Acetylated pachymaran as well as preparation method and application thereof

Abstract

The invention discloses acetylated pachymaran as well as a preparation method and application thereof, and belongs to the technical field of biochemical engineering. The method comprises the following steps: grinding dried poria cocos into powder, sieving, dissolving in a NaOH solution until the pH value is 12, heating, extracting and centrifuging to obtain supernate; adjusting the pH value to 7.0 by hydrochloric acid to obtain a polysaccharide solution; concentrating, adding an ethanol solution, discarding the supernate, suspending the precipitate in distilled water, dialyzing and freeze-drying to obtain crude poria cocos polysaccharide; the method comprises the following steps: dissolving pachymaran in dimethyl sulfoxide, stirring at room temperature, heating to 40 DEG C, adding NBS and acetic anhydride, adding absolute ethyl alcohol, filtering, discarding supernatant, dissolving with distilled water, carrying out ultrafiltration concentration, and freeze-drying to obtain acetylated pachymaran. The invention discloses acetylated pachymaran and a preparation method thereof, the acetylated pachymaran prepared by the method has high water solubility, the technical problem of low solubility of traditional pachymaran in water is effectively solved, and the bioavailability of the acetylated pachymaran is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemical engineering, and specifically relates to acetylated pachymaran and a preparation method and application thereof. Background Art

[0002] Poria polysaccharide 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 activity in enhancing immune function, anti-tumor and antioxidant. However, due to the low solubility of β(1→3)-D-glucan in water, its application in the field of health care is limited. Improving the solubility of Poria polysaccharide can significantly improve its bioavailability, promote drug delivery and release, broaden its application fields, improve preparation efficiency and reduce costs, so that it has a wider application prospect and higher value in the fields of medicine, food, and health products.

[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 be carried out at a specific temperature, pH value and reaction time to ensure the efficiency of the reaction and the quality of the product, and some by-products will be produced, 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 a preparation method and application thereof. The acetylated pachymaran prepared by the method has high water solubility, effectively solves the technical problem of low solubility of traditional pachymaran in water, and improves the bioavailability of acetylated pachymaran.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows: A method for preparing acetylated pachymaran comprises the following steps: S1. Grind the dried Poria cocos into powder, pass through a 60-mesh sieve, dissolve in 0.05 M NaOH solution, pH 12, stir, heat and extract, centrifuge to obtain a supernatant; S2, adding 0.1 M hydrochloric acid to the supernatant obtained in step S1 to adjust the pH to 7.0 to obtain a polysaccharide solution; S3, centrifuging the polysaccharide solution obtained in S2, adding 10% ethanol solution by mass, discarding the supernatant, suspending the precipitate in distilled water, dialyzing for 3 days, and freeze-drying to obtain crude Poria cocos polysaccharide; S4, dissolving the crude Poria cocos polysaccharide obtained in step S3 in dimethyl sulfoxide, stirring at room temperature for 30 min, heating to 40° C., adding NBS and acetic anhydride to react, and obtaining reactant A; S5. Add anhydrous ethanol to the reactant A obtained in step S4, filter, discard the supernatant, dissolve the precipitate with distilled water, perform ultrafiltration and concentration three times using a 3kD ultrafiltration tube, and freeze-dry to obtain acetylated Poria polysaccharide.

[0006] Preferably, in step S1, the dried Poria cocos is ground into powder, passed through a 60-mesh sieve, dissolved in a NaOH solution, stirred for 2 hours, heated and extracted at 60° C. for 30 to 60 minutes, extracted twice, and centrifuged at 10,000 to 12,000 rpm for 20 to 40 minutes to obtain a supernatant.

[0007] Preferably, in step S3, the polysaccharide solution obtained in S2 is centrifuged at 10,000 to 12,000 rpm for 20 to 40 min, a 10% ethanol solution is added, the supernatant is discarded, the precipitate is suspended in distilled water, and dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 8k to 1.4KDa.

[0008] Preferably, in step S3, the freeze-drying temperature is -45 to -60°C, and the freeze-drying time is 15 to 20 hours.

[0009] Preferably, in step S4, the crude Poria cocos polysaccharide obtained in step S3 is dissolved in dimethyl sulfoxide, stirred at room temperature for 30 min, heated to 40° C., and NBS and acetic anhydride are added and reacted at 40° C. for 4 h to obtain reactant A.

[0010] Preferably, in step S5, the precipitate is dissolved in distilled water, ultrafiltration and concentration are performed three times using a 3kD ultrafiltration tube, and freeze-dried at -45 to -60°C for 15 to 20 hours to obtain acetylated Pachymaran.

[0011] The invention also provides acetylated pachymaran prepared by the preparation method.

[0012] The present invention also provides an acetylated pachymaran prepared by the preparation method or application of the acetylated pachymaran in the preparation of medicines, foods or health products.

[0013] The present invention also provides a pharmaceutical composition comprising one or more acetylated pachymaran.

[0014] The pharmaceutical composition comprises the acetylated pachymaran as an active ingredient and a pharmaceutically acceptable carrier.

[0015] A pharmaceutical preparation comprises a therapeutically effective amount of any one of the above acetylated pachymaran and a pharmaceutically acceptable excipient.

[0016] The pharmaceutical preparations 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 by adding injection water before injection) and local preparations (such as ointments or solutions).

[0017] The carriers used in the pharmaceutical composition of the present invention are common carriers available in the pharmaceutical field, including: binders, lubricants, disintegrants, solubilizers, diluents, stabilizers, suspending agents, pigments, flavoring agents, etc. for oral preparations; preservatives, solubilizers, stabilizers, etc. for injectable preparations; bases, diluents, lubricants, preservatives, etc. for local preparations. The pharmaceutical preparations can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally or topically), and if some drugs are unstable under gastric conditions, they can be formulated into enteric-coated tablets.

[0018] Compared with the prior art, the present invention has the following advantages and technical effects: (1) The present invention provides a method for preparing acetylated pachymaran. The acetylated pachymaran prepared by the method has significantly improved water solubility, thereby effectively solving the technical problem of low solubility of traditional pachymaran in water.

[0019] (2) The method for preparing acetylated tuckahoe polysaccharide 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, the present method not only improves the solubility of tuckahoe polysaccharide, but also avoids the by-products that may be produced during the carboxymethylation process, thereby improving the purity and quality of the product. In addition, the reagents and conditions used in the preparation process of the present method are more mild, reducing the impact on the environment, and conforming to the development trend of green environmental protection.

[0020] (3) The acetylated tuckahoe polysaccharide prepared by the method of the present invention has an acetylation degree of up to 0.796. This precise degree of substitution control provides consistent and predictable biological activity for the product. In the field of medicine, the immune-enhancing, anti-tumor and antioxidant effects of acetylated tuckahoe polysaccharide have been better exerted, providing an important material basis for the development of new drugs. In the field of food and health products, due to its good water solubility and biological activity, it can be added to various products as a functional ingredient to meet consumers' demand for healthy food.

[0021] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The monosaccharide analysis results of the acetylated pachymaran provided in Example 1 of the present invention; Figure 2This is the test result of the antioxidant capacity of acetylated pachymaran provided in Example 1 of the present invention. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below through the accompanying drawings and embodiments.

[0024] Unless otherwise defined, technical or scientific terms used in the present invention shall have the common meanings understood by one having ordinary skills in the field to which the present invention belongs.

[0025] In the present invention, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0026] Example 1 This example provides an acetylated pachymaran, and the preparation method comprises the following steps: 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, pH 12, stir for 2 h, heat and extract at 60 °C for 45 min, extract twice, centrifuge at 12000 rpm for 30 min, and obtain the supernatant; S2, adding 0.1 M hydrochloric acid to the supernatant obtained in step S1 to adjust the pH to 7.0 to obtain a polysaccharide solution; S3, add the polysaccharide solution obtained in S2 into a 50 mL ultrafiltration tube, centrifuge at 12000 rpm for 20 min to concentrate, add 10% ethanol solution by mass, discard the supernatant, suspend the precipitate in distilled water, dialyze with a dialysis bag with a molecular weight cutoff of 8k KDa for 3 days, and freeze-dry at -50°C for 18 h to obtain crude Poria cocos polysaccharide; S4, weigh 0.5 g of the crude Poria cocos polysaccharide obtained in step S3, dissolve it in 10 mL of dimethyl sulfoxide, stir it at room temperature for 30 min, heat it to 40° C., add 0.5 mL of NBS and 2 mL of acetic anhydride, and react at 40° C. for 4 h to obtain reactant A; S5. Add 3 times the volume of anhydrous ethanol to the reactant A obtained in step S4, filter, discard the supernatant, dissolve the precipitate with distilled water, perform ultrafiltration and concentration three times using a 3kD ultrafiltration tube, and freeze-dry at -50°C for 18 hours to obtain acetylated Poria polysaccharide.

[0027] The effect of the acetylated Pachymaran provided in Example 1 was verified by the following test.

[0028] 1. Use ultraviolet spectrophotometry, take β-D-pentacetyl glucose as the standard, measure the acetyl content at a wavelength of 500nm, and calculate the degree of substitution. The specific steps are as follows: (1) Liquid preparation: Weigh 0.5 g of β-D-pentacetyl glucose, add distilled water to dissolve, place in a 100 mL volumetric flask, and then add distilled water to make up to volume. Take 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the standard solution respectively, place in a 15 mL centrifuge tube, and add distilled water to make up to 1 mL. Weigh 0.25 g of acetylated tuckahoe polysaccharide provided in Example 1, add distilled water to dissolve, place in a 50 mL volumetric flask, add distilled water to make up to volume, and take 1 mL to a 15 mL centrifuge tube.

[0029] (2) Testing: Add 2 mL of freshly prepared hydroxylamine hydrochloride solution to the above standard and the acetylated pachymaran sample provided in Example 1, add 5 mL of 1 mol / L sodium hydroxide solution, vortex and mix, let stand for 20 min, add 4 mL of 2 mol / L hydrochloric acid to neutralize the excess alkali, vortex and mix, let stand for 20 min. Add 1 mL of 0.37 mol / L ferric chloride solution dropwise, shake and mix, take 200 μL into a microplate, and detect the absorbance value at 540 nm on an ELISA instrument.

[0030] (3) The degree of substitution refers to the average number of hydroxyl groups substituted by acetyl groups on each dehydrated glucose unit. The degree of acetylation of acetylated pachymaran was obtained according to the standard curve. The percentage of acetyl groups in pachymaran was first calculated according to formula 1, and then the degree of substitution of acetylated pachymaran was calculated according to formula 2. The specific value was obtained according to the following formula: Formula 1: Formula 2: In the formula: M1 is the mass of polysaccharide (mg), M2 is the mass of acetyl group in polysaccharide (mg), 162 is the relative molecular mass of a monosaccharide in polysaccharide, 43 is the relative molecular mass of acetyl group, and 1 is the relative molecular mass of hydrogen atom.

[0031] Monosaccharide analysis: The monosaccharide composition of AX was analyzed using high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-IPAD) (Diane-ICS-5000+, USA). The specific process is as follows: (1) Accurately weigh 1.5 mg of the acetylated pachymaran sample provided in Example 1 and put it into an ampoule. Add 1.5 ml of trifluoroacetic acid and seal the ampoule.

[0032] (2) The sample was hydrolyzed at 120 °C for 3 hours.

[0033] (3) After the hydrolysis is complete, take out the sample and place it in a 2 ml centrifuge tube. Dry the hydrolyzate at 55 °C using a vacuum centrifugal concentrator. 4. After the dried sample, add methanol (0.5 mL) several times (5 times) to completely remove TFA in the sample.

[0034] (5) Dissolve the sample completely in 1.5 mL of ultrapure water for monosaccharide composition testing.

[0035] (6) Preparation before injection: Prepare 5 μg / mL monosaccharide standards (glucose, xylose, galactose, galacturonic acid, glucuronic acid, mannose, arabinose, and rhamnose) for HPAEC-PAD analysis. The samples were diluted after passing through the ultrafiltration tube. The injection concentration of PCP-K was 40 ppm, and the injection concentration of other samples was 20 ppm.

[0036] (7) High performance anion exchange chromatography-pulsed amperometric detection was used for analysis. The results are as follows Figure 1 .

[0037] Depend on Figure 1 It can be seen that Poria cocos polysaccharide is a glucan, mainly composed of glucose, with a purity greater than 99.99%.

[0038] 2. Antioxidant capacity verification: (1) Prepare DPPH solution. Dissolve an appropriate amount of DPPH in ethanol and shake well to fully dissolve it. Finally, a 0.1 mM DPPH solution is obtained. (2) Prepare tuckahoe polysaccharide solution and acetylated tuckahoe polysaccharide solution respectively, and follow the same procedure as step (1) of experiment 1 above. The concentrations are set to 1 mg / mL, 0.8 mg / mL, 0.6 mg / mL, 0.4 mg / mL, and 0.2 mg / mL, respectively. Vitamin C is used as a positive control, and the DPPH free radical scavenging rate is determined.

[0039] (3) Take a certain amount of DPPH solution and add it to a test tube. Then add the above polysaccharide solution and antioxidant respectively. Quickly shake the test tube to mix it thoroughly. Place the test tube at room temperature for 15 minutes to allow the reaction to complete.

[0040] (4) Use an ELISA instrument to measure the absorbance value of each reaction solution, and calculate the DPPH free radical scavenging rate based on the absorbance value; the calculation formula is as follows: Where A0 is the absorbance of distilled water + DPPH solution; A1 is the absorbance of sample solution + DPPH solution; A2 is the absorbance of sample solution + anhydrous ethanol. Figure 2 .

[0041] Depend on Figure 2It can be seen that acetylated Poria cocos polysaccharide can greatly improve its antioxidant rate.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing acetylated pachymaran, characterized in that: The following steps are involved: S1. Grind the dried Poria cocos into powder, pass through a 60-mesh sieve, dissolve in 0.05 M NaOH solution, pH 12, stir, heat and extract, centrifuge to obtain a supernatant; S2, adding 0.1 M hydrochloric acid to the supernatant obtained in step S1 to adjust the pH to 7.0 to obtain a polysaccharide solution; S3, centrifuging the polysaccharide solution obtained in S2, adding 10% ethanol solution by mass, discarding the supernatant, suspending the precipitate in distilled water, dialyzing for 3 days, and freeze-drying to obtain crude Poria cocos polysaccharide; S4, dissolving the crude Poria cocos polysaccharide obtained in step S3 in dimethyl sulfoxide, stirring at room temperature for 30 min, heating to 40° C., adding NBS and acetic anhydride to react, and obtaining reactant A; S5. Add anhydrous ethanol to the reactant A obtained in step S4, filter, discard the supernatant, dissolve the precipitate with distilled water, perform ultrafiltration and concentration three times using a 3kD ultrafiltration tube, and freeze-dry to obtain acetylated Poria polysaccharide.

2. The preparation method according to claim 1, characterized in that: In step S1, the dried Poria cocos is ground into powder, passed through a 60-mesh sieve, dissolved in a NaOH solution, stirred for 2 hours, heated at 60° C. for extraction for 30 to 60 minutes, extracted twice, and centrifuged at 10,000 to 12,000 rpm for 20 to 40 minutes to obtain a supernatant.

3. The preparation method according to claim 1, characterized in that: In step S3, the polysaccharide solution obtained in S2 is centrifuged at 10000-12000 rpm for 20-40 min, a 10% ethanol solution is added, the supernatant is discarded, the precipitate is suspended in distilled water, and dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 8k-1.4KDa.

4. The preparation method according to claim 1, characterized in that: In step S3, the freeze-drying temperature is -45 to -60°C, and the freeze-drying time is 15 to 20 hours.

5. The preparation method according to claim 1, characterized in that: In step S4, the crude Poria cocos polysaccharide obtained in step S3 is dissolved in dimethyl sulfoxide, stirred at room temperature for 30 minutes, heated to 40°C, and NBS and acetic anhydride are added to react at 40°C for 4 hours to obtain reactant A.

6. The preparation method according to claim 1, characterized in that: In step S5, the precipitate is dissolved in distilled water, ultrafiltration and concentration are performed three times using a 3kD ultrafiltration tube, and freeze-dried at -45 to -60°C for 15 to 20 hours to obtain acetylated pachymaran.

7. Acetylated pachymaran prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the acetylated pachymaran prepared by the preparation method according to any one of claims 1 to 6 or the acetylated pachymaran according to claim 7 in the preparation of medicines, foods or health products.

9. A pharmaceutical composition comprising one or more acetylated Pachymaran as claimed in claim 7.

10. A pharmaceutical composition according to claim 9, characterized in that: The invention comprises the acetylated pachymaran as an active ingredient and a pharmaceutically acceptable carrier.

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