JPS6153330B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION BACKGROUND OF THE INVENTION Technical Field The present invention relates to a novel polysaccharide T25G and a method for producing the same. More specifically, the present invention relates to a polysaccharide T25GI isolated and purified from a hot water extract of a 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. Prior Art Tsuruna is a Japanese folk medicine, also known as Hamadishiya, and is said to be good for stomach cancer and stomach ulcers. but,
Its ingredients and pharmacological effects are unknown, and a book on medicinal plants published in recent years states that it is said to be ``effective against stomach cancer, but it is doubtful.'' 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. 57−193416). The present inventors further purified this extract using a molecular sieve, and as a result,
The present invention was completed by successfully isolating a polysaccharide with antitumor activity. OBJECT OF THE INVENTION Therefore, 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 with a molecular weight of about 61,000. DETAILED DESCRIPTION OF THE INVENTION The present invention consists first of all of polysaccharide T25G obtained from the whole Tulna plant 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 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 [α] ²² _D = +54.9° (C = 0.5, H ₂ O) (f) Infrared absorption spectrum As shown in Figure 1. IRν ^KBr _nax cm ^-1 :3350-3400, 2920, 1660 (g) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum but shows terminal absorption. The second aspect of the present invention comprises a method for producing the polysaccharide T25G described above, which comprises subjecting a hot water extract of the whole plant of Tuluna to molecular sieve treatment and collecting a compound having a molecular weight of about 61,000. Thirdly, the present invention provides that the above-mentioned molecular sieve treatment is first performed using a molecular sieve agent having a molecular weight cut-off in the range of 1000 to 50000 to 1000 to 100000. The sugar fraction is further subjected to molecular sieving using a molecular sieving agent with a molecular weight cut-off in the range of 1000 to 100,000 to 1000 to 200,000, and the polysaccharide is separated from the sugar fraction that elutes first among the two fractions of the resulting polysaccharide. It consists of a method for producing the above-mentioned polysaccharide T25G obtained by collecting body tissue. Fourthly, the present invention comprises a method for producing the polysaccharide T25G, wherein the molecular sieve treatment is gel filtration, and the molecular sieve agent is a gel filtration agent. Fifth, the present invention comprises a method for producing the polysaccharide T25G, wherein the gel filtration agent is dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel, or porous glass beads. When producing the polysaccharide of the present invention, a hot water extract of the whole plant of Thurna is subjected to molecular sieve treatment, especially gel filtration,
A compound with a molecular weight of 61,000 is collected. Trunna is a member of the Trunaceae family (Garnetaceae,
Aizoaceae), the scientific name is Tetragonia
tetragonoides (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. Extracting the whole Trunna plant with hot water is carried out by adding hot water of 80℃ or higher to the Trunna whole plant, or by adding water to the Trunna whole plant and heating the mixture to boiling. Ru. 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 membrane method before subjecting it 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 membrane, dialyzed by dipping in water, and the dialyzed solution is concentrated to dryness or freeze-dried as desired to obtain the desired extract. The dialysis membrane used is regenerated cellulose, such as Spectra Pore (product of Spectrum Medical Industries) or Visking Tube (product of Union Carbide). 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, it is preferable to remove unnecessary components in advance by extracting the Trunna whole plant with an organic solvent and/or water at room temperature. Solvents used for extraction pretreatment include polar organic solvents such as methanol, ethanol, propanol, pyridine, and acetone, benzene, toluene, xylene, n-hexane, chloroform, carbon tetrachloride,
Examples include non-polar organic solvents such as ethyl acetate. 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, a hot water extract of the whole plant of Tulna is first passed through a column packed with a gel filtration agent having a molecular weight cut-off in the range of 1,000 to 50,000 to 1,000 to 100,000, and eluted with distilled water. When the sugar in the eluate is quantified by 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 passed through a column packed with a gel filtration agent having a molecular weight cut-off in the range of 1,000-100,000 to 1,000-200,000, and eluted with distilled water. When the sugar in the eluate is quantified in the same manner as above and the elution curve is drawn, a curve with two peaks is obtained. The first eluting sugar fraction is collected and the desired polysaccharide is collected 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. are suitably used. Fractional molecular weight is 1000~
Examples of gel filtration agents in the range of 50,000 to 1,000 to 100,000 include dextran gels Cephadex G-75 and G-100 (Pharmacia products, Sweden), polyacrylamide gels Biogel P-100 and P-150 ( BioRad products,
(USA), polyvinyl polymer gel Toyopearl HW-50 (Toyo Soda Kogyo Co., Ltd., Japan), porous beads CPG-10 (Electro Nucleonics Co., USA), etc. are preferably used. In addition, the molecular weight cut-off is from 1000 to 100000 to 1000 to 1000.
Examples of gel filtration agents in the 200,000 range include Cephadex G-100, G-150 Biogel P-
150 Toyopearl HW-50, CPG-10, etc. are preferably used. The polysaccharide of the present invention thus obtained
T25G has the following characteristics. (b) 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 A single peak is given by high performance liquid chromatography with a molecular weight cutoff of 10,000 to 100,000, and the molecular weight is determined by the Park & Johnson method.
J. Biol. Chem. 181, 149 (1949)), it is approximately 61,000. (e) Specific rotation [α] ²² _D = +54.9° (C = 0.5, H ₂ O) (f) Infrared absorption spectrum As shown in Figure 1. IRν ^KBr _nax cm ^-1 :3350-3400, 2920, 1660 (g) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum but 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 route of administration, age, weight, and symptoms of the patient, but is approximately 1 to 5 g per day for an adult male. 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, in the case of a water-in-oil (W/O) emulsion, a mineral oil such as liquid paraffin, a vegetable oil such as sesame oil or peanut oil, and a surfactant such as sorbitan fatty acid ester are used in combination. Next, the present invention will be explained in more detail with reference to Reference Examples, Examples, and Formulation Examples. Reference example Production of hot water extract of whole plant of trunna (1) 2 kg of dried whole plant of trunna was mixed with methanol (20×
3) was used for extraction pretreatment at room temperature for 24 hours, and the resulting extraction residue was extracted three times with hot water 20°C.
The obtained extracts were combined and concentrated to dryness using a rotary evaporator to obtain 130.3 g of powder. (2) Dissolve 10.0 g of the powder obtained in (1) above in 500 ml of water, slowly add 99% ethanol to this aqueous solution, stop adding when the ethanol concentration in the aqueous solution reaches 80%, It was stirred for a while.
The generated precipitate was collected by centrifugation to obtain 7.22 g of a brown powdered Trunna whole plant extract. (3) 10.0 g of the powder obtained in (1) above was dissolved in 500 ml of water, and this aqueous solution was placed in a Visking tube and dialyzed against water. Concentrate the dialysis fluid to dryness,
6.37 g of a brown powdered Tulna whole plant extract was obtained. Example 1.092 g of the hot water extract of the whole plant of Trunna obtained in Reference Example (2) or (3) above was dissolved in 20 ml of distilled water, and a column packed with Cephadex G-75 (molecular weight cut off 1000-50000) was prepared. (diameter 7.0 cm, length 35.0 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 fractions.
145 mg of polysaccharide was obtained from the first eluted sugar fraction. The polysaccharide thus obtained was used as Cephadex G.
When gel filtration is further performed using -100 (molecular weight cutoff 1000 to 100000), the polysaccharide is separated into two fractions. Desired polysaccharide from the first eluting sugar fraction
45mg of T25G was obtained. Formulation Example 1 1000 mg of polysaccharide T25G 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, a formulation containing 10 mg of polysaccharide T25G in one vial was obtained. Before use, dissolve in distilled water for injection. Formulation Example 2 A vial preparation was prepared in the same manner as in Formulation Example 1 above. However, sterile 5% glucose solution for injection 500
500 ml of physiological saline was used instead of ml. When using,
Use by dissolving in distilled water for injection. Test example 1 Effect on Sarcoma 180 solid gun (sample preparation) Phosphate buffered saline (manufactured by Gibco, phosphoric acid 9.5m
The sample was dissolved in a solution of 0.5% carboxymethylcellulose (CMC) suspended in PBS) to a predetermined concentration. (Sarcoma 180 cancer cell transplantation) Sarcoma subcultured in the peritoneal cavity of ICR mice
180 cancer cells were taken out together with ascites fluid and diluted appropriately with physiological saline so that the number of cells was 1.0 x 10 ⁸ cells/ml. 0.1 ml of this suspension was subcutaneously transplanted into the back of a 4-week-old male ICR mouse using a syringe. Therefore, the number of transplanted cells per animal is 1×
There are 10 ⁷ pieces. (Sample Administration) Starting from the next day after transplanting the Sarcoma 180 cancer cells, 0.1 ml of the sample prepared above was administered into the abdominal cavity once a day for 4 consecutive days using a syringe. Six mice were used per sample per concentration. As a control, the above-mentioned PBS containing CMC, which was used as a solvent for the sample, was administered in the same manner. The dosage was expressed as mg per kg of mouse body weight. (Efficacy evaluation method) Cancer tissue that had grown on the 21st day after cancer cell transplantation was removed and its weight was measured (average value of 6 animals per group). The effect was determined by calculating the ratio (T/C) between this weight and that of the control. The control cancer tissue weight is
It was 3.11g. Ratio values of 100 to 71% are invalid (-), ratios of 70 to 51% are slightly valid (+), and 50 to 51% are invalid (-).
A response of 21% was considered effective (++), and a response of 20% to 0% was considered an excellent response (+++). The results are shown in Table 1.

[Table] Test Example 2 An acute toxicity test was conducted by administering the polysaccharide of the present invention to ICR male mice weighing 20±1 g.
The LD ₅₀ value was more than 250 mg/Kg when administered intraperitoneally. Specific Effects of the Invention As detailed above, according to the present invention, firstly,
Polysaccharide T25G is provided. This polysaccharide is a new compound that has not been described in any literature, and as shown in the test example, it exhibits a strong inhibitory effect on Sarcoma 180 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 can be 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 61,000. According to the present invention, thirdly, an advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide is subjected to the above molecular sieve treatment to have a molecular weight cut-off of 1000 to 50000 to 1000 to 1000.
Molecular sieves in the range of 100,000 and molecular weight cutoff of 1,000
High purity can be obtained by combining with a molecular sieve in the range of ~100,000 to 1,000 to 200,000. According to the present invention, fourthly, a more advantageous method for producing the above polysaccharide is provided. That is, the polysaccharide is
It can be obtained more easily 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 is
High purity can be easily obtained by using dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel or porous glass beads as a gel filtration agent.

[Brief explanation of the drawing]

FIG. 1 shows the infrared absorption spectrum of polysaccharide T25G of the present invention.

Claims (1)

[Claims] 1. Polysaccharide T25G 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 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 [α] ²² _D = +54.9° (C = 0.5, H ₂ O) (f) Infrared absorption spectrum As shown in Figure 1. IRν ^KBr _nax cm ^-1 :3350-3400, 2920, 1660 (g) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum but shows terminal absorption. 2. A method for producing polysaccharide T25G having the following characteristics, which comprises subjecting a hot water extract of the whole plant of trunna to molecular sieving to collect a polysaccharide having a molecular weight of about 61,000. (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 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 [α] ²² _D = +54.9° (C = 0.5, H ₂ O) (f) Infrared absorption spectrum As shown in Figure 1. IRν ^KBr _nax cm ^-1 :3350-3400, 2920, 1660 (g) Ultraviolet absorption spectrum When measured in an aqueous solution, it shows no absorption maximum but shows terminal absorption. 3 In the above molecular sieve treatment, first, the molecular weight cut-off is 1000~
It is carried out using a molecular sieve in the range of 50,000 to 1,000 to 100,000, and of the two fractions of the polysaccharide obtained, the first eluted sugar fraction is further eluted with a molecular weight cut-off of
A patent characterized in that a polysaccharide is collected from the sugar fraction that elutes first among two fractions of the obtained polysaccharide by performing molecular sieve treatment using a molecular sieve agent in the range of 1,000 to 100,000 to 1,000 to 200,000. A method for producing polysaccharide T25G according to claim 2. 4. The method for producing polysaccharide T25G according to claim 2 or 3, wherein the molecular sieving treatment is gel filtration, and the molecular sieving agent is a gel filtration agent. 5. Claim 4, wherein the gel filtration agent is dextran gel, polyacrylamide gel, hydrophilic polyvinyl gel, or porous glass beads.
Method for producing polysaccharide T25G as described in Section 2.