An acetylated pachyman polysaccharide, its preparation method and application

Abstract

The present invention discloses an acetylated Poria cocos polysaccharide, its preparation method and application, belonging to the technical field of bio-chemical engineering. The method comprises the following steps: drying Poria cocos, grinding it into powder, sieving, dissolving it in a NaOH solution with a pH of 12, heating for extraction, centrifuging to obtain a supernatant; adjusting the pH to 7.0 with hydrochloric acid to obtain a polysaccharide solution; concentrating, adding an ethanol solution, discarding the supernatant, suspending the precipitate in distilled water, dialyzing, and freeze-drying to obtain crude Poria cocos polysaccharide; dissolving it in dimethyl sulfoxide, stirring at room temperature, heating to 40 °C, adding NBS and acetic anhydride, adding absolute ethanol, filtering, discarding the supernatant, dissolving with distilled water, ultrafiltration and concentration, and freeze-drying to obtain the acetylated Poria cocos polysaccharide. The present invention discloses an acetylated Poria cocos polysaccharide and its preparation method. The acetylated Poria cocos polysaccharide prepared by this method has high water solubility, effectively solves the technical problem of low solubility of traditional Poria cocos polysaccharide in water, and improves the bioavailability of the acetylated Poria cocos polysaccharide.

Description

Technical Field

[0001] The present invention belongs to the technical field of biochemistry, and particularly relates to an acetylated pachymaran and its preparation method and application. Background Art

[0002] Pachymaran is an active ingredient extracted from the traditional Chinese medicine Poria cocos, mainly composed of linear β(1→3)-D-glucan. It shows significant biological activities in enhancing immune function, anti-tumor and antioxidant aspects. However, due to the low solubility of β(1→3)-D-glucan in water, this limits its application in the healthcare field. Improving the solubility of pachymaran can significantly improve its bioavailability, promote drug delivery and release, broaden the application fields, and improve the preparation efficiency and reduce costs, thus having a broader application prospect and higher value in the fields of medicine, food, health products, etc.

[0003] In the prior art, carboxymethylation is a commonly used method to improve the solubility of pachymaran in water. However, this method has some challenges, including the need to carry out the reaction at specific temperatures, pH values and reaction times to ensure the high efficiency of the reaction and the quality of the product, and at the same time, some by-products will be generated, affecting the purity and quality of the final product. In addition, carboxymethylation also involves technical challenges, cost issues and safety issues. Summary of the Invention

[0004] The present invention aims to provide an acetylated pachymaran and its preparation method and application. The acetylated pachymaran prepared by this method has high water solubility, effectively solves the technical problem of low solubility of traditional pachymaran in water, and improves the bioavailability of the acetylated pachymaran.

[0005] To solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A preparation method of an acetylated pachymaran, comprising the following steps:

[0007] S1. Grind the dried Poria cocos into powder, sieve it through a 60-mesh sieve, dissolve it in 0.05M NaOH solution with a pH of 12, stir, heat and extract, and centrifuge to obtain the supernatant;

[0008] S2. Add 0.1M hydrochloric acid to the supernatant obtained in step S1 to adjust the pH to 7.0 to obtain a polysaccharide solution;

[0009] S3. Centrifuge the polysaccharide solution obtained in S2, add an ethanol solution with a mass fraction of 10%, discard the supernatant, suspend the precipitate in distilled water, dialyze for 3 days, and freeze-dry to obtain crude pachymaran;

[0010] S4. Dissolve the crude Poria cocos polysaccharide obtained in step S3 in dimethyl sulfoxide, stir for 30 min at room temperature, raise the temperature to 40 °C, add NBS and acetic anhydride to react, and obtain reactant A;

[0011] S5. Add absolute ethanol to the reactant A obtained in step S4, filter, discard the supernatant, dissolve the precipitate in distilled water, perform ultrafiltration and concentration 3 times with a 3 kD ultrafiltration tube, and freeze-dry to obtain acetylated Poria cocos polysaccharide.

[0012] Preferably, in step S1, dry Poria cocos is ground into powder, passed through a 60-mesh sieve, dissolved in NaOH solution, stirred for 2 h, heated for extraction at 60 °C for 30 - 60 min, extracted 2 times, centrifuged at 10000 - 12000 rpm for 20 - 40 min, and obtain the supernatant.

[0013] Preferably, in step S3, the polysaccharide solution obtained in S2 is centrifuged at 10000 - 12000 rpm for 20 - 40 min, add an ethanol solution with a mass fraction of 10%, discard the supernatant, suspend the precipitate in distilled water, and dialyze with a dialysis bag with a molecular weight cut-off of 8 k - 1.4 kDa for 3 days.

[0014] Preferably, in step S3, the freeze-drying temperature is -45 - -60 °C, and the freeze-drying time is 15 - 20 h.

[0015] Preferably, in step S4, dissolve the crude Poria cocos polysaccharide obtained in step S3 in dimethyl sulfoxide, stir for 30 min at room temperature, raise the temperature to 40 °C, add NBS and acetic anhydride, and react at 40 °C for 4 h to obtain reactant A.

[0016] Preferably, in step S5, dissolve the precipitate in distilled water, perform ultrafiltration and concentration 3 times with a 3 kD ultrafiltration tube, and freeze-dry at -45 - -60 °C for 15 - 20 h to obtain acetylated Poria cocos polysaccharide.

[0017] The present invention also provides an acetylated Poria cocos polysaccharide prepared by the above-mentioned preparation method.

[0018] The present invention also provides an application of the acetylated Poria cocos polysaccharide prepared by the above-mentioned preparation method or the acetylated Poria cocos polysaccharide in the preparation of drugs, foods or health products.

[0019] The present invention also provides a pharmaceutical composition comprising one or more of the above-mentioned acetylated Poria cocos polysaccharides.

[0020] A pharmaceutical composition as described above, comprising the acetylated Poria cocos polysaccharide as an active ingredient and a pharmaceutically acceptable carrier.

[0021] A pharmaceutical preparation comprising a therapeutically effective amount of any one of the above-mentioned acetylated Poria cocos polysaccharides and a pharmaceutically acceptable excipient.

[0022] The pharmaceutical preparations described above include the following dosage forms: oral preparations (such as tablets, capsules, solutions or suspensions); injectable preparations (such as injectable solutions or suspensions, or injectable dry powders that can be used immediately after adding water for injection before injection); topical preparations (such as ointments or solutions).

[0023] The carriers for the pharmaceutical compositions of the present invention are common carriers available in the pharmaceutical field, including: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, colorless agents, flavoring agents, etc. for oral preparations; preservatives, solubilizing agents, stabilizers, etc. for injectable preparations; matrices, diluents, lubricants, preservatives, etc. for topical preparations. The pharmaceutical preparations can be administered orally or parenterally (such as intravenously, subcutaneously, intraperitoneally or topically). If certain drugs are unstable under gastric conditions, they can be formulated into enteric-coated tablets.

[0024] Compared with the prior art, the present invention has the following advantages and technical effects:

[0025] (1) The present invention provides a preparation method of acetylated pachymaran. The acetylated pachymaran prepared by this method has significantly improved water solubility, thus effectively solving the technical problem of low solubility of traditional pachymaran in water.

[0026] (2) The preparation method of acetylated pachymaran of the present invention ensures the high purity and stability of the product by precisely controlling key steps such as extraction, acetylation reaction and purification. Compared with the traditional carboxymethylation method, this method not only improves the solubility of pachymaran, but also avoids the by-products that may be generated during the carboxymethylation process, thereby improving the purity and quality of the product. In addition, the reagents and conditions used in this method during the preparation process are milder, reducing the impact on the environment and conforming to the development trend of green environmental protection.

[0027] (3) For the acetylated pachymaran prepared by the method of the present invention, the degree of acetyl substitution can reach 0.796. This precise control of the degree of substitution provides consistent and predictable biological activities for the product. In the pharmaceutical field, the immunomodulatory, antitumor and antioxidant effects of acetylated pachymaran are better exerted, providing an important material basis for the development of new drugs. In the fields of food and health products, due to its good water solubility and biological activities, it can be added as a functional ingredient to various products to meet the needs of consumers for healthy foods.

[0028] The technical solutions of the present invention will be further described in detail below with reference to the accompanying drawings and examples. Description of the Drawings

[0029] Figure 1The monosaccharide analysis results of the acetylated Poria cocos polysaccharide provided in Example 1 of the present invention;

[0030] Figure 2 The antioxidant capacity test results of the acetylated Poria cocos polysaccharide provided in Example 1 of the present invention. Detailed implementation manners

[0031] The technical solutions of the present invention will be further described below with reference to the accompanying drawings and examples.

[0032] Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.

[0033] In the present invention, unless otherwise specified, other test materials and instrument equipment are all conventional test materials in this field and can be obtained through commercial channels.

[0034] Example 1 This example provides an acetylated Poria cocos polysaccharide, and the preparation method includes the following steps:

[0035] S1. Grind the dried Poria cocos into powder, pass through a 60-mesh sieve, weigh 50 g of Poria cocos powder and dissolve it in 0.5 L of 0.05 M NaOH solution with a pH of 12, stir for 2 h, heat and extract at 60 °C for 45 min, extract twice, centrifuge at 12000 rpm for 30 min to obtain the supernatant;

[0036] S2. Add 0.1 M hydrochloric acid to the supernatant obtained in step S1 to adjust the pH to 7.0 to obtain a polysaccharide solution;

[0037] S3. Add the polysaccharide solution obtained in S2 into a 50 mL ultrafiltration tube, centrifuge at 12000 rpm for 20 min for concentration, add an ethanol solution with a mass fraction of 10%, discard the supernatant, suspend the precipitate in distilled water, dialyze with a dialysis bag with a molecular weight cut-off of 8 k KDa for 3 days, and freeze-dry at -50 °C for 18 h to obtain crude Poria cocos polysaccharide;

[0038] S4. Weigh 0.5 g of the crude Poria cocos polysaccharide obtained in step S3 and dissolve it in 10 mL of dimethyl sulfoxide, stir at room temperature for 30 min, raise the temperature to 40 °C, add 0.5 mL of NBS and 2 mL of acetic anhydride, react at 40 °C for 4 h to obtain reactant A;

[0039] S5. Add three volumes of absolute ethanol to the reactant A obtained in step S4, filter, discard the supernatant, dissolve the precipitate in distilled water, perform ultrafiltration and concentration three times with a 3 kD ultrafiltration tube, and freeze-dry at -50 °C for 18 h to obtain the acetylated Poria cocos polysaccharide.

[0040] The acetylated Poria cocos polysaccharide provided in Example 1 is verified by the following tests.

[0041] 1. Using ultraviolet spectrophotometry, with β-D-pentaacetylglucose as the standard, the acetyl group content was measured at a wavelength of 500 nm, and the degree of substitution was calculated. The specific steps are as follows:

[0042] (1) Solution preparation:

[0043] Weigh 0.5 g of β-D-pentaacetylglucose, dissolve it in distilled water, place it in a 100 mL volumetric flask, and then make up the volume with distilled water. Respectively take 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL of the standard solution and place them in 15 mL centrifuge tubes, and make up to 1 mL with distilled water. Weigh 0.25 g of the acetylated Poria cocos polysaccharide provided in Example 1, dissolve it in distilled water, place it in a 50 mL volumetric flask, and then make up the volume with distilled water. Take 1 mL and transfer it to a 15 mL centrifuge tube.

[0044] (2) Detection:

[0045] Add 2 mL of freshly prepared hydroxylamine hydrochloride solution to the above standard products and the acetylated Poria cocos polysaccharide sample provided in Example 1, add 5 mL of 1 mol / L sodium hydroxide solution, vortex and mix well, let it stand for 20 min, then add 4 mL of 2 mol / L hydrochloric acid to neutralize the excess alkali, vortex and mix well and then let it stand for 20 min. Add 1 mL of 0.37 mol / L ferric chloride solution, mix well by shaking, respectively take 200 μL and transfer it to a microplate, and measure the absorbance value at 540 nm on an enzyme-labeling instrument.

[0046] (3) The degree of substitution refers to the number of hydroxyl groups substituted by acetyl groups on each anhydroglucose unit on average. According to the standard curve, the acetyl substitution degree of the acetylated Poria cocos polysaccharide is obtained. First, calculate the percentage content W of acetyl groups in the Poria cocos polysaccharide according to Formula 1, and then calculate the degree of substitution of the acetylated Poria cocos polysaccharide according to Formula 2. The specific values are obtained according to the following formulas:

[0047] Formula 1:

[0048]

[0049] Formula 2:

[0050]

[0051] In the formula: M1 is the mass of the polysaccharide (mg), M2 is the mass of the acetyl group in the polysaccharide (mg), 162 is the relative molecular mass of a monosaccharide unit in the polysaccharide, 43 is the relative molecular mass of the acetyl group, and 1 is the relative molecular mass of the hydrogen atom.

[0052] Monosaccharide analysis: The monosaccharide composition of AX was analyzed by high performance anion exchange chromatography - pulsed amperometric detector (HPAEC - IPAD) (Diane - ICS - 5000 +, USA). The specific process is as follows:

[0053] (1) Accurately weigh 1.5 mg of the acetylated Poria cocos polysaccharide sample provided in Example 1 and put it into an ampoule bottle, add 1.5 ml of trifluoroacetic acid, and seal the bottle.

[0054] (2) Hydrolyze the sample at 120 °C for 3 hours.

[0055] (3) After complete hydrolysis, take out the sample and place it in a 2 - ml centrifuge tube. Dry the hydrolysis solution with a vacuum centrifugal concentrator at 55 °C. 4. For the dried sample, add methanol (0.5 mL) multiple times (5 times) to completely remove TFA in the sample.

[0056] (5) Completely dissolve the sample in 1.5 mL of ultrapure water for monosaccharide composition testing.

[0057] (6) Preparation before injection: Prepare a 5 μg / mL monosaccharide standard (glucose, xylose, galactose, galacturonic acid, glucuronic acid, mannose, arabinose, rhamnose) for HPAEC - PAD analysis. After the sample passes through an ultrafiltration tube, it is diluted. The injection concentration of PCP - K is 40 ppm, and the injection concentration of other samples is 20 ppm.

[0058] (7) Analyze using high performance anion exchange chromatography - pulsed amperometric detector. The results are as Figure 1 .

[0059] It can be Figure 1 seen that Poria cocos polysaccharide is a glucan, mainly composed of glucose, with a purity greater than 99.99%.

[0060] 2. Verification of antioxidant capacity:

[0061] (1) Prepare a DPPH solution. Dissolve an appropriate amount of DPPH in ethanol, shake well to make it fully dissolve, and finally obtain a 0.1 mM DPPH solution;

[0062] (2) Prepare Poria cocos polysaccharide solution and acetylated Poria cocos polysaccharide solution respectively. Follow the steps (1) of the above - mentioned experiment 1, and set the concentrations to 1 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, 0.2 mg / mL respectively. Use vitamin C as a positive control to measure the DPPH free radical scavenging rate.

[0063] (3) Take a certain amount of DPPH solution and add it to a test tube. Then add the above-mentioned polysaccharide solution and antioxidant respectively. Quickly shake the test tube to mix it well. Place the test tube at room temperature for 15 minutes to complete the reaction fully.

[0064] (4) Use an enzyme-labeled instrument to measure the absorbance value of each reaction solution, and calculate the DPPH free radical scavenging rate according to the absorbance value. The calculation formula is as follows:

[0065]

[0066] Among them, A0 is the absorbance value of distilled water + DPPH solution;

[0067] A1 is the absorbance value of the sample solution + DPPH solution; A2 is the absorbance value of the sample solution + absolute ethanol. The results are as Figure 2 .

[0068] It can be Figure 2 seen that the acetylated Poria cocos polysaccharide can greatly improve its antioxidant rate.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions of the present invention or make equivalent replacements, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A preparation method of acetylated poria polysaccharide, characterized in that, It includes the following steps: S1. Grind the dried Poria cocos into powder, sieve it through a 60-mesh sieve, dissolve it in 0.05M NaOH solution with a pH of 12, stir, heat for extraction, and centrifuge to obtain the supernatant; S2. Add 0.1M hydrochloric acid to the supernatant obtained in step S1 to adjust the pH to 7.0 to obtain a polysaccharide solution; S3. Centrifuge the polysaccharide solution obtained in S2, add an ethanol solution with a mass fraction of 10%, discard the supernatant, suspend the precipitate in distilled water, dialyze for 3 days, and freeze-dry to obtain crude Poria cocos polysaccharide; S4. Dissolve the crude Poria cocos polysaccharide obtained in step S3 in dimethyl sulfoxide, stir at room temperature for 30 min, raise the temperature to 40°C, add NBS and acetic anhydride to react to obtain reactant A; S5. Add anhydrous ethanol to the reactant A obtained in step S4, filter, discard the supernatant, dissolve the precipitate in distilled water, perform ultrafiltration and concentration 3 times with a 3kD ultrafiltration tube, and freeze-dry to obtain acetylated Poria cocos polysaccharide; In step S1, grind the dried Poria cocos into powder, sieve it through a 60-mesh sieve, dissolve it in NaOH solution, stir for 2 h, heat for extraction at 60°C for 30 - 60 min, extract 2 times, centrifuge at 10000 - 12000 rpm for 20 - 40 min to obtain the supernatant; In step S3, centrifuge the polysaccharide solution obtained in S2 at 10000 - 12000 rpm for 20 - 40 min, add an ethanol solution with a mass fraction of 10%, discard the supernatant, suspend the precipitate in distilled water, and dialyze for 3 days with a dialysis bag having a molecular weight cut-off of 8k - 1.4KDa; In step S3, the freeze-drying temperature is -45 - -60°C, and the freeze-drying time is 15 - 20 h; In step S4, dissolve the crude Poria cocos polysaccharide obtained in step S3 in dimethyl sulfoxide, stir at room temperature for 30 min, raise the temperature to 40°C, add NBS and acetic anhydride, and react at 40°C for 4 h to obtain reactant A; In step S5, dissolve the precipitate in distilled water, perform ultrafiltration and concentration 3 times with a 3kD ultrafiltration tube, and freeze-dry at -45 - -60°C for 15 - 20 h to obtain acetylated Poria cocos polysaccharide.

2. The acetylated Poria cocos polysaccharide prepared by the preparation method according to claim 1.

3. The application of the acetylated Poria cocos polysaccharide prepared by the preparation method according to claim 1 or the acetylated Poria cocos polysaccharide according to claim 2 in the preparation of drugs, foods or health products.

4. A pharmaceutical composition comprising one or more of the acetylated Poria cocos polysaccharides as described in claim 2.

5. A pharmaceutical composition according to claim 4, characterized in that, It includes using the acetylated Poria cocos polysaccharide as an active ingredient and a pharmaceutically acceptable carrier.

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