialysis membrane method is dissolved in water to obtain a hot water extract of the whole plant of Trunna, which is a raw material for the method of the present invention. Furthermore, prior to the hot water extraction treatment of the whole trunna plant, by extracting the trunna whole plant with an organic solvent and/or room temperature water,
It is also desirable to remove unnecessary components in advance. Solvents used for extraction pretreatment include methanol, ethanol,
polar organic solvents such as propatool, pyridine, acetone, benzene, toluene, xylene, n-hexane,
Nonpolar organic solvents such as chloroform, carbon tetrachloride, and ethyl acetate can be used.

The molecular sieve treatment for isolating the polysaccharide of the present invention is preferably carried out by gel filtration using a gel filtration agent.

It is desirable to perform gel filtration twice using gel filtration agents with different molecular weight cutoffs.

That is, the hot water extract of the whole plant of trunna is first extracted with a molecular weight cut-off of 1.0.
The sample is passed through a column packed with a gel filtration agent ranging from 0.00 to 50,000 to 1.000 to too, ooo, and eluted with distilled water. When the sugar in the eluate is quantified using the phenol-sulfuric acid method according to a conventional method and the sugar elution curve is drawn, a curve with two peaks is obtained. Collect the sugar fraction that elutes first.

Next, the sugar fraction thus obtained is further reduced to a molecular weight fraction i.
,ooo~100,000~1.000~200.
The sample is passed through a column packed with a gel filtration agent in the range of 0.000 and eluted with distilled water. When the sugar in the eluate is quantified in the same manner as above and the elution curve is plotted, a curve with two peaks is obtained. The first eluting sugar fractions are collected and the desired polysaccharide is recovered by suitable means such as evaporation to dryness or freeze drying.

As the gel filtration agent used in the above gel filtration, dextran gel, polyacrylamide gel, polyvinyl polymer gel, porous glass beads, etc. can be suitably used. Molecular weight cutoff is 1.000-50,000-1,00
0-100. Examples of gel filtration agents in the range of Qθo include Sephadex G-75, G, which is a dextran gel.
-100 (Pharmacia product, Sweden), polyacrylamide gel Biogel P-100, P-1
50 (Bio-Rad product, USA), polyvinyl polymer gel Toyopearl Hw-5o (Toyo Soda Kogyo product 9 Japan), porous beads CPG-10 (Electro Nucleonics product, USA), etc. are suitable. used.

In addition, the molecular weight cut-off is 1,000 to 100,000 to 1
Examples of gel filtration agents in the range of , 000 to 200.000 include Sephadex G-100, G-150 Biogel P-150 Toyopearl HW-50, CPG-10
etc. are preferably used. The thus obtained polysaccharide T25GI of the present invention has the following properties. (a) Color and shape White powder (b) Solubility Soluble in water, insoluble in organic solvents such as alcohol, benzene, ethyl acetate, etc. (c) Sugar composition Mainly composed of glucose, and further contains arabinose and galactose.

(d) Molecular weight The fractional molecular weight is 10,000 to ioo, ooo. A single peak is given by high performance liquid chromatography, and the molecular weight is determined by the Park & Johnson method (Park & Johnson: J, Biol
, Chew, 181.149 (1848)), it is approximately 81,000.

(e) Specific rotation (f) Infrared absorption spectrum As shown in FIG.

2820.1660 (g) Ultraviolet absorption spectrum Does not show absorption maximum when measured in aqueous solution, Shows terminal absorption.

The polysaccharide of the present invention is useful for various malignant tumors, and its administration form includes parenteral administration such as subcutaneous injection, intravenous injection, and intramuscular injection, or tablet, capsule, granule, powder, and syrup. Oral administration can be mentioned. The dosage of this polysaccharide varies depending on the administration route, age, weight, and symptoms of the patient, but for adult males, it is approximately 1 to
It is 5g.

The polysaccharide of the present invention is formulated and administered according to conventional methods. For example, put the dry powder of this polysaccharide in a container such as a vial, prepare physiological saline, glucose solution, or carboxymethyl cellulose (CMC) suspension in a separate container such as an ampoule, and suspend and dissolve the powder before use. Inject. Alternatively, it may be injected in the form of an emulsion. For example, water in oil (Wl
In the case of type o) emulsion, a combination of mineral oil such as liquid paraffin, vegetable oil such as sesame oil or peanut oil, and a surfactant such as sorbitan fatty acid ester is used.

Next, the present invention will be explained in more detail with reference to Reference Examples, Examples, and Formulation Examples.

Reference example Tsuruna heat output - 0, (1) 2 kg of dried Tsuruna whole plant was mixed with methanol (20 sentences x
3) at room temperature for 24 hours, and the resulting extraction residue was extracted three times with hot water 201. The obtained extracts were combined and concentrated to dryness using a rotary evaporator.
30.3 g of powder was obtained. (2) Dissolve 10.0 g of the powder obtained in (1) above in 500 ml of water, and add 9 ml to this aqueous solution.
Add 9% ethanol slowly, stop adding when the ethanol concentration in the aqueous solution reaches 80%, and wait for a while.
Stirred. The generated precipitate was collected by centrifugation to obtain 7.22 g of a brown powdered whole plant extract.

(3) Add 10.0g of the powder obtained in (1) above to 500 g of water.
This aqueous solution was placed in a Visking tube and dialyzed against water. The dialyzed solution was concentrated to dryness to obtain 6.37 g of a brown powdered whole plant extract of Tuluna.

Example 1-1 1.092 g of the hot water extract of the whole plant of Tulna obtained in Reference Example (2) or (3) was dissolved in 20 ml of distilled water,
Sephadex G-75 (molecular weight cut off 1.000-50
,000) (diameter 7.0 cm, length 3
5.0 cm), and gel filtration was performed using distilled water. When gel filtration is performed while quantifying the sugar content in the eluate using the phenol-sulfuric acid method, the polysaccharide is separated into two parts. 145 mg of polysaccharide was obtained from both sugars that were eluted first.

The polysaccharide thus obtained was treated with Sephadex G-100 (
When gel filtration is further performed using the molecular weight cutoff i, ooo to too, ooo), the polysaccharide is divided into two halves. 45 mg of the desired polysaccharide T25GI from both sugars that elute first
was gotten.

Formulation Example 1 1000 mg of polysaccharide T25G I was dissolved in 500 ml of a sterile 5% glucose solution for injection, and 5 ml of this solution was aseptically dispensed into vials and freeze-dried.

In this way 10 mg of polysaccharide T25G in 1 vial
A formulation containing I was obtained. Example 2 of two formulations to be dissolved in distilled water for injection before use A vial formulation was prepared in the same manner as in Formulation Example 1 above. However, 500 ml of physiological saline was used instead of 500 ml of sterile 5% glucose solution for injection.

Before use, dissolve in distilled water for injection.

Test Example 1 Sarcoma 180 J gun (sample preparation) Phosphate buffered saline (manufactured by Gibco, phosphoric acid 9.511IM)
The sample was dissolved in a solution of 0.5% carboxymethylcellulose (C'MC) suspended in PBS) to a predetermined concentration.

(Sarcoma 180 cancer cell transplantation) Sarcoma 180 cancer cells subcultured in the peritoneal cavity of ICR mice were taken out together with ascites fluid, and diluted appropriately with physiological saline to adjust the cell number to 1.0×IO8 cells/1. . 011ml of this suspension was subcutaneously transplanted into the back of a 4-week-old male ICR mouse using a syringe. Therefore 1
The number of transplanted cells per animal is 1 x 107 cells.

(Sample administration) Once a day from the day after transplanting Sarcoma 180 cancer cells
For four consecutive days, 0.1 ml of the sample prepared above was administered intraperitoneally using a syringe. Six mice were used per sample liter concentration. The control is the above CM used as a solvent for the sample.
PBS containing C was administered in the same manner. The dosage was expressed in mg per kg of mouse body weight. (Efficacy evaluation method) On the 21st day after cancer cell transplantation, the cancer tissue that had grown I was excised and its weight was measured (average of 6 mice per group). value). The effect was determined by taking the ratio (T/C) between this weight and that of the control. The control cancer tissue weight was 3.11 g. Ratio values of 100 to 71% are invalid (-), ratios of 70 to 51% are slightly valid (+), and ratios of 50 to 21% are valid (+).
+), and those of 20 to 0% were considered to be markedly effective (+ + +).

The results are shown in Table 1.

Table 1 Effect on Sarcoma 180 solid tumor (mice) Test Example 2 An acute toxicity test was conducted by administering the polysaccharide of the present invention to ICR male mice weighing 20±Ig. It was 250 mg/kg or more.

■Specific Effects of the Invention As detailed above, according to the present invention, firstly, polysaccharide T
25G I is provided. This polysaccharide is a new compound that has not been published in any literature, and as shown in the test example,
It has a strong suppressive effect on solid cancer and has very low toxicity, making it effective as an antitumor agent.

According to the present invention, secondly, a method for producing the above polysaccharide is provided. That is, the polysaccharide is obtained by treating a hot water extract of the whole plant of Tulna with a molecular sieve and collecting a polysaccharide having a molecular weight of about 81,000.

According to the present invention, thirdly, an advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide was subjected to the above molecular sieve treatment with a molecular weight cut-off of 1,000 to 50,000 to 1,000 to i
Molecular sieves in the range of 0.0 and 0.0 and molecular weight cut-off of 1.0.
000~10.0.000~1,000~200.0
High purity can be obtained by combining it with a molecular sieve in the 000 range.

According to the present invention, fourthly, a more advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide can be more easily obtained by performing gel filtration using a gel filtration agent as the molecular sieve treatment.

According to the present invention, fifthly, a more advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide can be easily obtained in high purity by using dextran gel, polyacrylamide gel, woody polyvinyl gel, or porous glass beads as a gel filtration agent.

[Brief explanation of drawings]

FIG. 1 shows the infrared absorption spectrum of polysaccharide T25G I of the present invention. Patent applicant Chirusen Co., Ltd. 5-12-2 Higashiei-cho, Mizuho-ku, Nagoya City

Claims (1)

[Scope of Claims] (1) Polysaccharide T25GI obtained from the whole plant of Tulna and having the following properties. (a) Color and shape White powder (b) Solubility Soluble in water, insoluble in organic solvents such as alcohol, benzene, ethyl acetate, etc. (c) Sugar composition Mainly composed of glucose and also contains arabinose and galactose. (d) Molecular weight cutoff molecular weight is 10. High performance liquid chromatography of Goo ~ 100,000 gives a single peak, and the molecular weight is approximately fil, 000. (e) Specific rotation (f) Infrared absorption spectrum As shown in FIG. 2920.1660 (1.) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum and shows terminal absorption. (2) Process for producing patented T2 5G I, characterized by subjecting a hot water extract of the entire Trunna plant to a molecular sieve to collect a polysaccharide having a molecular weight of approximately 61,000 (a) Color and shape: white powder (mouth ) Soluble: Soluble in water, insoluble in organic solvents such as alcohol, benzene, ethyl acetate, etc. (iii) Sugar composition: Mainly composed of glucose, and (iii) contains arabinose and galactose. (d) Molecular weight A single peak is obtained by high performance liquid chromatography with a molecular weight cutoff of 10,000 to 100,000, and the molecular weight is approximately 81,000. (E) Specific optical rotation 2 [α) = +54.8° (C = 0.5, H2O) (
) As shown in Figure 1 of the infrared absorption spectrum, 1A6 (4) 2820,
16BO (g) Ultraviolet absorption spectrum (5) When measured in an aqueous solution, it does not show an absorption maximum but shows terminal absorption. In the above molecular sieve treatment, first, the molecular weight cut-off is from 1.000 to 50,000 to 1,000 to 100.0.
Of the two fractions of the polysaccharide obtained, the first eluted sugar fraction is further divided into two fractions with a molecular weight of 1,000 to 100.000 to 1.0.
00 to 200.000, and the polysaccharide is collected from the sugar fraction that elutes first among the two fractions of the obtained polysaccharide. A method for producing polysaccharide T25GI according to Scope 2. 4. The method for producing polysaccharide 725GI according to claim 2 or 3, wherein the molecular sieve treatment is gel filtration, and the molecular sieve agent is a gel filtration agent. Polysaccharide T25G according to claim 4, wherein the gel filtration agent is dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel, or porous glass beads.
Method for producing I.