A method for purifying medicinal fusiformis polysaccharides using sequential simulated mobile chromatography

Abstract

The present invention discloses a method for purifying medicinal pore fungus polysaccharides by sequential simulated mobile chromatography, and relates to the field of biological extraction technology. The method comprises the following steps: (1) extracting medicinal pore fungus to obtain a polysaccharide extract; (2) purifying the polysaccharide extract by sequential simulated mobile chromatography to obtain a purified solution; (3) the purified solution is subjected to a refining and impurity removal treatment, concentrated, and dried to obtain the medicinal pore fungus polysaccharide. The method for purifying medicinal pore fungus polysaccharides provided by the present invention has low process operating costs, and the impurity removal rate and yield of the pore fungus polysaccharide are significantly improved, with the impurity removal rate reaching 80-90%, the yield reaching 80-90%, and the decolorization rate reaching 60%-80%. The method of the present invention is pollution-free, has low subsequent costs, simplifies the operating steps, uses simple equipment, reduces equipment investment, increases resin utilization, enables continuous production, and improves production efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of biological extraction, in particular to a method for purifying medicinal fusiformis polysaccharide using sequential simulated mobile chromatography. Background Art

[0002] Medicinal pore fungi are a type of fungi of the genus Fattya with medicinal functions, mainly including pine needle pore fungi, wood hoof pore fungi, red-margined pore fungi, red-fleshed pore fungi, bitter white hoof pore fungi, etc. Studies have found that the polysaccharides of this type of fungi have significant anti-cancer effects and can be used to treat esophageal cancer, gastric cancer, colon cancer, rectal cancer, lung cancer, breast cancer, uterine cancer, etc., and have the effect of improving patients' symptoms such as increasing appetite and weight, and reducing pain. At present, there is little research on medicinal phylloscopus polysaccharides. Chinese patent CN200810152030.9 discloses the use of a pine phylloscopus polysaccharide extract in the preparation of drugs for preventing and treating tumor metastasis. The prepared pine phylloscopus polysaccharide has not been removed by impurities and has low purity, but it shows obvious activation of polymorphonuclear leukocytes, improving their ability to kill tumor cells, thereby inhibiting tumor metastasis and postoperative recurrence; Chinese patent CN202110340598.9 discloses the use of extracellular polysaccharides of phylloscopus argentea, which can be used to prepare skin wound healing agents, especially for the preparation of skin burn healing agents; Chinese patent CN201210037925.4 discloses a method for extracting and purifying polysaccharides from the medicinal fungus Vani phylloscopus argentea. This method adopts a solvent extraction method, which has complex operation and low product purity. By reviewing the literature, it can be seen that large-scale research and production of medicinal pore fungus polysaccharides have not yet been carried out. With the deepening of research, the research on medicinal pore fungus polysaccharides will surely make significant progress, the demand and output will increase significantly, and the market prospects are broad.

[0003] Currently, the primary method for removing small molecules from polysaccharides is membrane separation. However, this method requires high equipment investment, is prone to membrane fouling, and requires frequent sterilization and cleaning, making it difficult to promote and apply industrially. Polysaccharide decolorization methods often use H₂O₂, but this method is inefficient, consumes a lot of solvent, and is highly polluting, making it unsuitable for industrial production. With increasingly fierce market competition, existing technologies for removing impurities from polysaccharides derived from phylloporoides are no longer able to meet current technical requirements, and there is an urgent need for efficient, industrial-scale polysaccharide impurity removal technologies.

[0004] Sequential simulated moving bed chromatography (SSMB) is currently the most advanced simulated moving bed chromatography separation technology in the world. Compared with SMB technology, it has the advantages of smaller footprint, higher feed concentration, larger feed volume, less eluent usage, higher product purity and yield, and higher outlet concentration. It is widely used in related fields abroad. At present, there are no reports on the research of SSMB polysaccharide impurity removal technology. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for purifying medicinal fusiformis polysaccharide by sequential simulated mobile chromatography to solve the problems existing in the above-mentioned prior art. The method of the present invention improves the impurity removal rate and yield of medicinal fusiformis polysaccharide, thereby significantly improving product quality.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for purifying medicinal fusiformis polysaccharide by sequential simulated mobility chromatography, comprising the following steps:

[0008] (1) extracting medicinal pore fungus to obtain a polysaccharide extract;

[0009] (2) purifying the polysaccharide extract using sequential simulated mobility chromatography to obtain a purified solution;

[0010] (3) The purified solution is purified and impurity-removed, concentrated, and dried to obtain the medicinal polysaccharide.

[0011] In step (2), the sequential simulated mobile chromatography consists of 6 chromatographic columns, numbered columns 1-6; column 1 has 1 feed port, 1 eluent feed port, 1 discharge port and 1 impurity component outlet, column 2, column 3 and column 6 have 1 feed port and 1 discharge port respectively, column 4 has 1 feed port, 1 polysaccharide raw material feed port and 1 discharge port, column 5 has 1 feed port, 1 discharge port and 1 polysaccharide component outlet; the discharge port of column 1 is connected to the feed port of column 2, the discharge port of column 2 is connected to the feed port of column 3, the discharge port of column 3 is connected to the feed port of column 4, the discharge port of column 4 is connected to the feed port of column 5, the discharge port of column 5 is connected to the feed port of column 6, and finally the discharge port of column 6 is connected to the feed port of column 1 to form a closed loop.

[0012] Furthermore, in step (1), the medicinal pore fungus is one or more of pine needle pore fungus, wood hoof pore fungus, red edge pore fungus, red flesh pseudo-pore fungus and bitter white hoof fungus.

[0013] Furthermore, in step (2), the concentration of the polysaccharide extract is 10-20 wt%.

[0014] Furthermore, in step (2), the purification step includes:

[0015] a) opening the impurity component outlet of the column 1 and the polysaccharide component outlet of the column 5, introducing eluent from the eluent inlet of the column 1, and discharging impurity components from the impurity component outlet of the column 1; while introducing eluent into the column 1, introducing the polysaccharide extract from the polysaccharide raw material inlet of the column 4, and discharging the first portion of polysaccharide components from the polysaccharide component outlet of the column 5;

[0016] b) closing the eluent inlet of column 1, the impurity component outlet of column 1, the polysaccharide raw material inlet of column 4, and the polysaccharide component outlet of column 5, so that no material enters or exits, and a large circulation is performed, and the liquid flow direction is from column 1 to column 6;

[0017] c) opening the eluent inlet of the column 1 and the polysaccharide component outlet of the column 5, introducing eluent from the eluent inlet of the column 1, and discharging the second portion of polysaccharide component from the polysaccharide component outlet of the column 5.

[0018] Furthermore, in step a), the amount of the eluent is 10-30% of the volume of the single column chromatography column; the feed amount of the polysaccharide extract is 5-10% of the volume of the single column chromatography column; in step b), the circulation volume of the large cycle is 20-40% of the volume of the single column chromatography column; in step c), the amount of the eluent is 5-20% of the volume of the single column chromatography column.

[0019] Furthermore, in step (2), the stationary phase of the chromatographic column is a strongly acidic hydrogen ion resin.

[0020] Furthermore, in step (2), the purification operating temperature is 50-70°C.

[0021] Furthermore, in step (3), the purification and impurity removal treatment adopts a column chromatography decolorization method, and the macroporous resin for column chromatography decolorization is AB-8, X-5 or ADS-4.

[0022] The present invention also provides a medicinal polysaccharide prepared according to the above method.

[0023] The present invention also provides the use of the medicinal fusiformis polysaccharide in the preparation of products that promote the production of NO by macrophages.

[0024] The present invention discloses the following technical effects:

[0025] The method for purifying medicinal pore polysaccharides provided by the present invention has low process operating costs and significantly improves the impurity removal rate and yield of pore polysaccharides, with the impurity removal rate reaching 80-90%, the yield reaching 80-90%, and the decolorization rate reaching 60%-80%. The method of the present invention is pollution-free, has low subsequent costs, simplifies the operating steps, uses simple equipment, reduces equipment investment, increases resin utilization, enables continuous production, and improves production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 The figure is a flow chart of the sequential simulated mobile chromatography purification process of the present invention. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] like Figure 1As shown, the sequential simulated mobile chromatography used in the following examples consists of 6 chromatographic columns (inner diameter 35 mm, length 1000 mm), numbered columns 1-6, each chromatographic column 1 L, a total of 6 L, and the feed and discharge are controlled by a solenoid valve; column 1 has 1 feed port, 1 eluent feed port, 1 discharge port and 1 impurity component outlet, columns 2, 3 and 6 have 1 feed port and 1 discharge port respectively, column 4 has 1 feed port, 1 polysaccharide raw material feed port and 1 discharge port, column 5 has 1 feed port, 1 discharge port and 1 polysaccharide component outlet; the discharge port of column 1 is connected to the feed port of column 2, the discharge port of column 2 is connected to the feed port of column 3, the discharge port of column 3 is connected to the feed port of column 4, the discharge port of column 4 is connected to the feed port of column 5, the discharge port of column 5 is connected to the feed port of column 6, and finally the discharge port of column 6 is connected to the feed port of column 1 to form a closed loop. The impurity removal and refining chromatographic column is a chromatographic column (inner diameter 100 mm, length 1000 mm), and the inlet and outlet are controlled by a solenoid valve.

[0034] The medicinal pore fungi of the present invention include one or more of the following: pine needle pore fungi, wood hoof pore fungi, red margin pore fungi, red flesh pore fungi, and bitter white hoof pore fungi. The purification method is described below using pine needle pore fungi as an example.

[0035] The preparation method of the extract used in the following examples is as follows: after the pine needle fungus is crushed, it is defatted with petroleum ether, and then the defatted pine needle fungus powder is added to deionized water (the mass ratio of the defatted pine needle fungus powder to deionized water is 1:15), followed by ultrasonic extraction with an ultrasonic power of 300W, an ultrasonic temperature of 40°C, and an ultrasonic time of 20min. Anhydrous ethanol is added to make the final ethanol mass concentration of 80%, and the protein is removed by alcohol precipitation to obtain the polysaccharide extract.

[0036] Example 1

[0037] (1) Preparation of polysaccharide extract

[0038] The polysaccharide extract was prepared with deionized water to a concentration of 10 wt%.

[0039] (2) Simulated mobile chromatography purification

[0040] The stationary phase in this step is the strong acid hydrogen ion resin 99H + 320, the operating temperature is 50℃, such as Figure 1 Perform the following operations as shown:

[0041] a) Open the impurity component outlet of column 1 and the polysaccharide component outlet of column 5, and simultaneously introduce the polysaccharide extract and eluent. That is, eluent D (deionized water) is introduced through the eluent inlet of column 1, and component AD (impurity component) is discharged from the impurity component outlet of column 1. Simultaneously with the eluent introduction into column 1, polysaccharide extract is added through the polysaccharide raw material inlet of column 4, and a portion of component BD (the first polysaccharide component) is discharged from the polysaccharide component outlet of column 5. The amount of eluent introduced is 136.88 mL, and the amount of polysaccharide extract introduced is 85.26 mL.

[0042] b) Close the eluent inlet and impurity component outlet of column 1, the polysaccharide raw material inlet of column 4, and the polysaccharide component outlet of column 5. No material enters or exits, and a large circulation is performed. The liquid flow direction is from column 1 to column 6, and the circulation volume is 273.00 mL;

[0043] c) Open the eluent inlet of column 1 and the polysaccharide component outlet of column 5, and introduce eluent from the eluent inlet of column 1. Another portion of component BD (the second portion of polysaccharide component) is discharged from the polysaccharide component outlet of column 5. The amount of eluent introduced is 168.74 mL.

[0044] After the operation of column 1 is completed, repeat the above three steps in sequence, and move the inlet and outlet positions to the next column along the direction of column 1 to column 6. Follow this procedure for all subsequent operations.

[0045] (3) The polysaccharide components (i.e., purified solution) obtained in step (2) a) and c) are combined and transferred to a transfer tank. They are then pumped into a refining chromatographic column and adsorbed and decolorized using AB-8 macroporous resin. The sample volume is 2BV and the flow rate is 2BV / h to obtain a refined purified solution. The solution is then vacuum concentrated (concentration temperature 45°C) and freeze-dried to obtain a medicinal polysaccharide. Note that after adsorption, elution is performed using a 60% volume fraction ethanol solution with an elution volume of 2BV and a flow rate of 1BV / h. Finally, deionized water is used for regeneration with an elution volume of 3BV and a flow rate of 2BV / h. After regeneration, the sample is repeatedly injected to achieve the effect of column chromatography decolorization.

[0046] The medicinal fusiformis polysaccharide prepared in this example had an impurity removal rate of 82.6%, a yield of 83.2%, a decolorization rate of 75.6%, and a purity of 76.8%.

[0047] Example 2

[0048] (1) Preparation of polysaccharide extract

[0049] The polysaccharide extract was prepared with deionized water to a concentration of 15 wt%.

[0050] (2) Simulated mobile chromatography purification

[0051] The stationary phase in this step is the strong acid hydrogen ion resin 106H +, the operating temperature is 65℃, such as Figure 1 Perform the following operations as shown:

[0052] a) Open the impurity component outlet of column 1 and the polysaccharide component outlet of column 5, and simultaneously introduce the polysaccharide extract and eluent. That is, eluent D (deionized water) is introduced through the eluent inlet of column 1, and component AD (impurity component) is discharged from the impurity component outlet of column 1. Simultaneously with the introduction of eluent into column 1, the polysaccharide extract is introduced through the polysaccharide raw material inlet of column 4, and a portion of component BD (the first polysaccharide component) is discharged from the polysaccharide component outlet of column 5. The amount of eluent introduced is 146.27 mL, and the amount of polysaccharide extract introduced is 106.25 mL.

[0053] b) Close the eluent inlet and impurity component outlet of column 1, the polysaccharide raw material inlet of column 4, and the polysaccharide component outlet of column 5. No material enters or exits, and a large circulation is performed. The liquid flow direction is from column 1 to column 6, and the circulation volume is 265.00 mL;

[0054] c) Open the eluent inlet of column 1 and the polysaccharide component outlet of column 5, and introduce eluent from the eluent inlet of column 1. Another portion of component BD (the second portion of polysaccharide component) is discharged from the polysaccharide component outlet of column 5. The amount of eluent introduced is 168.63 mL.

[0055] After the operation of column 1 is completed, repeat the above three steps in sequence, and move the inlet and outlet positions to the next column along the direction of column 1 to column 6. Follow this procedure for all subsequent operations.

[0056] (3) The polysaccharide components (i.e., purified solution) obtained in step (2) a) and c) are combined and transferred to a transfer tank. They are then pumped into a refining chromatographic column and adsorbed and decolorized using X-5 macroporous resin. The sample volume is 2BV and the flow rate is 2.5BV / h to obtain a refined purified solution. The solution is then vacuum concentrated (concentration temperature 45°C) and freeze-dried to obtain a medicinal polysaccharide. Note that after adsorption, elution is performed with a volume fraction of 70% ethanol solution, the elution volume is 2.5BV, and the flow rate is 1.5BV / h; finally, deionized water is used for regeneration, the elution volume is 4BV, and the flow rate is 3BV / h. After regeneration, the sample is repeatedly injected to achieve the effect of column chromatography decolorization.

[0057] The medicinal phyllopodia polysaccharide prepared in this example had an impurity removal rate of 86.7%, a yield of 81.7%, a decolorization rate of 78.4%, and a purity of 79.2%.

[0058] Example 3

[0059] (1) Preparation of polysaccharide extract

[0060] The polysaccharide extract was prepared with deionized water to a concentration of 20 wt%.

[0061] (2) Simulated mobile chromatography purification

[0062] The stationary phase in this step is the strong acid hydrogen ion resin 99H + 320, the operating temperature is 70℃, such as Figure 1 Perform the following operations as shown:

[0063] a) Open the impurity component outlet of column 1 and the polysaccharide component outlet of column 5, and simultaneously introduce the polysaccharide extract and eluent. That is, eluent D (deionized water) is introduced through the eluent inlet of column 1, and component AD (impurity component) is discharged from the impurity component outlet of column 1. Simultaneously with the introduction of eluent into column 1, polysaccharide extract is added through the polysaccharide raw material inlet of column 4, and a portion of component BD (the first polysaccharide component) is discharged from the polysaccharide component outlet of column 5. The amount of eluent introduced is 168.54 mL, and the amount of polysaccharide extract introduced is 110.35 mL.

[0064] b) Close the eluent inlet and impurity component outlet of column 1, the polysaccharide raw material inlet of column 4, and the polysaccharide component outlet of column 5. No material enters or exits, and a large circulation is performed. The liquid flow direction is from column 1 to column 6, and the circulation volume is 315.00 mL;

[0065] c) Open the eluent inlet of column 1 and the polysaccharide component outlet of column 5, and introduce eluent from the eluent inlet of column 1. Another portion of component BD (the second portion of polysaccharide component) is discharged from the polysaccharide component outlet of column 5. The amount of eluent introduced is 173.56 mL.

[0066] After the operation of column 1 is completed, repeat the above three steps in sequence, and move the inlet and outlet positions to the next column along the direction of column 1 to column 6. Follow this procedure for all subsequent operations.

[0067] (3) The polysaccharide components (i.e., purified solution) obtained in step (2) a) and c) are combined and transferred to a transfer tank. They are then pumped into a refining chromatographic column and adsorbed and decolorized using ADS-4 macroporous resin. The sample volume is 5BV and the flow rate is 3BV / h to obtain a refined purified solution. The solution is then vacuum concentrated (concentration temperature 45°C) and freeze-dried to obtain a medicinal polysaccharide. Note that after adsorption, elution is performed with a 90% volume fraction ethanol solution, the elution volume is 3BV, and the flow rate is 2BV / h; finally, deionized water is used for regeneration, the elution volume is 5BV, and the flow rate is 5BV / h. After regeneration, the sample is repeatedly injected to achieve the effect of column chromatography decolorization.

[0068] The medicinal phyllopodia polysaccharide prepared in this example had an impurity removal rate of 87.3%, a yield of 81.7%, a decolorization rate of 66.7%, and a purity of 79.8%.

[0069] Effect description:

[0070] Macrophages RAW264.7 were stimulated with the medicinal Familiarina polysaccharide prepared in Example 1-3, and the concentration of NO produced was measured. The results are shown in Table 1.

[0071] Specific experimental method: 100 μL RAW264.7 cells (1×10 6 Cells (100 cells / mL) were pre-incubated in a 96-well microplate at 37°C in a 5% CO2 incubator for 24 hours. The supernatant was discarded and 200 μL of a 5 μg / mL polysaccharide solution was added to the cells. The culture medium served as a negative control, and 1 μg / mL lipopolysaccharide (LPS) was used as a positive control. The cells were incubated for 24 hours. NO content in the supernatant was determined by adding 100 μL of Griess reagent to 100 μL of supernatant. The reaction was incubated in the dark for 10 minutes at room temperature. The absorbance was then measured at 540 nm using a microplate reader (BioTekinstruments, Winooski, VT, USA). Sodium nitrite was used as a standard, and NO production was calculated by fitting the sample absorbance values ​​to the standard curve.

[0072] Table 1

[0073]

[0074]

[0075] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A use of a medicinal polysaccharide from Familiar Fungus in the preparation of a product that promotes the production of NO by macrophages, characterized in that: The method for using medicinal fusiformis polysaccharide comprises the following steps: (1) extracting medicinal pore fungus to obtain a polysaccharide extract; (2) purifying the polysaccharide extract using sequential simulated mobility chromatography to obtain a purified solution; (3) The purified solution is purified and impurity-removed, concentrated, and dried to obtain the medicinal polysaccharide. In step (2), the sequential simulated mobile chromatography consists of 6 chromatographic columns, numbered columns 1-6; column 1 has 1 feed port, 1 eluent feed port, 1 discharge port and 1 impurity component outlet, column 2, column 3 and column 6 have 1 feed port and 1 discharge port respectively, column 4 has 1 feed port, 1 polysaccharide raw material feed port and 1 discharge port, column 5 has 1 feed port, 1 discharge port and 1 polysaccharide component outlet; the discharge port of column 1 is connected to the feed port of column 2, the discharge port of column 2 is connected to the feed port of column 3, the discharge port of column 3 is connected to the feed port of column 4, the discharge port of column 4 is connected to the feed port of column 5, the discharge port of column 5 is connected to the feed port of column 6, and finally the discharge port of column 6 is connected to the feed port of column 1 to form a closed loop; In step (2), the purification step comprises: a) opening the impurity component outlet of the column 1 and the polysaccharide component outlet of the column 5, introducing eluent from the eluent inlet of the column 1, and discharging impurity components from the impurity component outlet of the column 1; while introducing eluent into the column 1, introducing the polysaccharide extract from the polysaccharide raw material inlet of the column 4, and discharging a first portion of polysaccharide components from the polysaccharide component outlet of the column 5; b) closing the eluent inlet of column 1, the impurity component outlet of column 1, the polysaccharide raw material inlet of column 4, and the polysaccharide component outlet of column 5, so that no material enters or exits, and a large circulation is performed, and the liquid flow direction is from column 1 to column 6; c) opening the eluent inlet of the column 1 and the polysaccharide component outlet of the column 5, introducing eluent from the eluent inlet of the column 1, and discharging the second portion of polysaccharide component from the polysaccharide component outlet of the column 5; The eluent is deionized water; The medicinal pore fungus is pine needle pore fungus.

2. The use according to claim 1, characterized in that In step (2), the concentration of the polysaccharide extract is 10-20 wt%.

3. The use according to claim 2, characterized in that In step a), the amount of the eluent is 10-30% of the volume of the single column chromatography column; the feed amount of the polysaccharide extract is 5-10% of the volume of the single column chromatography column; in step b), the circulation volume of the macrocycle is 20-40% of the volume of the single column chromatography column; in step c), the amount of the eluent is 5-20% of the volume of the single column chromatography column.

4. The use according to claim 1, characterized in that In step (2), the stationary phase of the chromatographic column is a strongly acidic hydrogen ion resin.

5. The use according to claim 1, characterized in that In step (2), the purification operating temperature is 50-70°C.

6. The use according to claim 1, characterized in that In step (3), the purification and impurity removal treatment adopts a column chromatography decolorization method, and the macroporous resin for column chromatography decolorization is AB-8, X-5 or ADS-4.

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