JPS6121481B2 - - Google Patents

Description

[Detailed description of the invention]

Pullulan is a glucan obtained by aerobically cultivating Aureobasidium pullulans in a nutrient medium containing sugars such as monosaccharides and oligosaccharides, and its chemical structure is mainly malt-based. It is a linear polymer in which triose is repeatedly bonded to α-1,6-glucoside. Pullulan is manufactured and sold industrially with a molecular weight of about 80,000 to 300,000, and its excellent properties such as water solubility, edibility, adhesiveness, and film-forming properties are widely used in the food industry, chemical industry, etc. It's being used. The present invention relates to a method for producing pullulan for pharmaceutical use, and in particular to a method for producing pullulan for plasma volume expansion. The pullulan of the present invention is suitable as a plasma expander for the prevention and treatment of traumatic shocks caused by bleeding. When the pullulan of the present invention is administered into a living body, it is decomposed by tissue enzymes to the extent that it can be excreted by the kidneys, so it is suitable for producing an injection for increasing plasma volume with excellent safety. Recently, due to the shortage of blood for transfusion and the risk of occurrence of serum hepatitis, methods have been taken to actively use plasma expanders to reduce blood transfusions. For this purpose, the present inventors previously published a patent application No. 63976 of 1974 (see Japanese Patent Application Laid-Open No. 157513/1983) in which the weight average molecular weight (hereinafter referred to as w) is
An application was filed regarding a plasma expander containing 30,000 to 90,000 pullulan. In order to further investigate the manufacturing method of pullulan for plasma volume expansion, the present inventor investigated the weight average molecular weight/number average molecular weight (hereinafter referred to as w/n) of various commercially available pullulan by gel filtration method. As a result, it was found that all of them had a wide molecular weight distribution of 2.0 or more. Therefore, the present inventor conducted an experiment by intravenously injecting pullulan with a w of 60,000 and various w/n values into domestic rabbits, and found that pullulan with a w/n of 1.5 or less caused an increase in venous pressure during intravenous injection. It was found that pullulan is suitable as a plasma expander because it causes less rapid urine excretion. Furthermore,
w/n is 1.5 or less and w is 30000 to 90000
When we investigated the production method of pullulan, we found that if the raw material pullulan is partially decomposed, the high molecular weight pullulan contained therein undergoes hydrolysis more easily than the lower molecular weight pullulan, and if it is then fractionated with an organic solvent, etc. The present invention was completed by discovering that the desired pullulan fraction could be collected in high yield. Next, a detailed explanation will be given based on experiments. Experiment 1 Intravenous injection of pullulan with various w/n values w 60000 w/n 2.8, 2.0, 1.5, 1.2
Four types of pullulan are added to physiological saline at 6%
(W/V) was dissolved and sterilized to make a plasma expander for intravenous injection experiments. 100 ml of these plasma expanders were rapidly intravenously injected into a rabbit weighing approximately 3.0 kg over a period of approximately 15 minutes, and changes in venous pressure during intravenous injection and excretion rate of the injected pullulan were measured. The venous pressure change is the venous pressure before the start of intravenous injection (mm
H ₂ O) and the venous pressure at the end of the intravenous injection were measured, and the increase multiple of the venous pressure at the end of the intravenous injection relative to the venous pressure before the start of the intravenous injection was determined, which was used as an index of the load on the circulatory system during the intravenous injection. In addition, the amount of pullulan excreted in the urine within 2 hours after the start of intravenous injection was measured, and the excretion rate (%) with respect to the amount of intravenous injection pullulan was determined, and the degree of retention in the body was used as an index of the plasma volume increase effect. The results of the experiment are shown in Table 1. The numerical values shown are the average values of two rabbits each.

[Table] As is clear from the results in Table 1, w/n
Pullulan with a value of 1.5 or less causes less increase in venous pressure during intravenous injection and less rapid urinary excretion of injected pullulan, making it suitable for plasma volume expansion. Next, pullulan with w/n of 2.8 and w of 60,000 was fractionated by the gel filtration method, and pullulan with a molecular weight of 15,000 or less and pullulan with a molecular weight of 150,000 or more were collected. In experiments, pullulan with a molecular weight of 15,000 or less had a venous pressure of approximately 1.2 times, but the urinary excretion rate reached approximately 80%; Although the rate was about 7%, it was found that the venous pressure was about three times as high. These results show that pullulan, which has a molecular weight of less than 15,000, has an extremely short half-life until it is excreted in the urine after intravenous injection, and this only increases the burden on the kidneys, making it less effective as a plasma volume expander. can hardly be expected. Furthermore, in the case of pullulan with a molecular weight of 150,000 or more, rapid intravenous injection has been found to significantly increase venous pressure, so there is concern that it may increase the load on the circulatory system. Therefore, in order to achieve the purpose of the present invention, it is necessary not only to say that w of pullulan is 30,000 to 90,000, but also to have a low molecular weight fraction with a molecular weight of 15,000 or less and a high molecular fraction with a molecular weight of 150,000 or more. Pullulan with a w/n of 1.5 or less, which is reduced as much as possible to narrow the molecular weight distribution width, is preferred. Experiment 2 Manufacture of pullulan Next, the w/n of the present invention is 1.5 or less,
A method for producing pullulan in which w is 30,000 to 90,000 was studied. Raw material pullulan with w/n of 2.5 and w of 150,000 was made into a 10% (w/v) aqueous solution, and (A) undecomposed, (B) heated to 80°C under acidic sulfuric acid (PH approximately 2) for 2 hours. (C) Enzyme agent (α
-Amylase agent (manufactured by Nagase Sangyo Co., Ltd., trade name Neospitase) was added at a rate of 550 units of glue purification activity per pullulan gram at pH 5.5 and temperature 60°C.
After keeping for 20 hours to partially decompose, the enzyme was inactivated by heating, and then, while keeping these (A), (B), and (C) at 30°C, methanol was added to each at a concentration of 43% (v/v). After removing the resulting lower layer, methanol was further added to the resulting upper layer to give a concentration of 60% (v/v), and the mixture was allowed to stand.The lower layer was fractionated and dried into a powder. A pullulan product of w60000 with /n of 1.4 was obtained. The yield (in terms of solids) of this product based on the raw material pullulan is summarized in Table 2.

[Table] As is clear from the results in Table 2, the yield is higher when the target pullulan is fractionated and collected after partial decomposition than when the raw material pullulan is fractionated and collected as it is. In order to investigate the cause of this, we sampled partially decomposed pullulan over time and examined changes in molecular weight distribution using a gel filtration method, and found that the polymeric pullulan in the raw material pullulan was better in both cases of acid and enzyme. It was found that it has properties that are more susceptible to hydrolytic action than low-molecular-weight pullulan. From this, by appropriately selecting the degree of partial decomposition to maximize the target pullulan fraction, approximately 40%
It has become easy to obtain high yields of ~60% or more. Furthermore, partial decomposition with an enzyme resulted in a slightly higher yield than partial decomposition with an acid. After partial decomposition, the raw material pullulan used in the present invention has a w/n of 1.5 or less and a w of
It is sufficient that pullulan having a molecular weight of 30,000 to 90,000 can be collected, and generally, a pullulan with w larger than that of the target pullulan is suitable. The conditions for partial decomposition are such that the raw material pullulan undergoes partial decomposition to increase the amount of the target pullulan fraction. Examples of the means include organic and inorganic acids such as lactic acid, citric acid, hydrochloric acid, and sulfuric acid, or cyclodextrin glucanotransferase (EC 2.4.1.19), α-amylase (EC 3.2.1.1),
Pullulanase (EC 3.2.1.41), Isopululanase (EC 3.2.1.57), Izoamylase (EC 3.2.1.68)
For industrial purposes, partial decomposition methods using acids or enzymes are suitable because they can be easily processed in large quantities. To obtain the desired pullulan fraction from the partially decomposed product, a fractionation method using a hydrophilic organic solvent such as methanol, ethanol, isopropanol, or acetone is usually used. A filtration method, a membrane separation method, etc. can be used. The obtained pullulan fraction is usually decolorized and desalted using activated carbon, ion exchange resin, etc., then filtered through a membrane filter, concentrated to form a colorless and transparent syrup, or further dried and powdered to form a white color. Make powdered pyrogen-free pullulan products. The pullulan of the present invention obtained in this way is
Suitable for plasma volume expansion. Usually, the pullulan of the present invention is made into an aqueous solution of about 4 to 10% (w/t) based on solid matter, to which isotonic agents such as salts and sugars are added and sterilized to produce an injection preparation for plasma volume expansion. Next, a few examples will be described. Example 1 200g of raw material pullulan with w/n of 2.3 and w of 300000 was made into a 10% (w/v) aqueous solution,
After adjusting the pH to about 2 using hydrochloric acid and keeping it at 80℃ for 2 hours to partially decompose pullulan, neutralize it with caustic soda, cool it and keep it at 30℃, and add 40% (v/v) methanol to this. ), and after removing the resulting lower layer, methanol was further added to the resulting upper layer to give a concentration of 55%, and after leaving it to stand, the lower layer was collected separately. Methanol is distilled off from this, activated carbon is added to the remaining pullulan aqueous solution to decolorize it, desalt is carried out using H-type and OH-type ion exchange resins, and the purified pullulan obtained is concentrated, dried, and then filtered through a membrane filter. Shatter it, lol/
Approximately 90 g of white powder of pullulan with n of 1.4 and w of 50,000 was obtained. Example 2 200g of raw material pullulan with w/n of 2.6 and w of 80000 is made into a 20g (w/v) aqueous solution,
Adjust the pH to about 2 using sulfuric acid, keep at 80℃ for 2 hours to partially decompose pullulan, neutralize with caustic soda, and add 50% ethanol to this while keeping at 40℃.
(v/v), remove the resulting lower layer, and then add ethanol to the resulting upper layer.
The solution was added to a concentration of 70% (v/v), and the resulting lower layer was collected. This was further treated and purified in the same manner as in Example 1 to obtain about 70 g of white powder of pullulan with w/n of 1.3 and w of 30,000. Example 3 200g of raw material pullulan with w/n of 2.1 and w of 200000 was made into a 5% (w/v) aqueous solution, partially decomposed in the same manner as in Example 1, neutralized, and kept at 30°C. Add 20% acetone to this.
(v/v), and after removing the resulting lower layer, add 45% acetone to the resulting upper layer.
% (v/v), the resulting lower layer was collected, and further treated and purified in the same manner as in Example 1 to obtain a white powder of pullulan with w/n of 1.5 and w of 85000. About 80g was obtained. Example 4 200g of raw material pullulan with w/n of 2.3 and w of 300000 was made into a 10% (w/v) aqueous solution,
In addition, cyclodextrin glucanotransferase (EC
2.4.1.19) was added at a rate of 150 units of glue refining power per gram of pullulan, kept at pH 6.0 and temperature 65℃ for 20 hours to partially decompose pullulan, and then kept at 90℃ for 15 minutes to decompose the enzyme. was inactivated by heating, and then Example 1
Fractionated with methanol in the same manner as in Example 1, purified, concentrated, and dried to obtain a product with a w/n of 1.4, which is the same as in Example 1.
Approximately 105 g of white powder of pullulan with w50000 was obtained. Example 5 200 g of raw material pullulan with w/n of 2.6 and w of 80,000 was made into a 20% (w/v) aqueous solution, and commercially available pullulanase (manufactured by Hayashibara Biochemical Research Institute, Ltd.) was added to this by 4 units per gram of pullulan. After partial decomposition of pullulan by keeping it at pH 6.0 and temperature 50°C for 20 hours, the enzyme was inactivated by heating, and then fractionated with ethanol in the same manner as in Example 2, purified, By concentrating and drying, about 100 g of white powder of pullulan having the same w/n of 1.3 and w30000 as in Example 2 was obtained. Example 6 200g of raw material pullulan with w/n of 2.1 and w of 200000 was made into a 5% (w/v) aqueous solution,
Commercially available α-amylase (EC 3.2.1.1) (manufactured by Nagase Sangyo Co., Ltd., trade name Neospitase) was added at a rate of 500 units of glue refining power per gram of pullulan, and kept at pH 6.4 and temperature 55°C for 24 hours. After partial decomposition of pullulan, the enzyme was inactivated by heating, and then fractionated with acetone in the same manner as in Example 3, purified,
After concentration and drying, the same w/n as in Example 3 was 1.5.
Approx. white powder of pullulan with w85,000 in
Obtained 110g. Example 7 200g of raw material pullulan with w/n of 2.3 and w of 300000 was made into a 10% (w/v) aqueous solution,
Commercially available isoamylase (manufactured by Hayashibara Biochemical Research Institute Co., Ltd.) was added to this at a rate of 200 units per gram of pullulan, and the pullulan was partially decomposed by keeping it at pH 4.3 and temperature 50°C for 20 hours. It was inactivated by heating, then fractionated with methanol in the same manner as in Example 1, purified, concentrated, and dried to obtain about 115 g of white powder of pullulan having the same w/n of 1.4 and w50000 as in Example 1. . Example 8 200g of raw material pullulan with w/n of 2.1 and w of 580000 was 10% mixed with 0.05M NaCl aqueous solution.
(w/v), and while keeping this at 30℃, output
50W, 45KHz Ultrasonic Generator (Product Name: Branson
ultrasonic cleaner, Model 12, manufactured by Yamato Scientific Co., Ltd., Tokyo) for 30 minutes, and then Example 1
Fractionated with methanol in the same manner as in Example 1, purified, concentrated, and dried to obtain a product with a w/n of 1.4, which is the same as in Example 1.
Approximately 70 g of white powder of pullulan with w50000 was obtained.

Claims (1)

[Claims] 1. A method for producing pullulan for plasma volume expansion, which comprises partially decomposing raw material pullulan and then fractionating it to collect pullulan having a weight average molecular weight/number average molecular weight of 1.5 or less and a weight average molecular weight of 30,000 to 90,000. .