JPS58140094A - Method for reducing molecular weight of polysaccharide with rod-like helical structure - Google Patents

Abstract

PURPOSE:To make the titled compound useful as a carcinostatic agent into a low-molecular weight polysaccharide by an industrial scale in high energy efficiency without generating heat and noise, by subjecting a solution of the polysaccharide having the cylindrical helix structure to high shear rate conditions so that the main chain is selectively cut. CONSTITUTION:A polysaccharide (e.g., beta-1,3-glucane, etc.) having a cylindrical helix structure is dissolved in water, etc. to give a solution, which is subjected to high shear rate conditions by a process wherein the aqueous solution is passed through a nozzle while the aqueous solution being pressurized by using a plunger pump, etc., so that only the main chain of the polysaccharide is selectively cut, to give a low-molecular weight polysaccharide having the same repeating unit and cylindrical helix structure as the original polysaccharide. The preparation of the low-molecular weight polysaccharide is preferably carried out at >=1X10<4>(sec<-1>) shear rate and at 0.1-10wt% concentration of the polysaccharide solution respectively.

Description

[Detailed description of the invention] relates to a method for reducing the molecular weight of its derivatives.

A number of highly viscous philosophies are already known.

Recently, research on the structure of these highly viscous multicysts has progressed, and it has been revealed that some of them have a double-stranded helical structure. (For example, T Norisuy.

*t. al; J, Polym@r seienc*
, Polym@r Physics Ed. /J
! rll7-3;! ;tc/9g? ? ), E. D
．． T. Atkins and K. D.

Park@r; J-Polymer 8ei*nee
Partc. Koza49-J/(/91a9)-T.
L. Bluhm and A Sarko; Can.
J. Oh@mriko93 ~ko99C/97ri),
R-H. Maraheiault, ate
．． ;Can. J. Oh@m jj 300 ~. 3
03 (/De77), E. R. Morrla;A. C.
S- Symposium Seri@s, n
, '73 g/(/9ri7), E.

R. Morria, at ml; J.
Mo1. Hiol. /10 / (/9
These polysaccharides have a wide range of uses in foods, medicines, etc., due to their high viscosity. Especially βl.

Since some 3-glucans have immune-enhancing properties, new research and development as anticancer agents is underway. However, these polyesters have extremely high viscosity depending on the conditions, so they have had various problems in terms of their handling and purpose of use.

In view of this difficult situation, the present inventors first developed β-l.

We have discovered a method for reducing the molecular weight of 3-glucan using ultrasound, and have already applied for a patent application. (% Kaisho! 738 pieces), that is, in this method of reducing molecular weight by ultrasonic irradiation, β - l
, only the main chain consisting of 3-glucoside bonds is selectively cleaved, resulting in a lower molecular weight, and +m has a chemical structure consisting of repeating units and a double-stranded helical structure, just like the original glucan. It was revealed.

(K Tabata, at al; OJLrb6
11ydrat@R@seareh. i? lko it3
Although the low-molecularization method using ultrasonic irradiation is a very unique method due to the specificity of its low-molecularization mechanism, it does not cause heat generation during mosquito treatment. Keion,
It has various disadvantages such as elution of metal particles from the chip and also has the disadvantage of low energy consumption, so it is strongly desired to eliminate or improve these various disadvantages in implementation on an industrial scale. Ta.

Therefore, as a result of intensive research into methods other than ultrasonic irradiation to reduce molecular weight, we have arrived at the method of the present invention, which enables the reduction of molecules by simple operations.

That is, the present invention has the same basic structure as the raw material used, that is, a rod-like helix structure, which is characterized by specifically cutting only the main chain of various solutions having a rod-like helix structure under high shear rate conditions. Concerning new methods for creating various small molecules with the following properties. In the present invention, a polysaccharide having a rod-shaped helical structure (hereinafter simply referred to as "the polysaccharide") is a polysaccharide in which several polysaccharide molecules having a linear main chain are twisted together to form a rod-like lobe. Most of them have a single-stranded or triple-helix structure. Specifically, examples thereof include schizophyllan, scleroglucan, lentinan, force-P, and landtan gum. Further, examples of derivatives of the polymorphism include alkylolamide derivatives of the polymorphism. Many of these are obtained from nature, and those obtained by culture or extraction have an average molecular weight of about a.
ooo Indicates less than 10,000. However, these polysaccharides can be dissolved in alkaline solutions or solvents such as nomethysulfoxide,
When heated, it may be dispersed into a single-stranded random coil, and even under conditions where it is re-strung into a helical structure after being dispersed in a random coil, it does not return to its original helical structure. It has been found that many multi-m chains become disorderly entangled multimers.

The method of the present invention is particularly applicable to solutions of polysaccharides having the above-mentioned specific structure, that is, a rod-like helical structure, in which polysaccharides are suitably dissolved among the various molecular forms mentioned above. Furthermore, the characteristics of the method for reducing molecular weight in the present invention are as follows:
The purpose of this method is to selectively cleave only the main chain of the polyester and maintain the same chemical structure and rod-like helix structure as the original polypropylene after it has been reduced to a low molecular weight. That is, when the polyester is hydrolyzed with an acid (therefore, the bonds in the main chain and side chains are randomly cut, and when a specific glycosidic bond is cut with an enzyme, the cut points are randomly selected,
In any case, it is impossible to avoid the formation of single vessels and oligosaccharides that constitute the polysaccharide, and as a result, very low-molecular fragments with an average molecular weight of about 300θ or less, which cannot form a helical structure, are produced. It is unavoidable to generate a considerable amount.

In contrast, the method of the present invention is characterized in that low-molecular oligosaccharides that cannot form such helical structures are not produced. That is, by carrying out the method of the present invention (by carrying out the method of the present invention), a low-molecular carcass having an average molecular weight of at least about io, oθθ or more is quantitatively produced.

Roughly speaking, the following methods are already known as this type of related technology.

(A treatment method of subjecting polyurethane to a high shear rate as exemplified in Japanese Published Patent Application No. 11/11/7/'10.

(2) A method of reducing the molecular weight of synthetic polymers by subjecting them to high shear rates.

(W, +1. John@on, C, C, prlcc
Jocrnol Polym@l5e1@uc* X
LV COLCU 2 COJ (/DE 40)) & METHYL RIMETHACRYLATE,/IJ Sf V 7 (A, Nakau
s, y.

Minoura, Journal of A ppl
ieel polym@rBe %@ne@ /?
2//9-2/30 (/975) ) (to Na
kano.

Y, Mlnoura, Ma@romol@cul*
s g: 03/577-180 (/91)) However, the former discloses hydrolyzing polysaccharides with # under high shear rates, and therefore the low-molecular-weight fraction produced is 7% of the polysaccharides. Since it is an acid hydrolyzate, the cut point is of course mainly determined by the percentage.

Also, the latter is, for example, vinyl polymer? Methyl remethacrylate, 4-listyrene, etc. are synthetic polymers that are themselves linear and therefore do not have a helical structure, and molecular cleavage is not specific to the main chain.

In short, as shown in the method of the present invention, there is no known method that can maintain the same helical structure even after the low-molecularization treatment, except that it consists of nine repeating units like the raw material used.

By the way, in order to carry out the method of the present invention, it is necessary to
It is necessary to pass the liquid through a microscopic space. Examples of such microspaces include nozzles, slits, packed layers, sintered plates, ceramic plates, and the like.

Additionally, important factors in implementing the method of the present invention include:
Examples include the 11 degree pressure of the solution applied to the solution, the cross-sectional area of the space through which it passes, and the a degree of the polyamorous solution.

However, it has been found that the shear rate of the nine solutions caused by these various factors (conditions) VC is basically the most important and dominant one. Therefore, it is thought that when the polysaccharide solution is flowed at high speed in a microspace in this way, a deviation in the flow occurs in the radial direction of the microspace, and this force causes the polysaccharide molecules to be cut. As a result, it is thought that the cutting depends on the flow velocity deviation, that is, the shear velocity vLK.

The method of the invention consists of subjecting a nuclear polysaccharide solution to high shear rate conditions. However, as the shear rate of the cough solution increases, the rate of molecular weight reduction increases, and eventually the molecular weight of the solution decreases. This relationship is a sawtooth one and therefore - it is not possible to define the shear coupling in general, but at least from the point of view of industrial implementation /
Substantially lower molecular weight cannot be achieved at a shear rate below #7' (see).

When carrying out the method of the present invention, the higher the pressure applied to the multi-t's solution, and the smaller the cross-sectional area of the microspace through which the multi-t's solution passes, the higher the %+! Produces breaking speed. However, increasing the pressure too much increases the danger, and there are limits to the scale of pressure-resistant equipment for industrial implementation. Furthermore, if the cross-sectional area of the microspace through which the solution passes is too small, stable operation will be difficult due to blockage by foreign matter, and conversely, if the cross-sectional area is too large, various harsh conditions will be required to provide the desired shear rate. Therefore, the predistribution pressure is generally 10 kg/m2~t.
rooyn1, the cross-sectional area of the microscopic space is /×/θ~/QQw
s'' is preferred, but the method of the present invention is not intended to be limited to these ranges. When carrying out the method of the present invention in 1! We discovered that it is very effective to add to this solution a solvent that is compatible with the solvent used and is also a poor solvent for the polysaccharide (hereinafter referred to as "poor solvent"). Regarding the former, it has been found that the higher the concentration of the polysaccharide in the solvent solution, even when treated at the same shear rate, the lower the molecular weight of the paper becomes possible, and therefore the efficiency of the method of the present invention is further improved. This effect is not critical and becomes somewhat noticeable when the concentration of the polysaccharide exceeds about 0./1 (weight).

However, the polysaccharides used in the method of the present invention are various, and many have relatively low boiling points in various solvents. Therefore, generally 70
Practical at a degree below tatami! It is desirable to use it at r4 or lower. In addition, from the operational point of view that the dissolution operation is much easier when carried out at a low concentration, a concentration of less than O1s may be adopted depending on the raw material used. This is desirable in order to avoid the disadvantage that undissolved portions are generated and block microscopic spaces. Therefore, there is no intention to limit it to 0. / Low 1 below -0,34! It is desirable to implement the If.

Since the amount of dissolved moT increases as the molecular weight of the compound progresses, it is desirable to appropriately concentrate the low-molecular-weight VC# and adjust it so that the effect of gradual reduction in molecular weight becomes more effective.

Next, the inventors also found that the larger the amount of solvent to be added for the latter, the more the effect of lowering the molecular weight is improved.

However, this passion is so overwhelming! Addition of the solvent at high temperatures must be avoided. As already mentioned, in this case as well, the amount of nuclear polysaccharide that can be dissolved increases as the molecular weight decreases, so the amount of this solvent added can be appropriately increased as the molecular weight decreases.

As described above, in the method of the present invention, as the polysaccharide becomes lower in molecular weight, the speed of low molecular □ flowering gradually decreases. It is extremely advantageous to measure the increase in the rate of deterioration.

In the above, an organic solvent is used as the solvent for the polysaccharide solution. Note that it is most desirable to use water from the viewpoints of stability during the molecular weight reduction treatment and toxicity when remaining in the polysaccharide after the treatment. In addition, examples of poor solvents for the polysaccharide that are miscible with solvents include acetone, methanol, ethanol,
It is possible to use compounds with a relatively low boiling point such as isoderonol, n-deronol, tetrahydrofuran, etc.

Desirable in terms of post-processing operations.

The present invention also includes the use of derivatives of polysaccharides having a rod-like helical structure, such as N-furkylamide derivatives, as starting materials. However, when using the 1114 polysaccharide, it is
It is preferable to use a suitable organic compound such as acetone, benzene, etc. as a solution. In such cases, the poor solvent for the polycycles is water,
Examples include hydrocarbon XS+.

The mIf of the solution when performing the method of this invention does not have a large effect because it is the effect of reducing the molecular weight. However, if the temperature is too low, the polysaccharide, cutting, and poor solvent may be separated, and reactions may occur with the amounts of these starting materials, so generally, a temperature range of 1 degree to approximately 00Qc is selected. .

Before carrying out the method of the present invention, it is desirable to remove some undissolved matter and suspended matter by passing through a suitable filtration means.

9. The polysaccharide bath solution is usually flow-like before being passed into the enemy's small space.
It is desirable to move KR. It has technotropic properties due to the rod-shaped molecular shape of the raw material used, the polysaccharide, so this simple process lowers the apparent viscosity of the solution and prevents it from flowing into the microscopic space. This is to make it easier.

In the treatment of the method of the present invention, generally the flow rate at which the polysaccharide solution passes through the microscopic space is high, so that the molecular weight may not be sufficiently reduced in the K, - times of treatment; The molecular weight is reduced to the desired molecular weight by repeated steps or by using a multistage device.

The low-molecular-weight polysaccharide obtained by the process of the present invention is an anticancer polysaccharide, and its pharmacological effects and toxicity are the same as those of the original polysaccharide. It facilitates in vivo administration and preparation as a pharmaceutical product. When used as an additive in foods, the extremely high viscosity of the original polysaccharide is reduced, the uniformity during food manufacturing is improved, and the technotropic aSS properties are reduced as expected. 0 Below, the implementation will be explained.

Example/ Average molecular weight approx. T, 400,000 schizophyllane was dissolved in water to form an aqueous solution of O8-glycerol. Pressure is applied to this aqueous solution using a Granger Pong. /l, ■ was passed through a nozzle to lower the molecular weight. 1. Change the rotation speed of the lanzier pump to change the pressure, and obtain lλ powder/x in each case.21. l s hour/4/2 at 12.3410, and f2ok/1)ll''e processing alt.

The drifting snow per nozzle is o and -5cR, respectively.
/ sec% 0.90J/sec,
lI1.3 m”/see s and 8.Hiroshi, B7-・C. From the radius of the nozzle and the drifting snow, the following formula,
Shear rate = 4 < x flow 1i / K x (nozzle radius)
3, the breaking velocity of 9W is 114 -s 7・lI10 s@a, ko, lX10m. . , da, 3-
x10 see s and Ha/X107. . . -1
It was 1.

taj-conditions, m residence time in the nozzle (
The relationship between θ) and molecular weight was determined. The results are shown in Figure/.

Next, the chemical structure of the low-molecular-weight schizophyllan flK finally obtained by the treatment under each condition was tested as follows, and it was confirmed that it was the same as the schizophyllan before the treatment.

(1) Schizophyllane and low-molecular-weight schizobyrane before treatment are methylated by the Hakomori method, then hydrolyzed with zeic acid and then trifluoroacetic acid, and this hydrolyzate is acetylated with acetic anhydride in pyridine, followed by gas chromatography. When the products were analyzed, all samples showed /, 5-di-0-acetel-13, Dow 6-chitler O-methyl-D-glucitol, Ha, ? ,j−
) So0-Aceteluko,! , 4-toIJ-o-methyl-D-glucitol, and l.

3.3.4-Tetra-0-acetate A/-2, 1I-no-0-methyl-D-glucitol was detected, with a molar ratio of about 1:co:/9.

(2) Each sample was coughed up with 0.0/N sodium metaperiodate, and the yellowing effect of sodium metaperiodate and the formation of formic acid were measured by the iodine adjustment method and titration with sodium hydroxide. Both are 0.0% relative to glucose residue/mol in resobuirane. dag~0,j
! rmol (1) Consumption of metaperiodic acid and O, co/~
O, formation of core mol of formic acid was observed.

Taj! The sample was treated with .XO-β-/3-glutinase, and glucose and rntheobise were added as products at a molar ratio of 21/21.

(4)/? ! The molecular weight of each sample was determined by the sedimentation method after dissolving it in water, dimetels, and sulfoxide.

From the above results, it was found that the polysaccharide had the same basic *aV even after being reduced to a low molecular weight as before the treatment.

Example IIa Coscleroglucan (average molecular weight: about 5 million) and Zufuta/gum (average molecular weight: about 1 million) were each added to water VC#
This made 16 liquids of O05 heavy water.

A pressure of 4/cs was applied to each solution Ks00, and the length was jl and the radius was 0. /■ was passed through a nozzle to reduce the molecular weight. When each solution passes through the nozzle, the snow drift is 1. scleroglucan. Qx/i@es dentan gum 7. It was axlo ex/■・C1. Therefore, find 9l from shear rate = da x flow rate / π (nozzle radius) 3! The new speed is scleroglucan/, tXlo”m@@-1s
9. with dentan gum. 'l
It was O30,000. For both polysaccharides, methylation was performed using Hakomori-doko before and after the low molecular weight treatment.
Gas chromatography of the product after photototal hydrolysis with acid and acetylation revealed that the same peaks were observed before and after the reduction of molecular weight, and the size of the valley peak was also the same, indicating that the chemistry of treatment is For the scleroglucan that was the same as the original variety, it is water'.
Since the ratio of molecular weights in P and methyl sulfoxide was 3 to 1 both before and after lowering the molecular weight, the same helix 4
Regarding 0 dentane gum, which clearly has 1114, the intrinsic viscosity before and after lowering the molecular weight is /1000, respectively.
(mol/I””) and 1070 (mar
l&), and the relationship between this molecular weight and intrinsic viscosity is dolx
varth et al. (G, ) Iolxwarth: Ca
rbohydrateR@s@areh, la4/
73-/g & (/97g)), it can be concluded that they have the same helical structure.

Implementation 993: Schizofine 2 with an average molecular weight of about 00,000 was mixed with water, -0, overlyzed acetone aqueous solution, and 10 times overly overlaid with ethanol-containing water.
l! Then, each θ was injected with a solution of Denka Nobuo. Take these liquids to an average mouth size of 3〇Ke/2'' ijk j
Processed through a sintered plate of 0 microns and thickness 11. Therefore, the 1rllllr fast skin in the sintered plate is calculated from the following equation:
It is calculated that the value is 0' or more.

The average and molecular weight of schizophylran in each solution after three treatments were as follows.

Schizofirane average molecular weight ethanol aqueous solution 30,000 ml
Aqueous acetone solution 600,000 water #l liquid
The average molecular weight of 7 million examples is approximately! #l in scleroglucan water with f coθ force
Each concentration is 0. l li meeting, O, l IS heavy weight, and 0.90 heavy heat whisper water #1 liquid! Made by A, pressure/
The samples were passed through a nozzle with a diameter of 70'14/2" and a radius of 0.16 cm. The molecular weight of each scleroglucan after the -0 treatment was as follows.

Solution Average molecular weight after treatment θ, 1oz
z饅 Szamano, 4's IIλ10,000.90 -g10,000 Example S ゛ After filtering the 0 weight water bath solution of Schizophyllan used in practical experiment 1 through a clay plate with a pore size of 0.1 wm, While flowing in the tank at a Reynolds number of 1
C! The solution was injected from a nozzle of length J ts and radius 0.1 attached to the bottom of the tank under a pressure of ``rOKe/CM''.The zero operation was able to continue stably for three hours. The molecular weight of Schizophyllane after 4 was about 37θ force in each case.After the 0 operation, there was a shallow area of untreated liquid of about 1θ0− at the upper part of the nozzle inlet. I went without it, but I had to close the nozzle in a room that processes quadruple solutions and release the pressure from the tank several times to avoid the nozzle's 5e clamp.

[Brief explanation of drawings]

Figure / shows the relationship between mwi time (0) and average molecular weight σ) in the nozzle when Schizophyllan is used as the raw material. Name: Method for reducing the molecular weight of polysaccharides with a rod-shaped helix structure 3, Relationship with the case of the person making the amendment Applicant Kaken Kagaku Co., Ltd. 4, Agent 5, Date of amendment order Voluntary 6, Amendment Subject Column 7 of the detailed explanation of the invention in the specification, Contents of amendment + 11 The specification is amended as follows. (2) Details! On page 15, line 5, "It is for the purpose of getting used to it." is corrected as follows. [Familiarize yourself. Furthermore, since the polysaccharide-soluble acid has a sticky cocoon, it is desirable to uniformly lower the molecular weight of the whole by appropriately fluidizing the wall of the container. By causing the entire device to flow in this manner, it is possible to reduce the amount of liquid remaining on the wall of the device after the liquid is discharged from the device. J 13) Page 22 of the specification, the 4th stroke “Nanadatta,’
Insert a -t statement after. [After the operation, after draining the liquid, I! The water that dripped from the wall remained at the bottom of the 2500p tank. The molecular weight of schizophyllan after treatment is approximately 37 in each case.
It was 0,000. ”

Claims (1)

[Scope of Claims] (1) A method for reducing molecular weight by subjecting a multilayer solution having a rod-like helical structure to high shear rate conditions. (2) A polysaccharide with a rod-shaped helical structure has a linear β-
/, 3-glucan. The method according to claim m. (3) The method according to claim (1), wherein the multilayer having a rod-like helix structure is a denta/fum. 14) / X 10' (see”) or more!IJ
Claim Fj! processing under Wr speed! (5) The solution concentration of the polysaccharide having a rod-like helix structure is 0.
．． / - / The method according to claim (1), which relates to 0 layers. (6) The method according to claim (1), which comprises using water as a solvent for the polysaccharide having a rod-like helical structure. (7) A patent claim that involves adding a solvent to a multilayer solution having a rod-shaped helical structure that is compatible with the solvent and is a poor solvent for the multilayer solution to reduce the molecular weight. (8) The method according to claim 11, which comprises soaking the solution once before subjecting it to high shear rate conditions. (9) A method according to claim (1), comprising rendering the solution free-flowing before subjecting it to high shear rate conditions.