JPS6317842B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing a vegetable polysaccharide sulfate. Specifically, the present invention relates to a method for producing a novel sulfate ester of a vegetable polysaccharide obtained from vegetable gum. Animal mucopolysaccharides such as heparin and chondroitin sulfate are sulfate esters of polysaccharides, but they have very complex structures, including glucosamine and carboxyl groups as sugar residues. For this reason, although heparin, chondroitin sulfate, etc. have a wide range of uses as pharmaceuticals that exhibit physiological activities such as anticoagulant and antilipidemic effects, they are still manufactured from the lungs and intestinal mucosa of animals. The disadvantage is that it has to rely on extraction, and its supply is limited. On the other hand, many of the rubbery substances secreted by plants are polysaccharides, and there are dozens of types that are currently known. Its structure is mostly D-
It is a type of polyuronide in which glucuronic acid or D-galacturonic acid and other neutral sugar residues are bonded, and it has a simpler structure than animal mucopolysaccharide and is easily available. In view of these circumstances, the present inventors conducted intensive research to obtain a new compound with blood coagulation effect, and found that a plant polysaccharide obtained by sulfuric acid esterification of a plant polysaccharide obtained from plant gums was found. It was discovered that sulfuric esters are suitable for this purpose, and the present invention was achieved. The present invention will be explained in detail below. Vegetable gum is a well-known substance that is secreted when the stem or fruit of a plant is injured, or is secreted naturally and solidifies to protect the wound, and its main component is polysaccharide. . Known vegetable gums include gum arabic, gum tragacanth, mesquite gum, damson gum, cherry gum, peach gum, almond gum, peach gum, lemon gum, grape gum, kola gum, gatsutei gum, karaya gum, kaya gum, and mil gum. Among vegetable gums, gum arabic and gum tragacanth are advantageous because they are easily available. These vegetable gums contain plant polysaccharides corresponding to their respective types; for example, gum arabic contains arabin (arabic acid), and gum tragacanth contains tragacanth (tragacanthic acid). Each of these vegetable polysaccharides can be separated and purified from vegetable rubber by well-known methods. Arabin is composed of 13.8% D-glucuronic acid, 36.8% D-galactose, 30.3% L-arabinose, 11.4% L-rhamnose, etc., and some of their bonding modes and structures are known. In addition, tragacanth is D-galacturonic acid 43
%, D-xylose 40% and other L-fucose. Sulfate esterification of these vegetable polysaccharides can be carried out by a conventional method. It is preferable that the vegetable polysaccharide is pulverized before sulfuric acid esterification. If it is pulverized, the reaction rate in the next step will be increased and a favorable result will be obtained. Sulfuric acid esterification is usually performed by reacting a vegetable polysaccharide with chlorosulfonic acid. When sulfuric acid esterification is performed using chlorosulfonic acid, the vegetable polysaccharide may be suspended in a tertiary amine such as pyridine, and then chlorosulfonic acid may be added. The amount of tertiary amine is usually 5% for vegetable polysaccharides.
~10 times the amount (by weight). If the amount of tertiary amine is too small, the reaction will proceed rapidly and the vegetable polysaccharide will be decomposed by chlorosulfonic acid, and if the amount of tertiary amine is too large, the complex of chlorosulfonic acid and tertiary amine will become a solvent. Since the amount is too small relative to the amount, the reaction progresses slowly and it is difficult to produce the desired sulfuric acid ester of polysaccharide, which is not preferable. The amount of chlorosulfonic acid is usually 1 to 2 times the amount (by weight) of the vegetable polysaccharide. If the amount of chlorosulfonic acid is too small, the production of polysaccharide sulfate ester will not proceed sufficiently, and if the amount of chlorosulfonic acid is too large, the reactants and products will be decomposed, which is not preferable. The temperature when adding chlorosulfonic acid to a tertiary amine suspension of vegetable polysaccharide is usually -10 to 10°C.
It is. If this temperature is too low, the reaction will not proceed sufficiently and sulfuric acid esterification will be difficult to proceed, and if the temperature is too high, the polysaccharide will be decomposed by chlorosulfonic acid, so neither is preferable. After the addition of chlorosulfonic acid is completed, the reaction temperature is raised, usually from 60 to 100°C. If this temperature is too low, the reactivity of the complex of chlorosulfonic acid and tertiary amine will be poor, and if the temperature is too high, the temperature will be close to the boiling point of the solvent, which will impede the reaction operation. It is preferable to stir vigorously during the reaction. Since the reaction time varies depending on various reaction conditions, it may be determined as appropriate while measuring the physiological activity of the product. The sulfuric acid ester of a vegetable polysaccharide thus produced can be post-treated, purified and isolated by a conventional method. That is, for example, by using an appropriate combination of gel filtration and ion exchange resin treatment, the sulfate ester of a vegetable polysaccharide is isolated, neutralized and concentrated with an alkali, and then alcohol is added to remove the alkali of the sulfate ester of a vegetable polysaccharide. A metal salt is obtained. The degree of sulfate esterification in the sulfate ester of the produced plant polysaccharide is determined as the ratio of the sulfone groups in the product to the hydroxyl groups in the raw material plant polysaccharide.
It is usually 0.3 to 1, preferably 0.33 to 0.67. It is preferable that the degree of sulfuric acid esterification is within the above range because the anticoagulant effect is high. The produced sulfate ester of plant polysaccharide has anticoagulant and antilipidemic effects and can be used as a pharmaceutical. The method of use as an anticoagulant and the like is the same as that of conventionally well-known heparin and the like. The present invention will be described in more detail with reference to Examples below, but the present invention is not limited by the Examples unless it exceeds the gist thereof. Example 1 2.0 g of commercial arabin are suspended in pyridine (20 ml). The suspension is cooled to −10° C. and stirred vigorously. While stirring, chlorosulfonic acid (6.8 g) was slowly added dropwise over 30 minutes, keeping the temperature at 0°C. The reaction mixture is left at room temperature and then heated to 80°C. After 3 hours, the reaction is completed and the temperature is returned to room temperature. Add 20 ml of water to the reaction mixture to make an aqueous solution.
This is passed through a 360 ml column of cellulose gel (Sephadex, a trademark of Pharmacia Fine Chemicals Co., Ltd.) G25, which has been prepared in advance, and eluted with a water solvent to separate into each fraction. Measure the antithrombin activity and collect the active part. The active part was concentrated under reduced pressure, and the concentrated solution (20 ml) was prepared using a strongly acidic ion exchange resin column (Mitsubishi Chemical Industries, Ltd. registered trademark "Diaion" SK).
The acidic portion of the aqueous eluate is collected and treated with decolorizing charcoal, and the decolorized solution is neutralized with sodium hydroxide to obtain the desired polysaccharide sulfate ester salt. Add 20 times the amount of alcohol such as methanol or ethanol to the neutralized solution to precipitate the product. The precipitate was collected by filtration and further mixed with alcohol,
The product is washed with ether, dried by heating, and pulverized to obtain the desired arabin sulfate ester salt. Yield 65%, elemental analysis C15.94% H2.62%
Ash content: 35.83% The infrared absorption spectrum is shown in Figure 1. Example 2 2.0 g of commercially available tragacanthic acid are ground well in a mortar, the powder is suspended in pyridine (40 ml) and stirred vigorously. The suspension was cooled to -10°C, and chlorosulfonic acid (6.3 g) was added dropwise over 30 minutes using a dropping funnel. At this time, the temperature is kept below 0°C and vigorous stirring is continued. After completing the dropwise addition, the reaction solution was returned to room temperature, and then
Heat to 100°C and react for 5 hours. After the reaction is completed, 40 ml of water is added to make an aqueous solution, and 800 ml of ethyl alcohol is further added to obtain a precipitate of the reaction mixture. The precipitate is collected by filtration and dissolved in 20 ml of water. A cellulose gel column (diameter 3.0 cm, length 37 cm) similar to that in Example 1 was prepared in advance.
Pass the reaction mixture through the column, elute with water, and take 10 ml of each fraction. Only the antithrobin active part is taken from each fraction.
Further concentrate under reduced pressure to bring the total volume to 20 ml. 20 ml of this aqueous solution is passed through an ion exchange resin (Mitsubishi Chemical Industries, Ltd. registered trademark "Diaion"SK#1, H type) column (diameter 3.5 cm, length 20 cm) and eluted with water, and the acidic eluted portion is taken. Neutralize the acidic solution with sodium hydroxide, concentrate the neutralized solution under reduced pressure to a total volume of 20 ml, and treat with activated carbon. Add 20 times the volume of ethanol or methanol to the concentrate to precipitate the sulfate ester salt of tragacanthic acid. The product is collected by filtration, washed with methanol and ether, dried by heating, and ground in a mortar to obtain tragacanthic acid sulfate. Yield 63.5% Elemental analysis C: 10.56% H2.27
% Ash content 45.85% The infrared absorption spectrum is shown in Figure 2. The physiological activity of the vegetable polysaccharide sulfate ester obtained as described above is as shown below. That is, in order to investigate heparin-like activity, the polysaccharide derivative obtained above was examined in a blood coagulation reaction system using bovine plasma and thrombin. Antithrombin effect and effect on whole blood clotting time Bovine citrate plasma to investigate antithrombin effect
Add 0.1 ml of sample aqueous solution to 0.1 ml, keep warm at 37℃ for 30 seconds, and add 0.1 ml of thrombin solution manufactured by Mochida Pharmaceutical Co., Ltd.
was added and kept at 37°C, and the time until fibrin threads precipitated was measured as the coagulation time. Assuming that the coagulation time without sample addition was 20 seconds, the sample concentration required to make the coagulation time 40 seconds was determined as _I50 . To measure the whole blood clotting time, 1 ml of fresh rabbit blood was added to 0.01 to 0.02 ml of the saline sample solution, and the clotting time was measured by the Lee-White method. The results are shown in the table below.

【table】

[Table] In Examples 1 and 2, similar results were obtained when neutralization was performed with barium hydroxide instead of with sodium hydroxide.

[Brief explanation of the drawing]

1 and 2 are infrared absorption spectra of the products of Examples 1 and 2.

Claims (1)

[Scope of Claims] 1. A method for producing a vegetable polysaccharide sulfate ester, which comprises sulfuric esterifying a vegetable polysaccharide obtained from plant gums. 2. The method for producing a sulfuric acid ester according to claim 1, wherein the sulfuric acid esterification is carried out by reacting a vegetable polysaccharide with chlorosulfonic acid. 3. The method for producing a sulfuric ester according to claim 1 or 2, wherein the vegetable polysaccharide is arabin. 4. The method for producing a sulfuric ester according to claim 1 or 2, wherein the vegetable polysaccharide is tragacanth.