JP5435538B2 - Method for producing low molecular weight product of chondroitin sulfate - Google Patents

Description

  The present invention relates to a method for producing a low molecular weight sulfated polysaccharide, specifically, a low molecular weight chondroitin sulfate. More specifically, the present invention relates to a method for producing a low molecular weight chondroitin sulfate, characterized by using a hydrothermal reaction to reduce the molecular weight of chondroitin sulfate without decomposing sugar residues and eliminating sulfate groups.

  Chondroitin sulfate is a naturally-occurring sulfated polysaccharide with uronic acid and acetylgalactosamine as a repeating unit, and a part of it is classified into chondroitin sulfate A to E depending on the substitution position and number of sulfate groups. Yes. Chondroitin sulfate is attracting attention for various physiological activities such as cartilage regeneration. For example, chondroitin sulfate is used for the treatment of low back pain, joint pain, shoulder periarthritis, etc. as an injection solution, and used as an ophthalmic solution for protecting the corneal surface layer. . It is also applied to health foods. It is known that these biological activities of chondroitin sulfate depend on molecular weight distribution and substitution degree of sulfate group. Chondroitin sulfate, which has been reduced in molecular weight to some extent, is considered better absorbed by the body. Furthermore, since chondroitin sulfate has a moisturizing action, it is also used in cosmetics and the like.

In order to investigate the correlation between the molecular weight of chondroitin sulfate and physiological activity, a method for reducing the molecular weight that allows easy control of the molecular weight and less elimination of sulfate groups is required. The acid hydrolysis method is generally used as a method for reducing the molecular weight of chondroitin sulfate, but this method has the advantage of having a short reaction time, but it is difficult to control the molecular weight and has a low sulfate group retention rate. Have There is also a method of reducing the molecular weight of chondroitin sulfate using an enzyme (see Patent Document 1), but it has the disadvantage that the reaction time is long and the cost is high. Under such circumstances, a method for easily controlling the molecular weight and rapidly reducing the molecular weight of chondroitin sulfate is desired. In addition, since the physiological activity of chondroitin sulfate is largely attributed to sulfate groups, a method for reducing the molecular weight of chondroitin sulfate while suppressing the elimination of sulfate groups is also desired. If a method for reducing the molecular weight of chondroitin sulfate that satisfies these conditions is realized, detailed research on the correlation between the molecular weight of chondroitin sulfate and physiological activity will proceed, and it will be possible to obtain low molecular weight chondroitin having high physiological activity. Be expected.
International Publication No. WO2004 / 97022

  In view of the above, the present invention provides a method that can reduce the molecular weight without decomposing sugar residues and eliminating sulfate groups, and that can easily control the molecular weight by changing reaction conditions. It was an object to provide a low molecular weight chondroitin sulfate obtained by a simple method.

  The inventors of the present invention have made extensive studies to solve the above problems and can solve the above problems by selecting temperature, reaction time and pH when treating chondroitin sulfate in high-temperature and high-pressure water (hydrothermal treatment). As a result, the present invention has been completed.

That is, the present invention provides the following method and a low molecular weight chondroitin sulfate obtained by the following method:
(1) A method for producing a low-molecular-weight chondroitin sulfate that does not involve decomposition of sugar residues and elimination of sulfate groups, wherein an aqueous solution of chondroitin sulfate is maintained under hydrothermal conditions in which sulfate groups are not substantially eliminated. A method characterized by:
(2) Unadjusted pH or an aqueous solution of chondroitin sulfate adjusted to a pH higher than about 2.5 to about 12.0 under hydrothermal conditions of 100 ° C. to less than about 160 ° C. for about 5 minutes to about 20 minutes The method according to (1), characterized in that it is kept below;
(3) An aqueous solution of chondroitin sulfate with no pH adjustment or a pH adjusted to a value higher than about 2.5 to about 12.0 under hydrothermal conditions of 100 ° C. to less than about 140 ° C. for about 5 minutes to about 60 minutes The method according to (1), characterized in that it is kept below;
(4) An aqueous solution of chondroitin sulfate having no pH adjustment or a pH adjusted to a value higher than about 2.5 to about 12.0 is maintained under hydrothermal conditions of 100 ° C. to about 140 ° C. for about 5 minutes to about 40 minutes. The method according to (1), characterized in that:
(5) Before the hydrothermal treatment, the pH of the aqueous solution of chondroitin sulfate is adjusted to a value higher than about 2.5 to less than about 6.0, and a hydrothermal reaction is carried out while promoting low molecular weight of chondroitin sulfate. And the method according to any one of (2) to (4);
(6) The pH of the aqueous solution of chondroitin sulfate is adjusted to about 3.4 to about 4.0 before hydrothermal treatment, and a hydrothermal reaction is carried out while promoting the low molecular weight of chondroitin sulfate. (2 ) To (4)
(7) The pH of the aqueous solution of chondroitin sulfate is adjusted to about 11.0 to about 12.0 before hydrothermal treatment, and a hydrothermal reaction is carried out while suppressing the low molecular weight of chondroitin sulfate (2 ) To (4)
(8) The method according to any one of (1) to (7), wherein the chondroitin sulfate is chondroitin sulfate C;
(9) The method according to any one of (1) to (7), wherein the chondroitin sulfate is chondroitin sulfate A;
(10) Low molecular weight chondroitin sulfate obtainable by the method according to any one of (1) to (9).

  According to the present invention, chondroitin sulfate can be rapidly reduced in molecular weight without decomposition of sugar residues and elimination of sulfate groups, and easily by changing reaction conditions such as reaction temperature, reaction time, and reaction pH. Molecular weight control can be performed. In addition, according to the present invention, a low molecular weight chondroitin sulfate whose molecular weight is controlled without accompanying decomposition of a sugar residue and elimination of a sulfate group is rapidly provided. Furthermore, the method of the present invention can keep the running cost low when implemented.

  The present invention provides a method for reducing the molecular weight of hydrolyzed chondroitin sulfate by hydrothermal treatment. Furthermore, the present invention also provides a method for facilitating the control of the molecular weight of low molecular weight chondroitin sulfate by controlling or lowering the low molecular weight by adjusting the pH of the aqueous solution before subjecting the aqueous solution of chondroitin sulfate to hydrothermal treatment. To do. By using hydrothermal treatment or by adjusting the pH of the chondroitin sulfate aqueous solution before hydrothermal treatment in addition thereto, disadvantages associated with other conventional methods, for example, enzyme treatment increases reaction time and costs. Acid hydrolysis has the disadvantage that the retention of sulfate groups is low, or that these methods make it difficult to control the molecular weight of the product. In other words, according to the method of the present invention, low-molecular-weight chondroitin sulfate can be obtained at low cost and in a short time, and the molecular weight of low-molecular-weight chondroitin sulfate can be easily controlled. And an excellent method that does not involve elimination of sulfate groups.

  In the method of the present invention, chondroitin sulfate may be a purified product, a crude product, a partially purified product, or an extract of material. The material of chondroitin sulfate is not particularly limited, and may be, for example, animal cartilage such as shark or animal skin, mollusk, seafood, yam, okra and other plants. Therefore, the chondroitin sulfate used in the present invention may contain contaminants such as proteins, lipids, and other saccharides. For example, an extract of the above material may be used as chondroitin sulfate in the method of the present invention. The extraction method of chondroitin sulfate from the material and the purification method of chondroitin sulfate are known. The obtained extract is purified by a known method, for example, treatment with a proteolytic enzyme such as pronase, ion exchange chromatography, chromatography such as gel filtration chromatography (GPC), etc., and then used in the present invention. Also good. In the present invention, chondroitin sulfate is dissolved in a suitable solvent such as water and then reduced in molecular weight. Preferably, an aqueous chondroitin sulfate solution is used. Chondroitin sulfate may be dissolved in water alone to form a chondroitin sulfate aqueous solution, or chondroitin sulfate may be dissolved in water containing other substances such as salts to prepare a chondroitin sulfate aqueous solution. The concentration of chondroitin sulfate in the solution is not particularly limited. Any of chondroitin sulfates A to E may be used as the type of chondroitin sulfate to be reduced in molecular weight by the method of the present invention.

  In general, hydrothermal treatment is carried out using subcritical water (temperature of 100 ° C. to 374 ° C., pressure of 1 atm to 22 MPa), but in the method of the present invention, the molecular weight is reduced by hydrothermal treatment. Therefore, it is necessary to set the temperature range and the processing time range so as not to cause decomposition of sugar residues and elimination of sulfate groups. In order to significantly reduce the molecular weight of chondroitin sulfate in the method of the present invention, it is preferable to hold at each temperature described below for about 5 minutes or more. In order to confirm the low molecular weight of chondroitin sulfate, a known molecular weight measurement method such as gel filtration column chromatography (GPC) can be used.

  In lowering the molecular weight of chondroitin sulfate by hydrothermal treatment, the higher the hydrothermal treatment temperature and the longer the hydrothermal treatment time, the lower the molecular weight of the chondroitin sulfate obtained and the lower the molecular weight of the resulting low molecular weight chondroitin sulfate. However, the higher the hydrothermal treatment temperature and the longer the hydrothermal treatment time, the easier the decomposition of sugar residues and the elimination of sulfate groups occur. Further, the lower the hydrothermal treatment temperature and the shorter the hydrothermal treatment time, the lower the molecular weight of the chondroitin sulfate obtained, and the lower the molecular weight of the resulting low molecular weight chondroitin sulfate. A person skilled in the art can easily determine the hydrothermal conditions for obtaining a low molecular weight chondroitin sulfate having a desired molecular weight without decomposition of sugar residues and elimination of sulfate groups by ordinary experiments.

In the present invention, if no significant change is observed in the entire IR spectrum pattern before and after the hydrothermal reaction, it is determined that the sulfate group of chondroitin sulfate is not eliminated and the sugar residue is not decomposed. In particular, it is important that there is no significant change in the pattern of the IR absorption spectrum derived from the pyranose ring (near 1100 cm ⁻¹ ) and the pattern of the IR absorption spectrum related to sulfur bonding (near 1400 cm ⁻¹ ). Furthermore, it is preferable to confirm the absence of sulfate groups by measuring the amount and substitution degree of sulfate groups. The amount of sulfate groups of chondroitin sulfate can be measured by a known method such as elemental analysis, barium precipitation, or a method using HPLC. It can also be determined that the sulfate group of the low molecular weight chondroitin sulfate is not eliminated when about 92% or more of the sulfate groups remain as measured by these methods. In addition, confirming that the substitution degree (DS) of the sulfate group shows a substantially constant value before and after the hydrothermal reaction is also one criterion for the absence of the sulfate group. Methods for measuring DS are known, and for example, elemental analysis, barium precipitation, and the like can be used.

  From the above criteria, as an example of hydrothermal conditions that do not involve decomposition of sugar residues of chondroitin sulfate and elimination of sulfate groups, an aqueous solution of chondroitin sulfate is used under hydrothermal conditions of 100 ° C. to less than about 160 ° C. And a condition of maintaining an aqueous solution of chondroitin sulfate under a hydrothermal condition of 100 ° C. to less than about 140 ° C. for about 5 minutes to less than about 60 minutes. However, it is not limited to these conditions. As an example of preferable hydrothermal conditions that do not involve decomposition of sugar residues and elimination of sulfate groups of chondroitin sulfate, an aqueous solution of chondroitin sulfate is maintained at 100 ° C. to about 140 ° C. for 5 minutes to about 40 minutes. Examples include conditions. However, it is not limited to these conditions. The hydrothermal conditions are the same even when the pH of the chondroitin sulfate aqueous solution is not adjusted before the hydrothermal reaction, or even when the pH is adjusted to a value higher than about 2.5 to about 12 as described below. It is.

  The pH of the aqueous solution of chondroitin sulfate is usually in the weakly acidic to neutral range (typically around pH 6) unless adjusted. In the method for reducing the molecular weight of chondroitin sulfate of the present invention, it is not necessary to adjust the pH of the aqueous solution of chondroitin sulfate before the hydrothermal reaction, but as described below, the pH of the aqueous solution of chondroitin sulfate is adjusted before the hydrothermal reaction. You may adjust to acidity-alkalinity. Therefore, in a further aspect, the present invention is characterized in that the pH of the aqueous solution of chondroitin sulfate is adjusted from acidic to alkaline, and then the aqueous solution of chondroitin sulfate is maintained under hydrothermal conditions in which the sulfate group is not eliminated, A method for producing a low molecular weight chondroitin sulfate is provided. By adjusting the pH of the aqueous solution of chondroitin sulfate to the acidic side and then subjecting it to a hydrothermal treatment, it is possible to carry out a low molecular weight reaction while promoting the low molecular weight of chondroitin sulfate. Further, by adjusting the pH of the aqueous solution of chondroitin sulfate to the alkaline side and then subjecting it to a hydrothermal treatment, it is possible to carry out a molecular weight reduction reaction while suppressing the molecular weight reduction of chondroitin sulfate. In the case where the pH is adjusted before the hydrothermal reaction, chondroitin sulfate having a desired molecular weight can be efficiently obtained by controlling the hydrothermal reaction temperature and time well.

  When the hydrothermal treatment is performed after adjusting the pH of the aqueous solution of chondroitin sulfate to the acidic side, if the hydrothermal treatment is performed after adjusting the pH to about 2.5 or lower, the IR spectrum can be obtained even at a treatment of 140 ° C. for 20 minutes. Therefore, it is preferable to perform hydrothermal treatment after adjusting the pH to a pH higher than about 2.5. In the present invention, the preferred pH range in the case of hydrothermal treatment after adjusting the pH of the aqueous solution of chondroitin sulfate to the acidic side is higher than about 2.5 to less than about 6.0, and more preferable pH range is About 3.4 to about 4.0. Thus, by adjusting the pH of the aqueous solution of chondroitin sulfate to the acidic side and then subjecting it to hydrothermal treatment, it is possible to rapidly obtain a relatively low molecular chondroitin sulfate.

  When the pH of the aqueous solution of chondroitin sulfate is adjusted to the alkaline side and then subjected to hydrothermal treatment, there is a tendency that the lowering of the molecular weight of the obtained chondroitin sulfate is slightly promoted in the range of about pH 7 to about 10. It's not a big change. Therefore, by adjusting the pH of the aqueous solution of chondroitin sulfate to the alkaline side and then subjecting it to hydrothermal treatment, it is easy to obtain a relatively high molecular weight because moderately low molecular weight is suppressed, and the molecular weight control is more precise. It is also possible to do this. In the present invention, when the aqueous solution of chondroitin sulfate is adjusted to the alkaline side before hydrothermal treatment and then subjected to hydrothermal treatment, the preferred pH range is about 11 to about 12. In this case, the IR spectrum does not change even when a hydrothermal reaction is performed at 140 ° C. for 20 minutes. However, for example, under a high pH condition where the pH exceeds about 12, the low molecular weight reaction is too slow to be practical.

  In the present invention, in the case where the pH of the aqueous solution of chondroitin sulfate is not adjusted before the hydrothermal reaction, no significant promotion or suppression of molecular weight reduction is observed, and the molecular weight can be controlled well while maintaining the processing speed to some extent. .

  In order to adjust the pH of the aqueous solution of chondroitin sulfate, an acid or a base is usually added to the aqueous solution. The acid and base used for adjusting the pH are well known to those skilled in the art and can be selected as appropriate. Typically, an aqueous solution of acid or base is prepared, and the pH can be gradually dropped to a chondroitin sulfate aqueous solution while monitoring the pH to obtain a desired pH. Examples of acids include hydrochloric acid, sulfuric acid, nitric acid, acetic acid, succinic acid, tartaric acid, malic acid, and other organic acids. However, taking into account its application to food and pharmaceuticals, it does not react undesirably with chondroitin sulfate. And those that do not have a coloring problem and do not impair the flavor are preferred, and examples of such acids include citric acid and ascorbic acid. Examples of the base include metal hydroxides such as sodium hydroxide and potassium hydroxide, metal carbonates such as sodium carbonate and potassium carbonate, and metal hydrogen carbonates such as sodium bicarbonate and potassium bicarbonate. In view of the application to the above, those that do not react undesirably with chondroitin sulfate, do not have toxicity and coloring problems, and do not impair the flavor are preferable. Examples of such bases include sodium hydroxide and potassium hydroxide. It is done.

  The apparatus used for the hydrothermal treatment of the present invention may be any apparatus as long as it can hold the sample in the above temperature range for the above time. For example, devices that can hold a sample at a constant temperature under pressure, such as various autoclave devices and pressure cookers, are known and commercially available, and may be used. Such a device is not very expensive and the running cost is relatively low. Moreover, since such an apparatus can be easily increased in size, according to the present invention, low-molecular-weight chondroitin sulfate can be obtained in a large amount at a low cost. Moreover, you may use apparatuses, such as a pressure cooker which can adjust temperature as apparatuses other than the above, for the method of this invention.

  As described above, in the method of the present invention, by changing the hydrothermal treatment temperature and / or hydrothermal treatment time, or in addition, by adjusting the pH of the aqueous chondroitin sulfate solution before the hydrothermal treatment, The molecular weight can be easily adjusted and controlled. The higher the hydrothermal treatment temperature, the lower the molecular weight of the chondroitin sulfate obtained, and the lower the hydrothermal treatment time, the lower the molecular weight of the chondroitin sulfate obtained. According to the method of the present invention, low molecular weight chondroitin sulfate having an average molecular weight in the range of about several thousand to several tens of thousands can be easily obtained by appropriately selecting the above conditions. Moreover, the low molecular weight chondroitin sulfate obtained by the method of the present invention has no unpleasant taste and odor, is substantially free from elimination of sulfate groups, and retains various activities as chondroitin sulfate. It can be suitably used for therapeutic agents such as low back pain, joint pain, shoulder periarthritis, corneal surface protective anti-inflammatory agent), food (especially functional food, health food, etc.), cosmetics and moisturizer. Therefore, in a further aspect, the present invention also provides a low molecular weight chondroitin sulfate obtained by the above low molecular weight method and foods, beverages, cosmetics and pharmaceuticals containing the same.

  The present invention will be described in more detail and specifically with reference to the following examples, but the examples are not intended to limit the present invention.

Example 1 Effect of Hydrothermal Treatment Temperature and Time on Molecular Weight A 1% aqueous solution of chondroitin sulfate C (manufactured by Wako Pure Chemical Industries, Ltd.) was used as a sample (pH 6.0 when dissolved in pure water). When investigating the influence of the hydrothermal treatment temperature, the treatment temperature was set to 120 ° C., 140 ° C., 160 ° C., and 180 ° C. and held at each temperature for 20 minutes. When investigating the influence of the hydrothermal treatment time, the treatment temperature was 140 ° C. and held for 5 minutes, 10 minutes, 20 minutes, 40 minutes, and 60 minutes. Pressure cooker VS-2416 (Kyoshin Engineering Co., Ltd.) was used as a hydrothermal device. The low molecular weight chondroitin sulfate C sample was analyzed by gel filtration column chromatography (GPC) using HPLC. The analysis conditions are as follows. Column: Shodex Asahipak GS520HQ + GS320HQ + GS220HQ, eluent: 0.1M NaNO ₃ , flow rate: 0.5 ml / min, detection: RI, column temperature: 40 ° C., calibration curve standard: PULRAN P800, P400, P200, P100, P50, manufactured by SHODEX , P20, P10, P5.

  The results are shown in FIG. 1, FIG. 2, FIG. 3 and Table 1. As the hydrothermal treatment temperature increased, the molecular weight of chondroitin sulfate C decreased, and the relationship between the treatment temperature and the molecular weight was smooth (FIG. 1). According to the method of the present invention, it was found that chondroitin sulfate C having a desired molecular weight can be easily obtained by changing the treatment temperature. It is also found that in any reaction system, the baseline of the GPC chart is low and flat, the shape of each peak is nearly symmetrical, and chondroitin sulfate C having a desired molecular weight can be fractionated using a chromatography apparatus or the like. It was. It was also confirmed that the molecular weight of chondroitin sulfate C decreased as the hydrothermal treatment time increased (FIG. 2). The sample kept at 140 ° C. for 5 minutes to 40 minutes was examined, and at any reaction time, there was one main peak, which was almost symmetrical.

  The IR spectrum of the low molecular weight chondroitin sulfate C sample at each reaction temperature (reaction time is 20 minutes) and reaction time (reaction temperature is 140 ° C.) was also examined (FIG. 3 left panel: reaction temperature dependence; FIG. 3 right panel: Reaction time dependence). In the hydrothermal reaction at 160 ° C. for 20 minutes, a slight change was confirmed in the IR spectrum pattern, and in the hydrothermal reaction at 180 ° C. for 20 minutes, the IR spectrum pattern changed significantly (left panel in FIG. 3). . A slight change was observed in the IR spectrum after a hydrothermal reaction at 140 ° C. for 60 minutes (the right panel in FIG. 3). This IR spectrum change was related to the pyranose ring, suggesting a change in the skeletal structure.

The above results are summarized in Table 1. The uppermost data in Table 1 is data of untreated chondroitin sulfate C. It was confirmed that the higher the hydrothermal reaction temperature (reaction time is 20 minutes), the lower the superimposed average molecular weight (Mw) and the number average molecular weight (Mn), and the lower the molecular weight. At 120 ° C. and 140 ° C., the substitution degree (DS) of Mw / Mn and sulfate groups was almost constant, and there was no IR spectrum change. At 160 ° C., a decrease in DS was observed with a slight change (Δ) in the IR spectrum. At 180 ° C., and a significant change in the value of DS in the 20 min reaction, a clear change in the IR spectrum (×) (in particular, 1200 cm ^-1 and around 1500cm around ^-1) was confirmed. In addition, it was confirmed that the longer the reaction time (reaction temperature is 140 ° C.), the lower the superimposed average molecular weight (Mw) and the number average molecular weight (Mn), and the lower the molecular weight. Until the reaction time was 40 minutes, the substitution degree (DS) of Mw / Mn and sulfate groups was almost constant, and there was no change in the IR spectrum. In the reaction for 60 minutes, Mw / Mn was increased, and a slight change (Δ) (near 1100 cm ⁻¹ ) was also observed in the IR spectrum.

Judging from the above results, hydrothermal treatment conditions in which decomposition of sugar residues and elimination of sulfate groups do not occur, as confirmed by the IR absorption spectrum pattern and DS value, are about 100 ° C. to less than about 160 ° C. Examples include conditions for holding from 5 minutes to less than about 20 minutes. Further, another hydrothermal treatment condition with little or no structural change including elimination of sulfate groups confirmed by IR absorption spectrum pattern and DS value is about 5 ° C. at about 100 ° C. to less than about 140 ° C. Examples include a condition of holding for a minute to less than about 60 minutes. As preferable hydrothermal conditions which are confirmed by the pattern of the IR absorption spectrum and the value of DS and in which decomposition of sugar residues and elimination of sulfate groups do not occur, about 100 to about 140 ° C. for about 5 to about 40 minutes The condition to hold | maintain is mentioned.

Example 2 Effect of pH of hydrothermal treatment on molecular weight Chondroitin sulfate C (manufactured by Wako Pure Chemical Industries) 1% aqueous solution was used as a sample. The pH was adjusted with aqueous acetic acid or aqueous sodium hydroxide. The pH was adjusted to 2.5, 3.4, 4.0, 6.0, 7.0, 10.0, 11.0, 12.0, and a hydrothermal reaction was performed at 140 ° C. for 20 minutes. Pressure cooker VS-2416 (Kyoshin Engineering Co., Ltd.) was used as a hydrothermal device. The low molecular weight chondroitin sulfate C sample was analyzed by gel filtration column chromatography (GPC) using HPLC. The analysis conditions are as follows. Column: Shodex Asahipak GS520HQ + GS320HQ + GS220HQ, eluent: 0.1M NaNO ₃ , flow rate: 0.5 ml / min, detection: RI, column temperature: 40 ° C., calibration curve standard: PULRAN P800, P400, P200, P100, P50, manufactured by SHODEX , P20, P10, P5.

The results are shown in FIG. The blank in DS in Table 2 indicates that no measurement was performed. It was found that at a pH lower than 6.0, the lower the pH, the lower the superimposed average molecular weight (Mw) and the number average molecular weight (Mn), and the lower the molecular weight (Table 2). In pH 7.0-10.0, compared with the case of pH 6.0, although there existed a tendency for low molecular weight reduction to some extent, it was not a significant difference. In pH 11.0-12.0, the fall of superposition | polymerization average molecular weight (Mw) and number average molecular weight (Mn) blunted, and it was confirmed that low molecular weight is suppressed. At all pH tested, the Mw / Mn and DS values were approximately the same. At pH 2.5, a slight change in the IR spectrum (near 1100 cm ⁻¹ ) was observed (Δ in Table 2 and FIG. 4). It was considered that some change occurred in the structure of chondroitin sulfate C at pH 2.5 or lower. Therefore, it was found that the pH range in which the decomposition of the sugar residue of chondroitin sulfate C and the elimination of the sulfate group does not occur is higher than about 2.5 to about 12.0. The blank in DS in Table 2 indicates that no measurement was performed.

  From the above results, in the case where the pH of the aqueous solution of chondroitin sulfate C is adjusted to the acidic side and then subjected to hydrothermal treatment, it is preferable to adjust the pH to a pH value higher than about 2.5 and then subject to hydrothermal treatment. I can say that. When the pH of the aqueous solution of chondroitin sulfate C is adjusted to the acidic side and then subjected to hydrothermal treatment, a preferable pH range is from a value higher than about 2.5 to less than about 6.0, and a more preferable pH range is about 3. 4 to about 4.0. Even if hydrothermal treatment is carried out at 140 ° C. for 20 minutes after adjusting to the above preferred acidic pH range, no change in IR spectrum is observed, and the values of Mw / Mn and DS are also untreated with chondroitin sulfate C. It was almost the same. Thus, by adjusting the pH of the aqueous solution of chondroitin sulfate C to the acidic side and then subjecting it to hydrothermal treatment, relatively low molecular chondroitin sulfate C can be rapidly obtained.

Furthermore, from the above results, when the aqueous solution of chondroitin sulfate C is adjusted to the alkaline side before hydrothermal treatment and then subjected to hydrothermal treatment, the preferred pH range is about 11.0 to about 12.0. . In this case, even if hydrothermal reaction was performed at 140 ° C. for 20 minutes, no change in IR spectrum was observed, and the values of Mw / Mn and DS were almost the same as those of untreated chondroitin sulfate C. In this way, by adjusting the pH of the aqueous solution of chondroitin sulfate C to the alkaline side and then subjecting it to hydrothermal treatment, it is possible to obtain a relatively high molecular weight because moderately low molecular weight is suppressed, and the molecular weight control is further improved. It is also possible to perform precisely.

Example 3 Reduction of molecular weight by hydrothermal reaction of chondroitin sulfate A and C Chondroitin sulfate A was also reduced in molecular weight by hydrothermal reaction. Each of chondroitin sulfate A (CALBIOCHEM) and chondroitin sulfate C (manufactured by Wako Pure Chemical Industries) was made into a 1% aqueous solution (pH 6.0) and hydrothermally treated at 140 ° C. for 60 minutes. The reaction was performed twice for each system. It was found that chondroitin sulfate A is reduced in molecular weight as chondroitin sulfate C is. For both chondroitin sulfate A and chondroitin sulfate C, the Mw / Mn value slightly varied in two experiments, but the DS value did not vary. In both chondroitin sulfate A and chondroitin sulfate C, the value of Mw / Mn increased by the hydrothermal reaction, but no change was observed in the DS value.

  The present invention provides a method for producing a low molecular weight chondroitin sulfate that is inexpensive and can be processed in a short time, that is easy to control the molecular weight, and that is not accompanied by elimination of sulfate groups. Therefore, the method of the present invention and the low molecular weight chondroitin sulfate obtained thereby can be used in fields such as health foods, functional foods, supplements, cosmetics, and pharmaceuticals.

FIG. 1 is a HPLC chart showing the relationship between the hydrothermal treatment temperature and the molecular weight of the resulting low molecular weight fucoidan. The raw material (control) is chondroitin sulfate that has not been hydrothermally treated. The hydrothermal reaction was performed at 120 ° C, 140 ° C, 160 ° C, and 180 ° C for 20 minutes, respectively. retention time (min) is HPLC retention time (minutes). FIG. 2 is a HPLC chart showing the relationship between the hydrothermal treatment time and the molecular weight of the resulting low molecular weight fucoidan. The raw material (control) is chondroitin sulfate that has not been hydrothermally treated. Hydrothermal reaction was performed at 140 ° C. for 5 minutes, 10 minutes, 20 minutes and 40 minutes, respectively. retention time (min) is HPLC retention time (minutes). The left panel in FIG. 3 shows changes in the IR spectrum of the low molecular weight fucoidan depending on the hydrothermal treatment temperature. The hydrothermal reaction was performed at 120 ° C, 140 ° C, 160 ° C, and 180 ° C for 20 minutes, respectively. The right panel of FIG. 3 shows the change in IR spectrum of the low molecular weight fucoidan with hydrothermal treatment time. The hydrothermal reaction was carried out at 140 ° C. for 5 minutes, 10 minutes, 20 minutes, 40 minutes and 60 minutes, respectively. The top chart of each panel is for chondroitin sulfate that has not been hydrothermally treated. Dashed circles indicate slight changes in the IR spectrum and solid circles indicate obvious changes in the IR spectrum. FIG. 4 shows the change of IR spectrum of low molecular weight fucoidan by pH before hydrothermal treatment. The hydrothermal reaction was performed at 120 ° C. for 20 minutes. The dotted circle indicates that there was a slight change in the IR spectrum.

Claims (8)

A method for producing low molecular weight chondroitin sulfate without decomposition of sugar residues and elimination of sulfate groups, wherein the pH is not adjusted or the aqueous solution of chondroitin sulfate is adjusted to a value higher than 2.5 to 12.0 Is maintained under hydrothermal conditions of 100 ° C. to less than 160 ° C. for 5 minutes to less than 20 minutes . A method for producing low molecular weight chondroitin sulfate without decomposition of sugar residues and elimination of sulfate groups, wherein the pH is not adjusted or the aqueous solution of chondroitin sulfate is adjusted to a value higher than 2.5 to 12.0 5 minutes to the hydrothermal conditions of less than 140 ° C. to not 100 ° C. the to how you characterized by keeping less than 60 minutes. A method for producing low molecular weight chondroitin sulfate without decomposition of sugar residues and elimination of sulfate groups, wherein the pH is not adjusted or the aqueous solution of chondroitin sulfate is adjusted to a value higher than 2.5 to 12.0 free 100 ° C. to how you characterized by keeping 5 minutes to 40 minutes to hydrothermal conditions of 140 ° C.. Before the hydrothermal treatment, the pH of the aqueous solution of chondroitin sulfate is adjusted to a value higher than 2.5 to less than 6.0, and a hydrothermal reaction is performed while promoting low molecular weight of chondroitin sulfate. The method according to any one of 1 to 3 . Before the hydrothermal treatment to adjust the pH of the aqueous solution of chondroitin sulfate to 3.4 to 4.0, and carrying out hydrothermal reaction while facilitating the smaller molecules of chondroitin sulfate, any of claims 1 to 3, The method according to claim 1. Before the hydrothermal treatment to adjust the pH of the aqueous solution of chondroitin sulfate in 11.0 to 12.0, and performing hydrothermal reaction while suppressing the lowering the molecular weight of chondroitin sulfate, any of claims 1 to 3, The method according to claim 1. The method according to any one of claims 1 to 6 , wherein the chondroitin sulfate is chondroitin sulfate C. Chondroitin sulfate is chondroitin sulfate A, any one method according to claim 1-6.

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