Xylan and its preparation method and application

Abstract

The invention discloses a pharmaceutical-grade raw material xylan, which is a component prepared by reclassification of extracted natural xylan, characterized in that the mass content of branched oxygen-acetyl-4-methyl-glucuronic acid group is between 9.0%-14.0%. The preparation method of the xylan is based on the principle that the solubility of xylan with different branched oxygen-acetyl-4-methyl-glucuronic acid group content in alkali solution is different, and the preparation is graded, specifically, the crude xylan is first extracted with hot alkali solution, and then the crude xylan is dissolved and beaten with alkali solution for washing, and the xylan with different solubility, i.e., different branched oxygen-acetyl-4-methyl-glucuronic acid group content is graded, and finally purified to obtain. As a raw material, the xylan can meet the requirements of the production of drug pentosan polysulfate sodium for the content of xylan branched sugar units.

Description

Technical Field

[0001] The invention belongs to the technical field of biological medicines and relates to xylan and a preparation method and application thereof. Background Art

[0002] Xylan is a heterogeneous polysaccharide widely present in plant cell walls, accounting for about 15% to 35% of the dry weight of plant cells. It is a complex pentose polysaccharide with a main chain composed of multiple xylanose groups connected by β-1,4-glycosidic bonds and a 4-oxy-methyl-glucuronic acid group connected to the side chain.

[0003] In the pharmaceutical industry, xylan can be used as a raw material to prepare pentosan polysulfate sodium (PPS). Pentosan polysulfate sodium is a heparin-like macromolecular carbohydrate derivative prepared by artificial sulfonation and depolymerization of xylan. It has excellent anti-inflammatory effects. Pentosan polysulfate sodium capsules were approved for marketing in the United States as early as 1996. It is the first and only legal drug approved by the FDA to date to treat the pain and discomfort of interstitial cystitis (IC). It is an oral drug used to treat bladder pain caused by interstitial cystitis, or suprapubic pain caused by interstitial cystitis combined with chronic prostatitis, or suprapubic pain caused by simple chronic prostatitis syndrome. The trade name is: Ai Mi Luo, and the English drug name is:

[0004] The structural formula of pentosan polysulfate sodium is as follows:

[0005]

[0006] According to the structural description of sodium pentosan polysulfate and the quality standard of sodium pentosan polysulfate, the mass content of its branched sugar 4-oxy-methyl-glucuronic acid is 4%-8%. This branched sugar is brought by the raw material xylan, and in natural xylan, the sugar unit corresponding to the branched 4-oxy-methyl-glucuronic acid is 4-oxy-methyl-glucuronic acid. According to the molar ratio of the same sugar unit, the mass content of 4-oxy-methyl-glucuronic acid in xylan suitable as the raw material of this drug should be 8%-16%.

[0007] However, in the existing technology and market practice, there is almost no information on the ratio or content of specific branched 4-oxy-methyl-glucuronic acid groups in xylan. Conventional xylan is also used in large quantities as the preparation of food-grade xylose or oligoxylose, and as a high-value biochemical reagent for the detection substrate of xylanase. At present, the main method for extracting xylan is the alkali extraction method, which is simple and easy to operate. However, the amount of branched 4-oxy-methyl-glucuronic acid groups contained in the extracted and prepared xylan is more than the requirement of the pharmaceutical grade sodium pentosan polysulfate raw material. Taking the xylan sold by Sigma Company with the item number V900513 as an example, the mass content of branched 4-oxy-methyl-glucuronic acid groups therein is about 18%, which is not suitable for the preparation of pharmaceutical grade sodium pentosan polysulfate, and can only be used as a biochemical reagent or food grade material.

[0008] Therefore, in order to expand the preparation sources of pharmaceutical-grade pentosan polysulfate sodium, it is extremely important to obtain xylan with specific branched 4-oxy-methyl-glucuronic acid content characteristics and its preparation method. Summary of the invention

[0009] In view of the above-mentioned defects in the prior art, the technical problem to be solved by the present invention is to provide a xylan and a preparation method and application thereof. The preparation method is simple and easy to operate. The mass content of branched 4-oxy-methyl-glucuronic acid groups in the prepared xylan is between 9.0% and 14.0%, and the xylan is suitable as a raw material for pharmaceutical-grade pentosan polysulfate sodium.

[0010] As mentioned above, xylan is a hybrid polymer molecule, the main chain of which is composed of multiple pyranose groups connected by β-1,4-glycosidic bonds (i.e., polyxylose), and the side chains are connected with 4-oxy-methyl-glucuronic acid groups. This polyhydroxy neutral polysaccharide has a very low solubility in pure aqueous solution, but can be well dissolved in hot alkali solution, and has a certain solubility in room temperature alkali solution. In addition to the main chain polyxylose, the degree of modification of the 4-oxy-methyl-glucuronic acid groups of the side chains, that is, the number (ratio) of different branches, is also an important factor in its solubility. The xylan component with more branches has a high solubility in room temperature alkali solution, and the xylan component with more branches has a high solubility in room temperature alkali solution. The solubility of xylan components with fewer chains in hot alkali solution is low. Based on this, the present invention first extracts xylan from wood powder with hot alkali solution, and then uses alkali solution at room temperature to dissolve and pulp to remove xylan components with higher solubility and corresponding to more branches. This is a classification based on the different solubilities of xylan components with different branched 4-oxy-methyl-glucuronic acid content in room temperature alkali solution, thereby obtaining xylan with a branched 4-oxy-methyl-glucuronic acid mass content between 9.0% and 14.0%, which can meet the requirements of the production of drug pentosan polysulfate sodium for high-purity xylan raw materials.

[0011] The xylan with a branched 4-oxy-methyl-glucuronic acid content of 9.0%-14.0% is a new component prepared by re-classification of extracted natural xylan, and is suitable as a raw material for the production of drug pentosan polysulfate sodium.

[0012] The technical solution adopted by the present invention is: the mass content of the branched 4-oxy-methyl-glucuronic acid groups in the xylan is between 9.0% and 14.0%.

[0013] The method for preparing the above-mentioned xylan comprises the following steps:

[0014] S1. Crushing: Wood is crushed or sawn to produce wood powder and / or sawdust with a particle size not greater than 10 mm;

[0015] S2. Extraction: Prepare a suspension with a concentration of 2%-12% by mixing wood powder and / or sawdust with water, add solid sodium hydroxide and / or solid potassium hydroxide, the amount of which is 0.1-0.6 times the amount of wood powder and / or sawdust, stir and heat to not less than 50°C, maintain the temperature, and stir and extract for not less than 1 hour;

[0016] S3, decolorization: add hydrogen peroxide with a final concentration of 0.2%-5%, maintain the temperature not lower than 50°C, stir, decolorize for not less than 1 hour, filter, and take the filtrate;

[0017] S4, precipitation: cool the filtrate to below 50°C, add ethanol, the volume of ethanol is 0.2-0.6 times the volume of the filtrate, stir for not less than 5 minutes, filter or centrifuge to collect the precipitate, resuspend and wash with room temperature water, filter or centrifuge to obtain the wet solid;

[0018] S5. Resuspend the wet solid in a sodium hydroxide solution and / or potassium hydroxide solution with a concentration of 1%-6%, the mass of the sodium hydroxide solution and / or potassium hydroxide solution being 3-6 times the mass of the wet solid, slurry at room temperature for not less than 1 hour, collect the precipitate by filtration or centrifugation, resuspend and wash with room temperature water, filter or centrifuge, take the wet solid, and dry it to obtain xylan with a branched 4-oxy-methyl-glucuronic acid content of 9.0%-14.0%.

[0019] Furthermore, in the aforementioned method for preparing xylan, the wood in step S1 is beech and / or birch.

[0020] Furthermore, in the aforementioned method for preparing xylan, the concentration of the suspension in step S2 is 5%-6%.

[0021] Furthermore, in the aforementioned method for preparing xylan, the feeding amount of the sodium hydroxide solid and / or potassium hydroxide solid in step S2 is 0.3-0.5 times the feeding amount of the wood powder and / or sawdust.

[0022] Furthermore, in the aforementioned method for preparing xylan, the temperature heated and maintained in step S2 is 60°C-80°C.

[0023] Furthermore, in the aforementioned method for preparing xylan, the final concentration of hydrogen peroxide in step S3 is 1%-3%.

[0024] Furthermore, in the aforementioned method for preparing xylan, the temperature heated and maintained in step S3 is 60°C-80°C.

[0025] Furthermore, in the aforementioned method for preparing xylan, the filtrate is cooled to 35° C. in step S4.

[0026] Furthermore, in the aforementioned method for preparing xylan, the volume of ethanol in step S4 is 0.3-0.5 times the volume of the filtrate.

[0027] Furthermore, in the aforementioned method for preparing xylan, the mass of the sodium hydroxide solution and / or potassium hydroxide solution in step S5 is 4-5 times the mass of the wet solid.

[0028] Furthermore, in the aforementioned method for preparing xylan, the concentration of the sodium hydroxide solution and / or potassium hydroxide solution in step S5 is 3%-5%.

[0029] Furthermore, in the aforementioned method for preparing xylan, the drying in step S5 is performed by hot air drying or vacuum drying.

[0030] Further, in the aforementioned method for preparing xylan, in step S5, the wet solid is resuspended with a sodium hydroxide solution and / or a potassium hydroxide solution having a concentration of 1%-6%, the mass of the sodium hydroxide solution and / or the potassium hydroxide solution being 3-6 times the mass of the wet solid, the slurry is made at room temperature for not less than 1 hour, and the precipitate is collected by filtration or centrifugation, and the entire process from resuspension to precipitation is an alkali solution washing and purification process, and the alkali solution washing and purification process can be repeated multiple times until the mass content of the 4-oxy-methyl-glucuronic acid group is between 9.0%-14.0% (w / w). Usually, the alkali solution washing and purification process is repeated 0-4 times.

[0031] H NMR spectrum ( 1H-NMR) is an important tool for analyzing the structure of polysaccharides. Xylan is composed of xylose polymerized in its main chain and 4-oxy-methyl-glucuronic acid groups in its side chains. In the structure, there are characteristic methylene (-CH2-) located in the main chain xylose group and 4-oxy-methyl hydrogen (4-O-CH3) signals located in the side chain 4-oxy-methyl-glucuronic acid groups. However, as mentioned above, the solubility of xylan in pure aqueous solution is very low, and it is difficult to directly use nuclear magnetic hydrogen spectrum and other methods to check. In view of the purpose of the present invention is to provide a qualified xylan raw material for pharmaceutical grade pentosan polysulfate sodium, and xylan is sulfonated to form sulfated pentosan, which is easily soluble in water due to the sulfation modification, and the methylene (-CH2-) of the xylose group in the sugar chain main chain and the 4-oxy-methyl hydrogen (4-O-CH3) of the side chain are still retained, and the nuclear magnetic hydrogen spectrum can be used for direct analysis. After sulfonation, the characteristic methylene (-CH2-) signal of xylosyl in sulfated pentosan is split at about δ3.98ppm and δ3.88ppm, while the characteristic 4-oxy-methyl hydrogen (4-O-CH3) signal is about δ3.49ppm. Based on this, the molar ratio of xylosyl and branched 4-oxy-methyl-glucuronic acid (corresponding to the branched 4-oxy-methyl-glucuronic acid group in xylan) can be calculated according to the integral, and then the mass content of branched 4-oxy-methyl-glucuronic acid group can be calculated according to the molecular weight.

[0032] Xylan with a branched 4-oxy-methyl-glucuronic acid content of 9.0%-14.0% can meet the requirements of high-purity xylan raw materials for the production of drug pentosan polysulfate sodium. Therefore, the xylan described in the present application can be used as a raw material in the preparation of drug-grade pentosan polysulfate sodium.

[0033] The invention has the beneficial effects of providing a pharmaceutical-grade raw material xylan and an extraction method thereof. The xylan is a new component prepared by reclassifying natural xylan extracted from the xylan, and is characterized in that the mass content of branched 4-oxy-methyl-glucuronic acid groups is between 9.0% and 14.0%. In terms of the preparation method, the invention creatively utilizes the different solubility of xylans with different branched 4-oxy-methyl-glucuronic acid group contents in alkali solution to perform classification. First, crude xylan is extracted with hot alkali solution, and then the crude xylan is dissolved, beaten and washed with alkali solution. Xylans with different solubilities, i.e., different branched 4-oxy-methyl-glucuronic acid group contents, are classified to obtain high-purity xylan, whose mass content of branched 4-oxy-methyl-glucuronic acid groups is between 9.0% and 14.0%, meeting the requirements (8%-16%) for xylan raw materials in the production of drug pentosan polysulfate sodium. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the H NMR spectrum of the sulfonated xylan product in Example 4 ( 1H-NMR) schematic diagram.

[0035] Figure 2 is the H NMR spectrum of the sulfonated xylan sample in the control group of Example 4 ( 1 H-NMR) schematic diagram.

[0036] Figure 3 It is a comparative diagram of the relative molecular weight and molecular weight distribution of the sulfonated samples in Example 4. (80-ALR-018-22-01 is the sulfonated sample of the finished xylan in Example 4; 80-ALR-018-22-02 is the sulfonated sample of the removed xylan control group in Example 4; Heparin Sodium STD is a heparin sodium reference substance for system suitability inspection) DETAILED DESCRIPTION

[0037] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0038] Example 1

[0039] Take sawdust from beech wood and sieve it with a 2-mesh coarse screen. The particle size of the sawdust powder obtained at this time is less than 6.7 mm. Take 100g of sawdust sieved with a coarse screen, add 2L of water and 60g of potassium hydroxide, stir and heat to 75°C, maintain 75°C, and continue stirring for 3 hours. Keep stirring and temperature, slowly add 120mL30% hydrogen peroxide, pay attention to bubbles and foam, add slowly when bubbling, until all added, continue to stir slowly for 2 hours, at this time the reaction liquid and solid residue have faded from dark brown to yellow. Filter, take the filtrate, and then cool the filtrate, cool the reaction system in a cold water bath, when the temperature drops to 35°C, add 1.2L of ethanol, stir for 10 minutes, filter with filter cloth, and then rinse the wet solid with 300mL of water three times, filter with suction, and collect 74g of wet solid. The wet solid was resuspended in 250 mL of 3% sodium hydroxide solution, magnetically stirred for 1.5 hours, filtered through filter cloth, and then rinsed with 100 mL of water three times, filtered, and transferred to a hot air oven and dried at 50°C for 12 hours to finally obtain 12.7 g of xylan.

[0040] Example 2

[0041] Take sawdust from beech wood and sieve it with a 2-mesh coarse screen. The particle size of the sawdust powder obtained at this time is less than 6.7 mm. Take 100g of sawdust sieved with a coarse screen, add 840mL of water and 20g of sodium hydroxide, stir and heat to 55°C, maintain 55°C, and continue stirring for 4 hours. Keep stirring and temperature, slowly add 80mL of 30% hydrogen peroxide, continue to stir slowly for 1 hour, at this time the reaction liquid and solid residue have faded from dark brown to yellow. Filter, take the filtrate, and then cool the filtrate. Cool the reaction system in a cold water bath. When the temperature drops to 45°C, add 0.2L of ethanol, stir for 6 minutes, centrifuge, and wash the solid with 100mL of water respectively, centrifuge, repeat the water washing and centrifugation twice, and collect 46g of wet solid. The wet solid was resuspended in 270 mL of 1% sodium hydroxide solution, magnetically stirred for 3 hours, centrifuged, and then washed with 100 mL of water, centrifuged, and washed and centrifuged twice. The solid was transferred to a vacuum drying oven and vacuum dried at 70°C for 8 hours to finally obtain 10.7 g of xylan.

[0042] Example 3

[0043] Take 1 kg of commercially available birch wood powder, add it to a stainless steel cooking pot, add 50 L of water, start stirring, add 300 g of sodium hydroxide, stir and heat to 80 ° C, keep, and continue stirring for 1 hour. Keep stirring, the temperature is about 60 ° C in a water bath, slowly add 1.7 L of 30% hydrogen peroxide, and continue to stir slowly for 1.5 hours. Filter, take the filtrate, and then cool the filtrate, cool the reaction system in a cold water bath, when the temperature drops to 40 ° C, add 30 L of ethanol, stir for 20 minutes, filter, transfer the solid to a stainless steel bucket, add 5 L of water and stir for 10 minutes, filter, and collect 716 g of wet solid. The wet solid is resuspended with 4.3 L of 3% sodium hydroxide solution, stirred for 2 hours, filtered, and the solid is transferred to another stainless steel bucket, 5 L of water is added and stirred for 5 minutes, filtered, and the solid is transferred to a vacuum drying oven, 65 ° C vacuum drying for 14 hours, and finally 146.8 g of xylan is obtained.

[0044] Example 4

[0045] Take a beech wood segment, use electric sawdust, pass through a 2.5 mesh sieve, get sawdust with a particle size of no more than 8 mm, weigh 50Kg, add to a 1500L stainless steel reactor, add 1000L water, start stirring and heating, add 20Kg sodium hydroxide, stir and heat to 70°C, maintain, and continue stirring for 2 hours. Keep the temperature and stirring, add a total of 60L 30% hydrogen peroxide in six times (there will be foam after each addition, wait for the foam to disappear and do not affect the control, then continue to add until all are added), and continue stirring for 1.5 hours. Filter, take the filtrate, then cool the reactor, add 400L ethanol when the temperature drops to 35°C, stir for 10 minutes, discharge to a flat centrifuge for centrifugation, collect solids, return to the original reactor after cleaning, add 250L water, stir and wash for 15 minutes, continue centrifugation, collect wet solids, and weigh a total of 32.15Kg. The wet solid was resuspended with 145 L of 4% sodium hydroxide solution, stirred for 2 hours, centrifuged, and the solid was retained (while the solution was collected for subsequent recovery of the control component xylan). About 300 L of water was continuously injected into the centrifuge for flushing and centrifugation. The solid was taken out and spread on a drying plate, and vacuum dried in a vacuum drying oven at 70°C for 13 hours to finally obtain xylan. For the convenience of description, the final xylan is defined as the target xylan, which weighs 5.64 kg.

[0046] The collected solution was transferred to an alcohol precipitation tank, 80 L of ethanol was added, stirred for 10 minutes, discharged to a flat plate centrifuge for centrifugation, and about 300 L of water was continuously injected into the centrifuge for washing and centrifugation. The solid was taken and spread on a drying plate, and vacuum dried in a vacuum drying oven at 70°C for 13 hours to obtain xylan in the control group, weighing 9.43 kg.

[0047] Take 10g of each of the target xylan and the control xylan in Example 4. According to the method for preparing sodium pentosan polysulfate by sulfonation of xylan disclosed in Example 1 of U.S. Patent US 2689848, the sulfonated sodium pentosan sulfate was prepared by sulfonation of xylan under chlorosulfonic acid / pyridine conditions (without depolymerization). The only difference in the amount of materials is the same process and parameters. The products of the two groups of samples after sulfonation are 20.3g (batch number: 80-ALR-018-22-01, sodium pentosan sulfate obtained by sulfonation of the xylan finished product group) and 21.4g (batch number: 80-ALR-018-22-02, sodium pentosan sulfate obtained by sulfonation of xylan in the control group), and the yields of the two groups are similar.

[0048] The two groups of samples after sulfonation were examined by nuclear magnetic resonance spectroscopy ( 1 H-NMR) and relative molecular weight, based on 1The characteristic 4-O-methyl hydrogen (4-O-CH3) signal (δ3.49ppm) of the corresponding 4-O-methyl-glucuronic acid in the H-NMR spectrum and the characteristic methylene (-CH2-) signal (δ3.98ppm+δ3.88ppm) of the xylose group were integrated, i.e., the molar ratio, to calculate the mass content of the branched 4-O-methyl-glucuronic acid group in the corresponding raw material xylan.

[0049] The experimental results are shown in Table 1. Figure 1 , Figure 2 and Figure 3 .

[0050] Table 1. Analysis results of branched 4-oxy-methyl-glucuronic acid content and relative molecular weight of xylan samples

[0051]

[0052] Note: * Depending on the sulfonation before and after, the molar ratio of the corresponding sulfonated 4-oxy-methyl-glucuronic acid group is the same.

[0053] From the above experimental results, the percentage of branched 4-oxy-methyl-glucuronic acid groups in the target xylan finished product is 11.8%, which meets the established index requirement of 9.0%-14.0%, and also meets the requirement of 8%-16% for pharmaceutical grade pentosan polysulfate sodium. Combined with its molecular weight of about 17000Da, this product should be suitable for the subsequent preparation of the pharmaceutical pentosan polysulfate sodium; while the xylan component removed by grade in the control group has a percentage of branched 4-oxy-methyl-glucuronic acid groups of 18.4%, which far exceeds the index requirement of 8%-16% for pharmaceutical grade pentosan polysulfate sodium. This component product is not suitable for the production of pharmaceutical pentosan polysulfate sodium.

[0054] There are many implementation methods of the present invention, and all technical solutions formed by equivalent transformation or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A method for preparing xylan, characterized in that: The preparation method comprises the following steps: S1. Crushing: The wood is crushed or sawn to form wood powder and / or sawdust with a particle size not greater than 10 mm; the wood is beech and / or birch; S2. Extraction: Prepare a suspension with a concentration of 2%-12% by mixing wood powder and / or sawdust with water, add solid sodium hydroxide and / or solid potassium hydroxide, the amount of which is 0.1-0.6 times the amount of wood powder and / or sawdust, stir and heat to not less than 50°C, maintain the temperature, and stir and extract for not less than 1 hour; S3, decolorization: add hydrogen peroxide with a final concentration of 0.2%-5%, maintain the temperature not lower than 50°C, stir, decolorize for not less than 1 hour, filter, and take the filtrate; S4, precipitation: cool the filtrate to below 50°C, add ethanol, the volume of ethanol is 0.2-0.6 times the volume of the filtrate, stir for not less than 5 minutes, filter or centrifuge to collect the precipitate, resuspend and wash with room temperature water, filter or centrifuge to obtain the wet solid; S5. Resuspend the wet solid in a sodium hydroxide solution and / or potassium hydroxide solution with a concentration of 1%-6%, the mass of the sodium hydroxide solution and / or potassium hydroxide solution being 3-6 times the mass of the wet solid, slurry at room temperature for not less than 1 hour, collect the precipitate by filtration or centrifugation, resuspend and wash with room temperature water, filter or centrifuge, take the wet solid, and dry it to obtain xylan with a branched 4-oxy-methyl-glucuronic acid content of 9.0%-14.0%.

2. The method for preparing xylan according to claim 1, characterized in that: The concentration of the suspension described in step S2 is 5%–6%.

3. The method for preparing xylan according to claim 1 or 2, characterized in that: The feeding amount of the sodium hydroxide solid and / or potassium hydroxide solid described in step S2 is 0.3-0.5 times the feeding amount of the wood fine powder and / or sawdust.

4. The method for preparing xylan according to claim 1 or 2, characterized in that: The temperature heated and maintained in step S2 is 60°C-80°C.

5. The method for preparing xylan according to claim 1, characterized in that: The final concentration of hydrogen peroxide in step S3 is 1%–3%.

6. The method for preparing xylan according to claim 1 or 5, characterized in that: The temperature heated and maintained in step S3 is 60°C-80°C.

7. The method for preparing xylan according to claim 1, characterized in that: In step S4, the filtrate is cooled to 35°C.

8. The method for preparing xylan according to claim 1 or 7, characterized in that: The volume of ethanol in step S4 is 0.3–0.5 times the volume of the filtrate.

9. The method for preparing xylan according to claim 1, characterized in that: The mass of the sodium hydroxide solution and / or potassium hydroxide solution in step S5 is 4-5 times the mass of the wet solid.

10. The method for preparing xylan according to claim 1, characterized in that: The concentration of the sodium hydroxide solution and / or potassium hydroxide solution in step S5 is 3%-5%.

11. A method for preparing xylan according to claim 1, 9 or 10, characterized in that: In step S5, the drying is performed by hot air drying or vacuum drying.

12. A xylan, characterized in that: Xylan is prepared by the method for preparing xylan according to any one of claims 1 to 11, wherein the mass content of branched 4-oxy-methyl-glucuronic acid groups in the xylan is between 9.0% and 14.0%.

13. Use of the xylan as claimed in claim 12 as a raw material in the preparation of pharmaceutical grade pentosan polysulfate sodium.

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