CN110590969B - Preparation method of uniform acidic Ganoderma Applanatum polysaccharide capable of identifying and regulating lymphocyte - Google Patents
Preparation method of uniform acidic Ganoderma Applanatum polysaccharide capable of identifying and regulating lymphocyte Download PDFInfo
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Abstract
The invention relates to a preparation method of uniform acidic Ganoderma Applanatum polysaccharide capable of identifying and regulating lymphocyte, belonging to the field of preparation of functional components of natural products. The preparation method comprises the steps of preparing ganoderma applanatum fermented crude polysaccharide and ganoderma applanatum fermented crude polysaccharide solution by taking ganoderma applanatum fermented extract, water and ethanol as raw materials, adding a mixed solution of chloroform and n-butyl alcohol into the ganoderma applanatum fermented crude polysaccharide solution, concentrating and drying to obtain ganoderma applanatum fermented deproteinized polysaccharide, performing biomacromolecule separation and purification by using an ion exchange column chromatography method to obtain a purified ganoderma applanatum fermented deproteinized polysaccharide solution, eluting, collecting and combining to obtain ganoderma applanatum fermented acidic polysaccharide, eluting again, collecting and freezing to obtain ganoderma applanatum acidic uniform polysaccharide. The method can be used for separating and purifying specifically to obtain Ganoderma Applanatum neutral heteropolysaccharide with specificity for recognizing intestinal cancer cells, and has the advantages of accuracy, simplicity and effectiveness.
Description
Technical Field
The invention relates to a preparation method of uniform acidic Ganoderma Applanatum polysaccharide capable of identifying and regulating lymphocyte, belonging to the field of preparation of functional components of natural products.
Background
The Ganoderma Applanatum belongs to Basidiomycota, Aphyllophorales, Ganoderma of Polyporaceae, and is an important medicinal fungus resource. The Ganoderma Applanatum polysaccharide is its main active ingredient, has effects of enhancing immunity, resisting oxidation, etc., and can be used for treating gastritis, hepatitis, and digestive tract cancer. The Ganoderma Applanatum polysaccharide as biological macromolecule has functional activity closely related to its chemical structure, and the chemical structure of Ganoderma Applanatum polysaccharide has close relationship with its preparation method and evaluation mode. At present, the preparation method of the Ganoderma Applanatum polysaccharide takes the extraction amount of the Ganoderma Applanatum polysaccharide as the starting point, and obtains the Ganoderma Applanatum polysaccharide with the maximum extraction amount by optimizing process conditions. However, the increase in the extraction amount often causes other impurities to be mixed in the extracted polysaccharide or destroys the active structure of the polysaccharide, thereby affecting the active function of the Ganoderma Applanatum polysaccharide.
Disclosure of Invention
The invention aims to overcome the defects of the existing research and technology and aims to provide a preparation method of uniform acidic Ganoderma Applanatum polysaccharide capable of identifying and regulating lymphocytes.
The technical scheme of the invention is as follows: a method for preparing an acidic homopolysaccharide from Ganoderma Applanatum capable of recognizing and regulating lymphocytes, comprising:
step one, preparing a Ganoderma Applanatum fermentation crude polysaccharide by taking Ganoderma Applanatum fermentation extract, water and ethanol as raw materials, and adding water into the Ganoderma Applanatum fermentation crude polysaccharide to prepare a Ganoderma Applanatum fermentation crude polysaccharide solution with the mass volume concentration of 5%;
step two, adding a mixed solution of chloroform and n-butyl alcohol into the Ganoderma Applanatum fermentation crude polysaccharide solution, and concentrating and drying to obtain Ganoderma Applanatum fermentation deproteinized polysaccharide;
step three, preparing the fermentation deproteinized polysaccharide of the Ganoderma Applanatum into fermentation deproteinized polysaccharide solution with the concentration of 20g/L, and separating and purifying biomacromolecules by adopting an ion exchange column chromatography method to obtain the fermentation deproteinized polysaccharide solution with uniform charges;
step four, sequentially taking distilled water, 0.1mol/L NaCl solution and 0.2mol/L NaCl solution as eluents, eluting at the flow rate of 1mL/min, eluting the purified fermentation deproteinized polysaccharide solution of the Ganoderma Applanatum, collecting the eluted part with 0.2mol/L NaCl solution as eluent, collecting 1 tube every 5 minutes, and collecting the total elution volume V1Detecting the collected liquid components in each tube, combining the liquid components with sugar content, and freeze-drying to obtain the ganoderma applanatum fermentation acidic polysaccharide;
wherein, tube separation collection is not carried out after the first two times of elution;
adding a NaCl solution into the Ganoderma Applanatum fermented acidic polysaccharide to completely dissolve the Ganoderma Applanatum fermented acidic polysaccharide;
and the molecular weight of the Ganoderma Applanatum fermentation acidic polysaccharide is homogenized by gel column chromatography to obtain Ganoderma Applanatum acidic homogeneous polysaccharide solution;
wherein 2mL of NaCl solution with the molar concentration of 0.15mol/L is added into every 10mg of the Ganoderma Applanatum fermented acidic polysaccharide;
step six, eluting the acidic homogenized polysaccharide solution obtained by fermenting the Ganoderma Applanatum with 0.15mol/L NaCl solution as eluent at the elution flow rate of 4mL/15min, collecting 1 tube per 4mL, and the total elution volume is V2And combining the eluates of the 46 th to 60 th tubes, and freeze-drying to obtain the uniform acidic polysaccharide capable of identifying and combining with lymphocytes.
The relative density of the Ganoderma Applanatum fermented extract is 1.4.
The preparation process of the Ganoderma Applanatum fermented crude polysaccharide in the first step comprises the following steps:
step 1, soaking the Ganoderma Applanatum fermented extract in 94-95% ethanol solution to obtain Ganoderma Applanatum fermented extract solution; wherein, the soaking temperature is as follows: soaking for 12-24 hours at 3-6 ℃;
step 2, adding water with the volume of 3-6 times of that of the Ganoderma Applanatum fermented extract to dissolve and precipitate, centrifuging at the rotating speed of 4000-5000 r/min, and filtering to obtain filtered Ganoderma Applanatum fermented extract dissolved solution, wherein the centrifuging time is 10 min;
step 3, extracting the filtered Ganoderma Applanatum fermentation extract solution for 3 hours in a water bath at 70 ℃, performing second centrifugation, extracting supernatant, collecting precipitate, performing water bath extraction on the collected precipitate part for 3 hours at 75 ℃, performing centrifugation, extracting supernatant again, collecting precipitate again, performing water bath extraction for 3 hours and centrifugation on the collected precipitate at 75 ℃, and collecting supernatant for the third time after precipitation;
mixing the supernatant liquid obtained by the three-time extraction to prepare an extracting solution, and performing reduced pressure concentration on the extracting solution to prepare a concentrated solution with the relative density of 1.3-1.5;
and 4, adding 95% ethanol into the concentrated solution until the final concentration of the ethanol in the solution is 70%, standing in a refrigerator at 4 ℃ for 12 hours, centrifuging at 5000r/m for 10min, and collecting precipitate to obtain the Ganoderma Applanatum fermented crude polysaccharide.
The preparation process of the fermentation deproteinized polysaccharide of the Ganoderma Applanatum in the second step comprises the following steps:
step a, adding a mixed solution of chloroform and n-butanol into the Ganoderma Applanatum fermentation crude polysaccharide solution, and magnetically stirring for 60min to obtain a mixed polysaccharide solution;
wherein the volume ratio of the polysaccharide solution prepared in the Ganoderma Applanatum fermented crude polysaccharide solution to the mixed solution is 4:1, and the volume ratio of chloroform to n-butanol is 4: 1;
step b, carrying out first centrifugal treatment on the mixed polysaccharide solution, collecting an upper layer solution, carrying out second centrifugal treatment on the residual polysaccharide solution, collecting the upper layer solution, then carrying out third centrifugal treatment on the residual polysaccharide solution, and collecting the upper layer solution;
mixing the upper layer solution obtained by the three times of centrifugal treatment to obtain polysaccharide solution after protein removal;
and c, concentrating the polysaccharide solution after protein removal to 1/3 of the original volume under reduced pressure, and freeze-drying to obtain the fermented proteoglycan-removed dry powder of the Ganoderma Applanatum.
The chromatographic column in the third step adopts a DEAE52 cellulose ion exchange column.
The calculation formula of the elution volume in the fourth step is as follows:
wherein, the diameter of the chromatographic column and the height of the chromatographic column bed are both the DEAE52 cellulose ion exchange column.
The total elution volume in the sixth step is V2
V2= Vi+Vo
Wherein Vi is the volume of internal water, i.e., the sum of the volumes of liquids present in the meshes of the swollen gel particles, and Vo is the volume of external water, i.e., the sum of the volumes of liquid phases present in the gaps between the gel particles in the gel column.
The invention has the advantages that:
the invention relates to a preparation method of uniform Ganoderma Applanatum polysaccharide capable of identifying and regulating lymphocytes, which aims at the problems in the prior art, and establishes a preparation method of the Ganoderma Applanatum polysaccharide with strong targeting property, good specificity and strong activity from the important link of the Ganoderma Applanatum polysaccharide exerting immune enhancing activity, namely the specific identification of the Ganoderma Applanatum polysaccharide and lymphocytes.
Detailed Description
The present invention will be further described with reference to specific embodiments, but the scope of the invention as claimed is not limited to the following embodiments.
Example 1
Soaking the Ganoderma Applanatum fermented extract in 95% ethanol at 4 deg.C for 12 hr, dissolving precipitate in 3 times of distilled water, centrifuging at 4000r/min for 10min to remove insoluble substances, extracting at 70 deg.C in water bath for 3 hr, centrifuging to collect extractive solution, repeatedly extracting precipitate for 2 times, mixing extractive solutions, concentrating under reduced pressure to obtain concentrated solution with relative density of 1.4, adding 95% ethanol into the concentrated solution until the final concentration of ethanol is 70%, centrifuging at 4 deg.C in refrigerator for 12 hr, and centrifuging at 5000r/min for 10min to collect precipitate to obtain Ganoderma Applanatum fermented crude polysaccharide. Adding Sevag reagent (chloroform: n-butyl alcohol = 4: 1) into 5% of the crude polysaccharide aqueous solution obtained by fermenting the Ganoderma Applanatum, magnetically stirring for 60min, centrifuging, collecting the upper-layer polysaccharide solution, repeating the above operation until the upper-layer polysaccharide solution is centrifuged and denatured-free protein layer is obtained, combining the polysaccharide solutions, concentrating under reduced pressure until the volume of the solution is 1/3, and freeze-drying to obtain dry powder, namely the polysaccharide removed by fermenting the Ganoderma Applanatum. Accurately weighing 100mg of the fermentation deproteinized polysaccharide of the Ganoderma Applanatum, dissolving the fermentation deproteinized polysaccharide in 5mL of distilled water, loading the solution on an ion exchange column, eluting with ionic water, 0.1mol/L NaCl solution and 0.2mol/L NaCl solution in sequence at flow rates of 1mL/min, eluting the purified fermentation deproteinized polysaccharide solution of the Ganoderma Applanatum, collecting the eluted part with 0.2mol/L NaCl solution as eluent, collecting 1 tube every 5 minutes, and the total volume of elution is V1And detecting the collected liquid components in each tube, combining the components with sugar content, and freeze-drying to obtain the Ganoderma Applanatum fermented acidic polysaccharide. Precisely weighing 10mg of the acidic polysaccharide fermented by the tongue, fully dissolving 2mL of 0.15mol/L NaCl, loading the acidic polysaccharide into a gel chromatography column, eluting with 0.15mol/L NaCl at an elution flow rate of 4mL/15min, collecting 1 tube per 4mL, determining the sugar content of each tube by a phenol-sulfuric acid method, combining 46-60 tubes of eluates, and freeze-drying to obtain the acidic uniform polysaccharide of the tongue. The obtained uniform acidic polysaccharide of Ganoderma Applanatum is beta-type pyranose, contains 0.539% protein and 3.401% uronic acid, has a molecular weight of 4.6kDa, and is composed of mannose, glucuronic acid, glucose and galactose at a molar ratio of 2.997:2.140:12.097:1, and the glycosidic linkage is 1 → 4,6, 1 → 2, 6. The acidic homopolysaccharide of Ganoderma Applanatum can recognize with lymphocyte cell membrane, regulate immunity, andand the acidic polysaccharide of the Ganoderma Applanatum does not have nuclear translocation with the prolonging of the action time.
Example 2
Soaking Ganoderma Applanatum fermented extract in 94% ethanol at 6 deg.C for 24 hr, dissolving precipitate in 6 times of distilled water, centrifuging at 5000r/min for 10min to remove insoluble substances, extracting in 75 deg.C water bath for 3 hr, centrifuging to collect extractive solution, repeatedly extracting precipitate for 3 times, mixing extractive solutions, concentrating under reduced pressure, adding 95% ethanol into concentrated solution to final ethanol concentration of 70%, standing at 4 deg.C for 12 hr, centrifuging at 5000r/min for 10min, and collecting precipitate to obtain Ganoderma Applanatum fermented crude polysaccharide. Adding Sevag reagent (chloroform: n-butyl alcohol = 4: 1) into 5% of the crude polysaccharide aqueous solution obtained by fermenting the Ganoderma Applanatum, magnetically stirring for 60min, centrifuging, collecting the upper-layer polysaccharide solution, repeating the above operation until the upper-layer polysaccharide solution is centrifuged and denatured-free protein layer is obtained, combining the polysaccharide solutions, concentrating under reduced pressure until the volume of the solution is 1/4, and freeze-drying to obtain dry powder, namely the polysaccharide removed by fermenting the Ganoderma Applanatum. Accurately weighing 100mg of the fermentation deproteinized polysaccharide of the lingua, fully dissolving the fermentation deproteinized polysaccharide with 5mL of distilled water, loading the lingua to an ion exchange column, eluting with distilled water and 0.1M sodium chloride solution respectively at an elution flow rate of 1mL/min, eluting with 0.2M sodium chloride solution at a flow rate of 1mL/min, collecting 1 tube per 5mL, determining sugar content of each tube according to a phenol-sulfuric acid method, combining the polysaccharide solutions, and freeze-drying to obtain the lingua fermentation acidic polysaccharide. Precisely weighing 10mg of the Ganoderma Applanatum fermentation acidic polysaccharide, 2mL of 0.15mol/L NaCl for fully dissolving, loading to a gel chromatography column, eluting with 0.15mol/L NaCl at a flow rate of 4mL/15min, collecting 1 tube per 4mL, measuring the sugar content of each tube by a phenol-sulfuric acid method, combining 46-60 tubes of eluates, and freeze-drying to obtain the Ganoderma Applanatum acidic homogeneous polysaccharide. The obtained uniform acidic polysaccharide of Ganoderma Applanatum is beta-type pyranose, contains 0.507% protein and 3.531% uronic acid, has a molecular weight of 3.4kDa, and is composed of mannose, glucuronic acid, glucose and galactose at a molar ratio of 3.042:2.019:12.251:1, and glycosidic linkages are connected in a manner of 1 → 4,6, 1 → 2, 6. The acidic uniform polysaccharide of the Ganoderma Applanatum can be recognized with cell membrane of lymphocyte and regulate its immune function, and the acidic polysaccharide of the Ganoderma Applanatum has no nuclear translocation with prolonged action time.
Example 3
Soaking Ganoderma Applanatum fermented extract in 95% ethanol at 3 deg.C overnight, dissolving precipitate in 4 times of distilled water, 4500r/min, centrifuging for 10min to remove insoluble substances, extracting in 75 deg.C water bath for 3h, centrifuging to collect extractive solution, repeatedly extracting precipitate for 3 times, mixing extractive solutions, concentrating under reduced pressure, adding 95% ethanol into concentrated solution until ethanol concentration reaches 70%, standing at 4 deg.C overnight for 12h, and centrifuging at 5000r/min for 10min to collect precipitate to obtain Ganoderma Applanatum fermented crude polysaccharide. Adding Sevag reagent (chloroform: n-butyl alcohol = 4: 1) into 5% of the crude polysaccharide aqueous solution obtained by fermenting the Ganoderma Applanatum, magnetically stirring for 60min, centrifuging, collecting the upper-layer polysaccharide solution, repeating the above operation until the upper-layer polysaccharide solution is centrifuged and denatured-free protein layer is obtained, combining the polysaccharide solutions, concentrating under reduced pressure until the volume of the solution is 1/4, and freeze-drying to obtain dry powder, namely the polysaccharide removed by fermenting the Ganoderma Applanatum. Accurately weighing 100mg of the fermentation deproteinized polysaccharide of the lingua, fully dissolving the fermentation deproteinized polysaccharide with 5mL of distilled water, loading the lingua to an ion exchange column, eluting with distilled water and 0.1M sodium chloride solution respectively at an elution flow rate of 1mL/min, eluting with 0.2M sodium chloride solution at a flow rate of 1mL/min, collecting 1 tube per 5mL, determining sugar content of each tube according to a phenol-sulfuric acid method, combining the polysaccharide solutions, and freeze-drying to obtain the lingua fermentation acidic polysaccharide. Precisely weighing 10mg of the Ganoderma Applanatum fermentation acidic polysaccharide, 2mL of 0.15mol/L NaCl for fully dissolving, loading to a gel chromatography column, eluting with 0.15mol/L NaCl at a flow rate of 4mL/15min, collecting 1 tube per 4mL, measuring the sugar content of each tube by a phenol-sulfuric acid method, combining 46-60 tubes of eluates, and freeze-drying to obtain the Ganoderma Applanatum acidic homogeneous polysaccharide. The obtained uniform acidic polysaccharide of Ganoderma Applanatum is beta-type pyranose, contains 0.570% protein and 3.375% uronic acid, has a molecular weight of 5.8kDa, and is composed of mannose, glucuronic acid, glucose and galactose at a molar ratio of 2.867:2.111:11.097:1, and the glycosidic linkages are connected in a manner of 1 → 4,6, 1 → 2, 6. The acidic uniform polysaccharide of the Ganoderma Applanatum can be recognized with cell membrane of lymphocyte and regulate its immune function, and the acidic polysaccharide of the Ganoderma Applanatum has no nuclear translocation with prolonged action time.
This concludes the description of the embodiments of the present invention.
Claims (3)
1. A method for preparing an acidic homopolysaccharide from Ganoderma Applanatum capable of recognizing and regulating lymphocytes, comprising:
step one, preparing a Ganoderma Applanatum fermentation crude polysaccharide by taking Ganoderma Applanatum fermentation extract, water and ethanol as raw materials, and adding water into the Ganoderma Applanatum fermentation crude polysaccharide to prepare a Ganoderma Applanatum fermentation crude polysaccharide solution with the mass volume concentration of 5%;
wherein the preparation process of the Ganoderma Applanatum fermented crude polysaccharide comprises the following steps:
step 1, soaking the Ganoderma Applanatum fermentation extract in 94-95% ethanol solution to obtain solution and precipitate; wherein, the soaking temperature is as follows: the soaking time is 12 to 24 hours at the temperature of between 3 and 6 ℃;
step 2, adding water with the volume of 3-6 times of that of the precipitate to dissolve the precipitate, centrifuging at the rotating speed of 4000-5000 r/min, and filtering to obtain a Ganoderma Applanatum fermentation extract dissolved solution, wherein the centrifugation time is 10 min;
step 3, extracting the Ganoderma Applanatum fermentation extract solution obtained in step 2 in water bath at 70 ℃ for 3h, performing second centrifugation, extracting supernatant, collecting precipitate, extracting the collected precipitate in water bath at 75 ℃ for 3h, performing centrifugation, extracting supernatant again, collecting precipitate again, performing water bath extraction at 75 ℃ for 3h and centrifugation on the collected precipitate again, and collecting supernatant for the third time;
mixing the supernatant liquid obtained by the three-time extraction to prepare an extracting solution, and performing reduced pressure concentration on the extracting solution to prepare a concentrated solution with the relative density of 1.3-1.5;
step 4, adding 95% ethanol into the concentrated solution until the final concentration of the ethanol in the solution is 70%, standing in a refrigerator at 4 ℃ for 12 hours, centrifuging at 5000r/m for 10min, and collecting precipitate to obtain the Ganoderma Applanatum fermentation crude polysaccharide;
step two, adding a mixed solution of chloroform and n-butyl alcohol into the Ganoderma Applanatum fermentation crude polysaccharide solution, and concentrating and drying to obtain Ganoderma Applanatum fermentation deproteinized polysaccharide;
step three, preparing the fermentation deproteinized polysaccharide of the Ganoderma Applanatum into fermentation deproteinized polysaccharide solution with the concentration of 20g/L, purifying by adopting an ion exchange column chromatography method, eluting by using distilled water, 0.1mol/L NaCl solution and 0.2mol/L NaCl solution as eluent at the flow rate of 1mL/min in sequence, and dissolving the fermentation deproteinized polysaccharide of the Ganoderma ApplanatumEluting with 0.2mol/L NaCl solution, collecting the eluted part, collecting 1 tube every 5min, and collecting total elution volume V1Detecting the collected liquid components in each tube, combining the liquid components with sugar content, and freeze-drying to obtain the Ganoderma Applanatum fermented acidic polysaccharide;
wherein, the tube separation collection is not carried out after the first two times of elution, and the ion exchange column is a DEAE52 cellulose ion exchange column;
step four, adding 2mL of NaCl solution with the molar concentration of 0.15mol/L into every 10mg of the Ganoderma Applanatum fermented acidic polysaccharide to completely dissolve the Ganoderma Applanatum fermented acidic polysaccharide;
and carrying out molecular weight homogenization on the acidic polysaccharide obtained by fermentation of the Ganoderma Applanatum by using gel column chromatography, eluting the acidic polysaccharide obtained by fermentation of the Ganoderma Applanatum with 0.15mol/L NaCl solution as an eluent at an elution flow rate of 4mL/15min, collecting 1 tube per 4mL, and collecting the total elution volume of V2And combining the eluates of the 46 th to 60 th tubes, and freeze-drying to obtain the uniform acidic polysaccharide capable of identifying and regulating lymphocytes.
2. The method for preparing the ganoderma lucidum acidic homopolysaccharide capable of identifying and regulating lymphocytes according to claim 1, wherein the relative density of the ganoderma lucidum fermented extract is 1.4.
3. The method for preparing the uniform acidic polysaccharide of Ganoderma Applanatum capable of identifying and regulating lymphocytes according to claim 1, wherein the step two comprises the following steps:
step a, adding a mixed solution of chloroform and n-butanol into the Ganoderma Applanatum fermentation crude polysaccharide solution, and magnetically stirring for 60min to obtain a mixed polysaccharide solution;
wherein the volume ratio of the Ganoderma Applanatum fermentation crude polysaccharide solution to the mixed solution is 4:1, and the volume ratio of chloroform to n-butanol is 4: 1;
step b, carrying out centrifugal treatment on the mixed polysaccharide solution, and collecting an upper-layer solution;
and c, repeating the step a and the step b until no denatured protein layer is formed by centrifugation, combining polysaccharide solutions, concentrating the polysaccharide solutions under reduced pressure to 1/3 with the original volume, and freeze-drying to obtain the fermented proteoglycan-removed dry powder of the Ganoderma Applanatum.
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