CN112079940A - Enrichment and purification method and application of agrocybe cylindracea polysaccharide with blood sugar reducing function - Google Patents
Enrichment and purification method and application of agrocybe cylindracea polysaccharide with blood sugar reducing function Download PDFInfo
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Abstract
The invention discloses an enrichment and purification method of agrocybe cylindracea polysaccharide with a blood sugar reducing function, which comprises the steps of extracting crude polysaccharide from fresh agrocybe cylindracea, and then sequentially carrying out enrichment and purification by a macroporous resin column, enrichment and purification by ODS resin, and enrichment and purification by a DEAE-cellulose DE-52 column to obtain the agrocybe cylindracea polysaccharide. The method enriches the strongest functional components of the agrocybe cylindracea to a molecular arrangement section as the effective raw material components of the corresponding functional product, has high extraction rate and less loss of the effective components, can shorten the extraction and purification period, reduces the production cost, has less organic residues and impurities of the product and has high product purity. The polysaccharide component obtained by enrichment and purification by the method has the advantages of less loss of effective components, high content of functional substances, better effect and low production cost.
Description
Technical Field
The invention belongs to the technical field of edible fungi polysaccharide extraction, and particularly relates to an enrichment and purification method and application of agrocybe cylindracea polysaccharide with a blood sugar reducing function.
Background
Agrocybe cylindracea (Agrocybe aegerita), also known as Pleurotus citrinopileatus, belongs to Basidiomycetes, Agaricales, and Farinoraceae, and is originally a wild mushroom growing in the stump of tea trees in mountain dense forest region in Richuan of Jiangxi, is a wood-rotting fungus growing in subtropical regions, has delicious taste and crisp mouthfeel, and is known as "Chinese mushroom".
The agrocybe aegerita is rich in nutrition, the protein content is up to 19.55%, the contained protein contains 18 amino acids, the highest content of methionine accounts for 2.49%, the second content of glutamic acid, aspartic acid, isoleucine, glycine and alanine, and the total amino acid content is 16.86%. Contains 18 kinds of amino acids essential for human body, especially 8 kinds of amino acids, dextran, mycoprotein, carbohydrate, etc. which are not synthesized by human body, and has crisp, tender and refreshing mushroom stem and fragrant taste. The mushroom has the effects of tonifying kidney, promoting urination, treating waist soreness and pain, excreting dampness, strengthening spleen, stopping diarrhea and the like, and is an ideal food for patients with hypertension, cardiovascular diseases and obesity. The edible fungus is delicious, crisp, tender and tasty, has better health care function, and is one of delicious and rare edible fungi.
The traditional Chinese medicine considers that the traditional Chinese medicine has better effects of tonifying kidney, promoting urination, strengthening spleen, stopping dirts, treating waist soreness, excreting dampness and the like, considers that the traditional Chinese medicine has the effects of tonifying kidney, strengthening yang, clearing stomach, moistening lung, reducing blood pressure, calming heart, restoring consciousness, preventing senility of middle-aged and elderly people, has obvious curative effects on frequent micturition, edema, asthma, child bedwetting, low fever, detoxification and the like caused by kidney deficiency, is an ideal food for patients with hypertension, cardiovascular diseases and obesity, and has very high edible and medicinal values. The agrocybe cylindracea contains a large amount of anticancer polysaccharide and has a good anticancer effect, and is called as 'China shiitake', 'health food' and 'anticancer keen'.
In recent years, researches on tea trees are more and more intensive, and modern pharmacological researches show that the tea mushroom polysaccharide has the effects of resisting tumors and aging, enhancing immunity, reducing cholesterol and the like. In addition, in recent years, many researches on extraction and biological activity of the agrocybe aegerita polysaccharide are reported, most of the activity researches are focused on the aspects of oxidation resistance and tumor resistance, and few researches are focused on the aspect of purification of active substances, and particularly, the researches are rarely reported on the aspect of blood sugar reduction of the agrocybe aegerita polysaccharide. However, the existing agrocybe aegerita polysaccharide extraction process mainly comprises the following steps: traditional hot water extraction, dilute acid (alkali) extraction, enzymolysis extraction, ultrasonic-assisted extraction, enzyme method, etc.
1. Patent application CN201811190569.3 discloses a high-efficiency agrocybe aegerita polysaccharide extraction method, which comprises the following steps: pretreating Agrocybe aegerita, freezing and pulverizing at ultralow temperature, extracting Agrocybe aegerita polysaccharide, microfiltering, ultrafiltering, concentrating for the first time, removing protein, and concentrating for the second time. The invention has simple process, low energy consumption, low cost and high polysaccharide yield; the invention adopts a series microporous filter membrane and an ultrafiltration membrane to filter the extracting solution, thereby achieving the purpose of refining and purifying crude polysaccharide; vacuum low-temperature drying and ultralow-temperature freezing crushing are adopted, the obtained agrocybe aegerita powder is obviously lighter in color compared with a product obtained by crushing at normal temperature, the polysaccharide loss rate of ultralow-temperature freezing crushing is less than 0.3%, and the polysaccharide loss rate is reduced by more than 15% compared with normal-temperature crushing. The method soaks the agrocybe aegerita superfine powder in a pressurized environment and is matched with normal-pressure ultrasonic soaking, so that more polysaccharides can be soaked at a lower temperature, and the polysaccharides can be effectively separated.
2. Patent application CN201611045179.8 discloses a method for extracting polysaccharide from agrocybe cylindracea, which comprises (1) cleaning raw materials, cutting into small pieces, drying, and grinding into powder; (2) rehydration of raw materials: weighing the powder, putting into a container, adding a proper amount of water, and soaking for 20-35 min to obtain a rehydrated slurry; (3) extraction: adding distilled water into the rehydrated slurry to ensure that the leaching ratio is 1:10-30 respectively, putting the container into a boiling water bath, steaming for 1-3h, cooling, pouring the leaching solution into a centrifuge tube, and centrifuging; (4) purifying polysaccharide; (5) alcohol precipitation and drying of the polysaccharide: pouring the extract into a container, adding 95% ethanol to reach 50% -80% saturation, standing for 24h, centrifuging, collecting precipitate, and freeze-drying to obtain polysaccharide finished product. The method shortens extraction time, increases extraction rate of effective components, and has the advantages of simple process, easy operation, and high extraction rate.
3. Patent application CN201510186384.5 discloses an extraction and separation method of agrocybe cylindracea polysaccharide, which comprises the following steps: (1) slicing fresh agrocybe aegerita, pre-cooling, freezing and drying after the agrocybe aegerita is frozen; (2) freeze-drying, rapidly pulverizing Agrocybe aegerita slices, adding ethanol water solution into Agrocybe aegerita powder, ultrasonic stirring for extraction, centrifuging the extractive solution, and collecting supernatant; (3) concentrating the supernatant until the weight of the supernatant is 2 times of that of the agrocybe cylindracea; (4) performing membrane filtration on the concentrated solution of the agrocybe cylindracea, passing through a membrane with the molecular weight of 1000, and collecting trapped fluid; (5) passing the trapped fluid through a membrane with the molecular weight of 200, and collecting the trapped fluid; (6) concentrating until no solvent exists; (7) crystallizing and purifying the concentrate with ethanol, filtering, and naturally drying to obtain Agrocybe aegerita polysaccharide. The extraction and separation method of the agrocybe cylindracea polysaccharide improves the extraction process and the separation process of the agrocybe cylindracea, adopts the ethanol water solution to extract the agrocybe cylindracea polysaccharide, can also remove impurities while ensuring the extraction rate, further separates the agrocybe cylindracea polysaccharide through membrane filtration, and finally improves the purity of the agrocybe cylindracea polysaccharide prepared while ensuring the yield through the mode of ethanol crystallization and purification.
However, the prior art separation and purification technology aims at all polysaccharides of agrocybe cylindracea and does not aim at a purification method of a certain function, so the prior art separation and purification technology has the defects of large loss of effective components and the like, and the yield and the purity of functional substances are reduced. The inventor of the invention enriches and purifies the hypoglycemic target component of the agrocybe aegerita on the basis of the hypoglycemic activity of the agrocybe aegerita crude polysaccharide determined in earlier research, and tries to provide a method for efficiently enriching and purifying the active polysaccharide, so that the method is popularized and applied and the industrial development is promoted.
Disclosure of Invention
The invention provides an enrichment and purification method and application of agrocybe cylindracea polysaccharide with the function of reducing blood sugar, aiming at solving the technical problems. According to the application, the functional components of the agrocybe cylindracea are enriched to a molecular arrangement section to serve as the effective raw material components of the corresponding functional product, and the polysaccharide component obtained through enrichment and purification by the method is high in purity, better in effect, less in loss of the effective components and low in production cost.
In order to achieve the above purpose, the invention adopts the following technical scheme:
an enrichment and purification method of agrocybe cylindracea polysaccharide with the function of reducing blood sugar is characterized in that crude polysaccharide is extracted from fresh agrocybe cylindracea, and then the crude polysaccharide is subjected to enrichment and purification by a macroporous resin column, ODS resin and DEAE-cellulose DE-52 column in sequence to prepare the agrocybe cylindracea polysaccharide, and specifically comprises the following steps:
(1) sample pretreatment: placing fresh agrocybe aegerita in an oven for drying, then sending the dried agrocybe aegerita into a grinder for grinding and sieving to obtain agrocybe aegerita powder;
(2) polysaccharide extraction: placing the agrocybe aegerita powder in the step (1) in a reaction device, adding distilled water into the reaction device, soaking at normal temperature, then carrying out reflux extraction in a water bath, filtering, adding distilled water into filter residues, repeatedly extracting for 2 times, and combining filtrates to obtain a polysaccharide extracting solution;
(3) alcohol precipitation: carrying out suction filtration on the polysaccharide extracting solution in the step (2), carrying out reduced pressure concentration to obtain an extract, completely dissolving the extract into a crude extract solution by using distilled water, adding absolute ethyl alcohol into the dissolved solution while stirring, standing overnight, filtering to remove a supernatant, collecting a precipitate, repeatedly washing for 2 times by using the absolute ethyl alcohol, and carrying out vacuum freeze drying to obtain crude polysaccharide;
(4) enrichment and purification: and (4) dissolving the crude polysaccharide obtained in the step (3) into a solution by using distilled water, and sequentially purifying by using a macroporous resin column, an ODS resin and a DEAE-cellulose DE-52 column to obtain the agrocybe aegerita polysaccharide.
Further, the macroporous resin column purification is: performing AB-8 type macroporous resin column chromatography, loading the crude polysaccharide and resin on a column by a wet method according to the mass ratio of 1: 29-31, standing and adsorbing for 55-65 min, eluting with distilled water, performing polysaccharide tracking detection by a phenol-sulfuric acid method until sugar-free components flow out, eluting with 10%, 30%, 50% and 70% ethanol at the elution flow rate of 1.9-2.1 mL/h, concentrating the distilled water eluate under reduced pressure, and performing vacuum freeze drying to obtain the macroporous resin column purified product (Fr-A).
Further, the ODS resin purification is: weighing dry powder purified by a macroporous resin column, dissolving the dry powder in distilled water to form a solution, loading Fr-A and resin on the column by a wet method according to the mass ratio of 1: 49-51, fully adsorbing the solution for 55-65 min by an ODS resin column, eluting with distilled water, performing polysaccharide tracking detection by adopting a phenol-sulfuric acid method until sugar-free components flow out, eluting with 20% by mass, 40% by mass and 60% by mass of ethanol at the elution flow rate of 1.9-2.1 mL/h, collecting the distilled water eluent, concentrating under reduced pressure, and performing vacuum freeze drying to obtain ODS resin purified product (Fr-B).
Further, the DEAE-cellulose DE-52 column purification is as follows: weighing dry powder purified by ODS resin, dissolving the dry powder in distilled water to form a solution, loading the solution on a DEAE-cellulose DE-52 column by a wet method, sequentially carrying out gradient elution by using distilled water, 0.1M NaCl solution, 0.3M NaCl solution and 0.5M NaCl solution at the flow rate of 1.9-2.1 mL/h, respectively collecting each fraction, tracking and detecting the eluent by using a phenol-sulfuric acid method until sugar-free groups flow out, respectively dialyzing each fraction by using distilled water, concentrating the solution in a dialysis bag under reduced pressure, and respectively carrying out vacuum freeze drying to respectively obtain Fr-B1, Fr-B2, Fr-B3 and Fr-B4, thus obtaining the agrocybe polysaccharides.
The Agrocybe aegerita polysaccharide is Fr-B1, Fr-B2, Fr-B3 or Fr-B4.
Further, in the step (1), the drying temperature is 50-53 ℃, the crushing time is 2-3 min, and the sieving is performed by a 50-70 mesh sieve.
Further, in the step (2), the material-liquid ratio of the agrocybe aegerita powder to the distilled water is 1: 19-21, the soaking time is 28-33 min, the extraction temperature is 98-103 ℃, and the extraction time is 115-125 min.
Further, in the step (3), the final concentration of the ethanol is 85%, the standing time is 11.5-13 h, and the temperature is 4 ℃.
Further, the temperature of the reduced pressure concentration is 48-53 ℃, the temperature of the freeze drying is-55-50 ℃, and the vacuum degree is 0-100 Pa.
The application of the agrocybe cylindracea polysaccharide with the function of reducing blood sugar, which is prepared by the enrichment and purification method, in preparing blood sugar-reducing medicines, health-care products or foods.
The application of the agrocybe cylindracea polysaccharide prepared by the extraction, enrichment and purification method in the product with the function of reducing blood sugar.
One of the purposes of the invention is to provide a high-purity enrichment and purification method of agrocybe cylindracea polysaccharide with effectiveness. The second purpose of the invention is to provide the agrocybe aegerita polysaccharide which can be applied to the preparation of hypoglycemic functional products such as hypoglycemic drugs and health care products; the Agrocybe aegerita polysaccharide is selected from one or more of Fr-B1, Fr-B2, Fr-B3 and Fr-B4.
Due to the adoption of the technical scheme, the invention has the following beneficial effects:
(1) according to the method for extracting, enriching and purifying the agrocybe aegerita polysaccharide, the agrocybe aegerita polysaccharide is obtained through pretreatment, hot water extraction and alcohol precipitation, four agrocybe aegerita polysaccharides with different components and a mixture are obtained through enrichment and purification, and the obtained mixture has more obvious hypoglycemic activity on the agrocybe aegerita polysaccharide.
(2) The polysaccharide component prepared by the method has more remarkable biological activity, the separation, enrichment and purification method is simple to operate, low in cost, less in organic residue and high in purity, resource waste caused by improper operation is avoided, the additional value of the agrocybe cylindracea is increased, the polysaccharide component obtained by purification is beneficial to subsequent deep development and utilization research on the edible and medicinal dual-purpose value of the agrocybe cylindracea, and the development of the agrocybe cylindracea polysaccharide industry is promoted.
(3) The method enriches the strongest functional components of the agrocybe cylindracea into a molecular arrangement section to serve as the effective raw material components of the corresponding functional product, and the polysaccharide component obtained by enrichment and purification by the method has the advantages of low loss of the effective components, high content of functional substances, better effect and low production cost.
(4) According to the method, the agrocybe cylindracea polysaccharide is extracted by adopting a reflux extraction and alcohol precipitation mode, the extraction rate of the crude polysaccharide is improved, and the crude polysaccharide obtained by extraction is enriched and purified by sequentially adopting a macroporous resin column, ODS resin and a DEAE-cellulose DE-52 column, so that the purity of the obtained agrocybe cylindracea functional polysaccharide is higher.
Detailed Description
The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.
Example 1
An enrichment and purification method of agrocybe cylindracea polysaccharide with the function of reducing blood sugar is characterized in that crude polysaccharide is extracted from fresh agrocybe cylindracea, and then the crude polysaccharide is subjected to enrichment and purification by a macroporous resin column, ODS resin and DEAE-cellulose DE-52 column in sequence to prepare the agrocybe cylindracea polysaccharide, and specifically comprises the following steps:
(1) sample pretreatment: placing fresh agrocybe aegerita in an oven for drying, then sending the dried agrocybe aegerita into a grinder for grinding and sieving to obtain agrocybe aegerita powder; the drying temperature is 50 ℃, the crushing time is 2min, and the sieving is a 50-mesh sieve;
(2) polysaccharide extraction: placing the agrocybe aegerita powder in the step (1) in a reaction device, adding distilled water into the reaction device, soaking at normal temperature, then carrying out reflux extraction in a water bath, filtering, adding distilled water into filter residues, repeatedly extracting for 2 times, and combining filtrates to obtain a polysaccharide extracting solution; the material-liquid ratio of the agrocybe cylindracea powder to the distilled water is 1:19, the soaking time is 28min, the extraction temperature is 98 ℃, and the extraction time is 115 min;
(3) alcohol precipitation: carrying out suction filtration on the polysaccharide extracting solution in the step (2), carrying out reduced pressure concentration to obtain an extract, completely dissolving the extract into a crude extract solution by using distilled water, adding absolute ethyl alcohol into the dissolved solution while stirring, standing overnight, filtering to remove a supernatant, collecting a precipitate, repeatedly washing for 2 times by using the absolute ethyl alcohol, and carrying out vacuum freeze drying to obtain crude polysaccharide; the concentration of the absolute ethyl alcohol is 85%, the standing time is 11.5h, and the temperature is 4 ℃;
(4) enrichment and purification: and (4) dissolving the crude polysaccharide obtained in the step (3) into a solution by using distilled water, and sequentially purifying by using a macroporous resin column, an ODS resin and a DEAE-cellulose DE-52 column to obtain the agrocybe aegerita polysaccharide.
The macroporous resin column purification comprises the following steps: performing AB-8 type macroporous resin column chromatography, loading the crude polysaccharide and resin on a column by a wet method according to the mass ratio of 1:29, standing and adsorbing for 55min, eluting with distilled water, performing polysaccharide tracking detection by a phenol-sulfuric acid method until sugar-free components flow out, eluting with 10%, 30%, 50% and 70% ethanol at the elution flow rate of 1.9mL/h, concentrating the distilled water eluate under reduced pressure, and performing vacuum freeze drying to obtain a macroporous resin column purified product (Fr-A).
The ODS resin purification is: weighing dry powder purified by a macroporous resin column, dissolving the dry powder in distilled water to form a solution, loading Fr-A and resin on the column by a wet method according to the mass ratio of 1:49, fully adsorbing the solution for 55min by an ODS resin column, eluting the solution by distilled water, performing polysaccharide tracking detection by a phenol-sulfuric acid method until sugar-free components flow out, eluting the solution by 20% ethanol, 40% ethanol and 60% ethanol at the elution flow rate of 1.9mL/h, collecting the distilled water eluent, concentrating the distilled water eluent under reduced pressure, and performing vacuum freeze drying to obtain the ODS resin purified product (Fr-B).
The DEAE-cellulose DE-52 column purification comprises the following steps: weighing dry powder purified by ODS resin, dissolving the dry powder in distilled water to form a solution, loading the solution on a DEAE-cellulose DE-52 column by a wet method, performing gradient elution by using distilled water, 0.1M NaCl solution, 0.3M NaCl solution and 0.5M NaCl solution in sequence at a flow rate of 1.9mL/h, respectively collecting each fraction, tracking and detecting eluent by using a phenol-sulfuric acid method until sugar-free groups flow out, respectively dialyzing each fraction by using distilled water, concentrating the solution in a dialysis bag under reduced pressure, and respectively performing vacuum freeze drying to respectively obtain Fr-B1, Fr-B2, Fr-B3 and Fr-B4, thus obtaining the agrocybe polysaccharides; the Agrocybe aegerita polysaccharide is Fr-B1, Fr-B2, Fr-B3 or Fr-B4.
The temperature of the reduced pressure concentration is 48 ℃, the temperature of the freeze drying is-50 ℃, and the vacuum degree is 10 Pa.
The application of the agrocybe cylindracea polysaccharide with the function of reducing blood sugar, which is prepared by the enrichment and purification method, in preparing blood sugar-reducing medicines, health-care products or foods.
Example 2
An enrichment and purification method of agrocybe cylindracea polysaccharide with the function of reducing blood sugar is characterized in that crude polysaccharide is extracted from fresh agrocybe cylindracea, and then the crude polysaccharide is subjected to enrichment and purification by a macroporous resin column, ODS resin and DEAE-cellulose DE-52 column in sequence to prepare the agrocybe cylindracea polysaccharide, and specifically comprises the following steps:
(1) sample pretreatment: placing fresh agrocybe aegerita in an oven for drying, then sending the dried agrocybe aegerita into a grinder for grinding and sieving to obtain agrocybe aegerita powder; the drying temperature is 53 ℃, the crushing time is 3min, and the sieving is a 70-mesh sieve;
(2) polysaccharide extraction: placing the agrocybe aegerita powder in the step (1) in a reaction device, adding distilled water into the reaction device, soaking at normal temperature, then carrying out reflux extraction in a water bath, filtering, adding distilled water into filter residues, repeatedly extracting for 2 times, and combining filtrates to obtain a polysaccharide extracting solution; the material-liquid ratio of the agrocybe cylindracea powder to the distilled water is 1:21, the soaking time is 33min, the extraction temperature is 103 ℃, and the extraction time is 125 min;
(3) alcohol precipitation: carrying out suction filtration on the polysaccharide extracting solution in the step (2), carrying out reduced pressure concentration to obtain an extract, completely dissolving the extract into a crude extract solution by using distilled water, adding absolute ethyl alcohol into the dissolved solution while stirring, standing overnight, filtering to remove a supernatant, collecting a precipitate, repeatedly washing for 2 times by using the absolute ethyl alcohol, and carrying out vacuum freeze drying to obtain crude polysaccharide; the concentration of the absolute ethyl alcohol is 85%, the standing time is 13h, and the temperature is 4 ℃;
(4) enrichment and purification: and (4) dissolving the crude polysaccharide obtained in the step (3) into a solution by using distilled water, and sequentially purifying by using a macroporous resin column, an ODS resin and a DEAE-cellulose DE-52 column to obtain the agrocybe aegerita polysaccharide.
The macroporous resin column purification comprises the following steps: performing AB-8 type macroporous resin column chromatography, loading the crude polysaccharide and resin on a column by a wet method according to the mass ratio of 1:31, standing and adsorbing for 65min, eluting with distilled water, performing polysaccharide tracking detection by a phenol-sulfuric acid method until sugar-free components flow out, eluting with 10%, 30%, 50% and 70% ethanol at the elution flow rate of 2.1mL/h, concentrating the distilled water eluate under reduced pressure, and performing vacuum freeze drying to obtain the macroporous resin column purified product (Fr-A).
The ODS resin purification is: weighing dry powder purified by a macroporous resin column, dissolving the dry powder in distilled water to form a solution, loading Fr-A and resin on the column by a wet method according to the mass ratio of 1:51, fully adsorbing the solution for 65min by an ODS resin column, eluting the solution by distilled water, performing polysaccharide tracking detection by a phenol-sulfuric acid method until sugar-free components flow out, eluting the solution by 20% ethanol, 40% ethanol and 60% ethanol at the elution flow rate of 2.1mL/h, collecting the distilled water eluent, concentrating the distilled water eluent under reduced pressure, and performing vacuum freeze drying to obtain the ODS resin purified product (Fr-B).
The DEAE-cellulose DE-52 column purification comprises the following steps: weighing dry powder purified by ODS resin, dissolving the dry powder in distilled water to form a solution, loading the solution on a DEAE-cellulose DE-52 column by a wet method, performing gradient elution by using distilled water, 0.1M NaCl solution, 0.3M NaCl solution and 0.5M NaCl solution in sequence at a flow rate of 2.1mL/h, respectively collecting each fraction, tracking and detecting eluent by using a phenol-sulfuric acid method until sugar-free groups flow out, respectively dialyzing each fraction by using distilled water, concentrating the solution in a dialysis bag under reduced pressure, and respectively performing vacuum freeze drying to respectively obtain Fr-B1, Fr-B2, Fr-B3 and Fr-B4, thus obtaining the agrocybe polysaccharides; the Agrocybe aegerita polysaccharide is Fr-B1, Fr-B2, Fr-B3 or Fr-B4.
The temperature of the reduced pressure concentration is 53 ℃, the temperature of the freeze drying is-55 ℃, and the vacuum degree is 100 Pa.
The application of the agrocybe cylindracea polysaccharide with the function of reducing blood sugar, which is prepared by the enrichment and purification method, in preparing blood sugar-reducing medicines, health-care products or foods.
Example 3
An enrichment and purification method of agrocybe cylindracea polysaccharide with the function of reducing blood sugar is characterized in that crude polysaccharide is extracted from fresh agrocybe cylindracea, and then the crude polysaccharide is subjected to enrichment and purification by a macroporous resin column, ODS resin and DEAE-cellulose DE-52 column in sequence to prepare the agrocybe cylindracea polysaccharide, and specifically comprises the following steps:
(1) sample pretreatment: placing fresh agrocybe aegerita in an oven for drying, then sending the dried agrocybe aegerita into a grinder for grinding and sieving to obtain agrocybe aegerita powder; the drying temperature is 50 ℃, the crushing time is 2.5min, and the sieving is a 60-mesh sieve;
(2) polysaccharide extraction: placing the agrocybe aegerita powder in the step (1) in a reaction device, adding distilled water into the reaction device, soaking at normal temperature, then carrying out reflux extraction in a water bath, filtering, adding distilled water into filter residues, repeatedly extracting for 2 times, and combining filtrates to obtain a polysaccharide extracting solution; the material-liquid ratio of the agrocybe cylindracea powder to the distilled water is 1:20, the soaking time is 30min, the extraction temperature is 100 ℃, and the extraction time is 120 min;
(3) alcohol precipitation: carrying out suction filtration on the polysaccharide extracting solution in the step (2), carrying out reduced pressure concentration to obtain an extract, completely dissolving the extract into a crude extract solution by using distilled water, adding absolute ethyl alcohol into the dissolved solution while stirring, standing overnight, filtering to remove a supernatant, collecting a precipitate, repeatedly washing for 2 times by using the absolute ethyl alcohol, and carrying out vacuum freeze drying to obtain crude polysaccharide; the concentration of the absolute ethyl alcohol is 85%, the standing time is 12h, and the temperature is 4 ℃;
(4) enrichment and purification: and (4) dissolving the crude polysaccharide obtained in the step (3) into a solution by using distilled water, and sequentially purifying by using a macroporous resin column, an ODS resin and a DEAE-cellulose DE-52 column to obtain the agrocybe aegerita polysaccharide.
The macroporous resin column purification comprises the following steps: performing AB-8 type macroporous resin column chromatography, loading the crude polysaccharide and resin on a column by a wet method according to the mass ratio of 1:30, performing static adsorption for 60min, eluting with distilled water, performing polysaccharide tracking detection by a phenol-sulfuric acid method until sugar-free components flow out, eluting with 10%, 30%, 50% and 70% ethanol at the elution flow rate of 2mL/h, concentrating the distilled water eluate under reduced pressure, and performing vacuum freeze drying to obtain a macroporous resin column purified product (Fr-A).
The ODS resin purification is: weighing dry powder purified by a macroporous resin column, dissolving the dry powder in distilled water to form a solution, loading Fr-A and resin on the column by a wet method according to the mass ratio of 1:50, fully adsorbing the solution for 60min by an ODS resin column, eluting by distilled water, performing polysaccharide tracking detection by a phenol-sulfuric acid method until sugar-free components flow out, eluting by 20%, 40% and 60% ethanol by mass at the elution flow rate of 2mL/h, collecting distilled water eluent, concentrating under reduced pressure, and performing vacuum freeze drying to obtain an ODS resin purified product (Fr-B).
The DEAE-cellulose DE-52 column purification comprises the following steps: weighing dry powder purified by ODS resin, dissolving the dry powder in distilled water to form a solution, loading the solution on a DEAE-cellulose DE-52 column by a wet method, performing gradient elution by using distilled water, 0.1M NaCl solution, 0.3M NaCl solution and 0.5M NaCl solution in sequence at a flow rate of 2mL/h, respectively collecting each fraction, tracking and detecting eluent by using a phenol-sulfuric acid method until sugar-free components flow out in groups, respectively dialyzing each fraction by using distilled water, concentrating the solution in a dialysis bag under reduced pressure, and respectively performing vacuum freeze drying to respectively obtain Fr-B1, Fr-B2, Fr-B3 and Fr-B4, thus obtaining the agrocybe polysaccharides; the Agrocybe aegerita polysaccharide is Fr-B1, Fr-B2, Fr-B3 or Fr-B4.
The temperature of the reduced pressure concentration is 50 ℃, the temperature of the freeze drying is-50 ℃, and the vacuum degree is 50 Pa.
The application of the agrocybe cylindracea polysaccharide with the function of reducing blood sugar, which is prepared by the enrichment and purification method, in preparing blood sugar-reducing medicines, health-care products or foods.
Example 4
An enrichment and purification method of agrocybe cylindracea polysaccharide with the function of reducing blood sugar is characterized in that crude polysaccharide is extracted from fresh agrocybe cylindracea, and then the crude polysaccharide is subjected to enrichment and purification by a macroporous resin column, ODS resin and DEAE-cellulose DE-52 column in sequence to prepare the agrocybe cylindracea polysaccharide, and specifically comprises the following steps:
(1) collecting fresh agrocybe aegerita, cutting off 2cm of roots, drying in an oven at 50 ℃, crushing by a crusher, and sieving by a 60-mesh sieve to obtain agrocybe aegerita powder;
(2) weighing 100g of agrocybe aegerita powder in the step (1), placing the agrocybe aegerita powder in a round-bottom flask, taking distilled water as a solvent, adding distilled water according to a material-liquid ratio of 1:20, soaking for 30min, then placing the mixture in a water bath kettle, performing reflux extraction for 2h at 100 ℃, filtering an extracting solution, continuously extracting residues for three times, combining extracting solutions for three times, performing suction filtration, vacuum-pumping, vacuum-reducing and concentrating the extracting solution at 50 ℃ to obtain an extract, wherein the extract yield is 70.5;
(3) adding distilled water into the extract obtained in the step (2), stirring while adding until the extract is completely dissolved, adding absolute ethanol into the solution while stirring until the ethanol concentration is 85%, standing overnight at 4 ℃ for 12h, filtering to remove supernatant, and collecting precipitate;
(4) carrying out vacuum freeze drying on the precipitate obtained in the step (3) at the temperature of-50 ℃ and the vacuum degree of 0-100Pa to obtain crude agrocybe cylindracea polysaccharide; the extraction rate of the crude polysaccharide is 13.4 percent, and the polysaccharide content is 36.5 percent;
(5) primary enrichment and purification by macroporous resin: soaking AB-type resin in 80% ethanol to fully expand, and filling resin with a volume of 100cm3Washing the ethanol in the resin completely with distilled water, weighing the dried crude agrocybe aegerita polysaccharide powder obtained in the step (4) to prepare a 10% solution, loading the sample and the resin on a column by a wet method according to the mass ratio of 1:30, standing and adsorbing for 60min, eluting with distilled water, performing polysaccharide tracking detection by adopting a phenol-sulfuric acid method until sugar-free components flow out, eluting with 10%, 30%, 50% and 70% ethanol at the elution flow rate of 2mL/h, concentrating the eluent of the distilled water under reduced pressure, and freeze-drying the eluent to obtain a distilled water fraction (Fr-A) with the polysaccharide content of 40.5%;
(6) enrichment and purification of ODS resin: filling the resin into a container of 100cm3Weighing Fr-A obtained in the step (5) to prepare a 10% sample solution, loading the sample solution on a wet method column according to the mass ratio of 1:50, fully adsorbing the sample solution for 1 hour through an ODS resin column, eluting the sample solution by using distilled water, performing polysaccharide tracking detection by using a phenol-sulfuric acid method until sugar-free components flow out, eluting the sample solution by using 20%, 40% and 60% ethanol in mass fraction at the elution flow rate of 2mL/h, collecting the distilled water eluent, concentrating the distilled water eluent under reduced pressure, and freeze-drying the distilled water eluent into powder to obtain a distilled water fraction (Fr-B) with the polysaccharide content of 45.6%;
(7) DEAE-cellulose DE-52 enrichment and purification: and (3) weighing the Fr-B powder obtained in the step (6), dissolving the Fr-B powder in distilled water to prepare a 5% solution, loading the solution onto a DEAE-cellulose DE-52 column by a wet method according to the mass ratio of 1:50, performing gradient elution by using distilled water and 0.1M, 0.3M and 0.5M NaCl solutions in sequence at the flow rate of 2mL/h, respectively collecting each fraction, tracking and detecting the eluent by using a phenol-sulfuric acid method until sugar-free groups flow out, dialyzing each fraction by using distilled water, concentrating the liquid in a dialysis bag under reduced pressure, and performing vacuum freeze drying to respectively obtain Fr-B1, Fr-B2, Fr-B3 and Fr-B4, wherein the polysaccharide content of Fr-B1, Fr-B2, Fr-B3 and Fr-B4 is 76.3%, 70.5%, 67.2% and 65.7%.
Comparative example 1
Agrocybe aegerita polysaccharides were obtained according to the method of the example in patent application CN 201811190569.3.
Comparative example 2
Agrocybe aegerita polysaccharides were obtained according to the method of the example in patent application CN 201611045179.8.
Comparative example 3
Agrocybe aegerita polysaccharides were obtained according to the method of the example in patent application CN 201510186384.5.
To further illustrate that the present invention can achieve the technical effects, the following experiments were performed:
the method for measuring the content of the agrocybe cylindracea polysaccharide comprises the following steps:
the phenol-sulfuric acid method is a commonly used polysaccharide detection method and is based on the principle that polysaccharide macromolecules are dehydrated to generate furfural and derivatives thereof and react with phenol at a high temperature when concentrated sulfuric acid is added into water.
Drawing a standard curve: weighing dried glucose with constant weight to prepare a glucose standard solution with the concentration of 50mg/mL, weighing solutions of standard products of 5uL, 15uL, 25uL, 35uL and 45uL to prepare solutions with the concentrations of 0.05mg/mL, 0.015mg/mL, 0.025mg/mL, 0.035mg/mL and 0.045mg/mL respectively in a 10mL volumetric flask, adding 1mL of the prepared standard solution into a test tube respectively, adding 1mL and 6% of phenol solution into each test tube respectively, adding 5mL of concentrated sulfuric acid into each test tube, shaking uniformly, standing for 10min, measuring each absorbance value at 490nm, and drawing a curve according to the glucose concentration to obtain a regression equation y of 8.829x +0.0144, R2 of 0.9993, wherein y is each absorbance value, and x is the glucose concentration (mg/mL).
Determination of polysaccharide content in samples: weighing a sample to prepare a sample solution of 5mg/mL, putting 20uL of the sample solution into a test tube, adding distilled water to complement to 1mL, then adding 1mL of 6% phenol solution and 5mL of concentrated sulfuric acid into the test tube, shaking uniformly, standing for 10min, measuring the absorbance at 490nm, measuring the absorbance of each sample for three times, and substituting the absorbance mean value into a regression equation to calculate the glucose content in the sample.
Secondly, the method for determining the hypoglycemic activity of the agrocybe cylindracea polysaccharide comprises the following steps:
(1) test article: the four polysaccharide components Fr-B1, Fr-B2, Fr-B3 and Fr-B4 of the agrocybe cylindracea prepared in the example 4, crude polysaccharide and component mixed polysaccharide (prepared by mixing Fr-B1, Fr-B2, Fr-B3 and Fr-B4 according to the mass ratio of 1:1:1: 1) are prepared into 3mg/mL solution for later use;
(2) determination of inhibition of alpha-amylase Activity by Components
The effect of each component on alpha-amylase activity was determined using the dinitrosalicylic acid (DNS) method: adding 0.5mL, 0.5% soluble starch solution and 4mL of DNS as blanks to 2mL of phosphate buffer solution (pH6.8), and taking 1mL of buffer solution, 1mL of 1% alpha-amylase solution, 0.5mL of 0.5% starch and 4mL of DNS as a control group to measure A;
taking 1mL of a sample solution to be tested, adding PBS (phosphate buffer solution) to a constant volume of 2mL, adding 1mL of alpha-amylase solution, shaking up, putting the mixture into a 37 ℃ water bath, preheating for 10min, adding 0.5mL of 0.5% starch solution, reacting for 10min in a 37 ℃ constant temperature water bath, taking out, adding 4mL of DNS solution, putting the mixture into a boiling water bath for 5min, and cooling to obtain a value A1 at 540 nm;
taking another group of sample solution to be tested with a proper concentration as a background group, adding buffer solution to fix the volume to 3mL, placing in a 37 ℃ water bath for preheating for 10min, adding 0.5mL of PBS, placing in a 37 ℃ water bath for 10min, adding 4mL of PBS solution, placing in a boiling water bath for 5min, cooling, and measuring the light absorption value A0 at 540 nm;
three parallels are made for each group of experiments, the average value of the final value is taken for three times, and the inhibition rate of the test object is calculated;
(3) determination of inhibition effect of each component on alpha-glucosidase activity
The influence of each component on the activity of the alpha-glucosidase is measured by a 4-nitrophenol-alpha-D-glucopyranoside (PNPG) colorimetric method: to 4mL of phosphate buffer (pH6.8) were added 1mL of 0.1mmol/L PNPG and 1mL of 0.1mol/L Na2CO3The solution is used as a blank zero adjustment group;
to add 1mL of buffer solution and 20mg/mL of alpha-glucanGlucosidase 1mL PNPG and 1mL Na2CO3The solution is a control group, and A is measured at 400 nm;
taking a sample solution, adding a buffer solution to a constant volume of 3mL, adding 1mL of alpha-glucosidase solution, shaking uniformly, placing in a 37 ℃ water bath kettle, heating for reaction for 10min, adding 1mL of PNPG, continuing the 37 ℃ water bath reaction for 10min, taking out, adding 1mL of Na2CO3Shaking the solution uniformly at 400nm to obtain an absorbance A1;
adding buffer solution into another sample solution to 4mL, shaking, heating in 37 deg.C water bath for 10min, adding 1mL PNPG, heating in 37 deg.C water bath for 10min, taking out, adding 1mL Na2CO3Solution, A0 is measured at 400 nm;
three parallels are made for each group of experiments, the average value of the final value is taken for three times, and the inhibition rate of the test object is calculated;
(4) the inhibition (%) of the test article was [1- (a1-a0)/a ] × 100%;
(5) results of the experiment
TABLE 1
TABLE 2
| Group of | On alpha-glucosidase inhibition% |
| Crude polysaccharide | 35.87 |
| Component mixed polysaccharide | 63.49 |
| Fr-B1 | 61.17 |
| Fr-B2 | 51.62 |
| Fr-B3 | 47.63 |
| Fr-B4 | 39.18 |
According to the experimental results shown in tables 1 and 2, the agrocybe aegerita polysaccharides (comprising Fr-B1, Fr-B2, Fr-B3 and Fr-B4 which are independent components) prepared by the invention have better inhibition rates than alpha-amylase and alpha-glucosidase of crude polysaccharides, the agrocybe aegerita polysaccharides prepared by the invention have better blood sugar reducing capability relative to the agrocybe aegerita crude polysaccharides, Fr-B1, Fr-B2, Fr-B3 and Fr-B4 prepared by the invention are obviously enhanced in blood sugar reducing capability after being mixed relative to Fr-B1, Fr-B2, Fr-B3 or Fr-B4 which are independent components, and Fr-B1, Fr-B2, Fr-B3 and Fr-B4 have synergistic effect on the blood sugar reducing capability.
Third, comparative experiment
The methods of the above examples 1 to 4 and comparative examples 1 to 3 were used to obtain agrocybe aegerita polysaccharides, and the comparison of the hypoglycemic function of the obtained agrocybe aegerita polysaccharides was carried out, and it was found that: the polysaccharide component obtained by enrichment and purification by the method has the advantages of less loss of effective components, high content of functional substances for reducing blood sugar, better effect and low production cost.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Claims (10)
1. An enrichment and purification method of agrocybe cylindracea polysaccharide with the function of reducing blood sugar is characterized by comprising the following steps: extracting crude polysaccharide from fresh agrocybe aegerita, and then sequentially carrying out macroporous resin column enrichment and purification, ODS resin enrichment and purification and DEAE-cellulose DE-52 column enrichment and purification on the crude polysaccharide to obtain the agrocybe aegerita polysaccharide.
2. The method for enriching and purifying agrocybe aegerita polysaccharides with the function of reducing blood sugar as claimed in claim 1, which is characterized by comprising the following steps:
(1) sample pretreatment: placing fresh agrocybe aegerita in an oven for drying, then sending the dried agrocybe aegerita into a grinder for grinding and sieving to obtain agrocybe aegerita powder;
(2) polysaccharide extraction: placing the agrocybe aegerita powder in the step (1) in a reaction device, adding distilled water into the reaction device, soaking at normal temperature, then carrying out reflux extraction in a water bath, filtering, adding distilled water into filter residues, repeatedly extracting for 2 times, and combining filtrates to obtain a polysaccharide extracting solution;
(3) alcohol precipitation: carrying out suction filtration on the polysaccharide extracting solution in the step (2), carrying out reduced pressure concentration to obtain an extract, completely dissolving the extract into a crude extract solution by using distilled water, adding absolute ethyl alcohol into the dissolved solution while stirring, standing overnight, filtering to remove a supernatant, collecting a precipitate, repeatedly washing for 2 times by using the absolute ethyl alcohol, and carrying out vacuum freeze drying to obtain crude polysaccharide;
(4) enrichment and purification: and (4) dissolving the crude polysaccharide obtained in the step (3) into a solution by using distilled water, and sequentially purifying by using a macroporous resin column, an ODS resin and a DEAE-cellulose DE-52 column to obtain the agrocybe aegerita polysaccharide.
3. The method for enriching and purifying agrocybe aegerita polysaccharides with hypoglycemic function according to claim 1 or 2, wherein the method comprises the following steps: the macroporous resin column purification comprises the following steps: performing AB-8 type macroporous resin column chromatography, loading the crude polysaccharide and resin on a column by a wet method according to the mass ratio of 1: 29-31, standing and adsorbing for 55-65 min, eluting with distilled water, performing polysaccharide tracking detection by a phenol-sulfuric acid method until sugar-free components flow out, eluting with 10%, 30%, 50% and 70% ethanol at the elution flow rate of 1.9-2.1 mL/h, concentrating the distilled water eluate under reduced pressure, and performing vacuum freeze drying to obtain the macroporous resin column purified product.
4. The method for enriching and purifying agrocybe aegerita polysaccharides with hypoglycemic function according to claim 1 or 2, wherein the method comprises the following steps: the ODS resin purification is: weighing dry powder purified by a macroporous resin column, dissolving the dry powder in distilled water to form a solution, loading Fr-A and resin on the column by a wet method according to the mass ratio of 1: 49-51, fully adsorbing the solution for 55-65 min by an ODS resin column, eluting with distilled water, performing polysaccharide tracking detection by adopting a phenol-sulfuric acid method until sugar-free components flow out, eluting with 20% by mass, 40% by mass and 60% by mass of ethanol at the elution flow rate of 1.9-2.1 mL/h, collecting the distilled water eluent, concentrating under reduced pressure, and performing vacuum freeze drying to obtain powder, thereby obtaining the ODS resin purified product.
5. The method for enriching and purifying agrocybe aegerita polysaccharides with hypoglycemic function according to claim 1 or 2, wherein the method comprises the following steps: the DEAE-cellulose DE-52 column purification comprises the following steps: weighing dry powder purified by ODS resin, dissolving the dry powder in distilled water to form a solution, loading the solution on a DEAE-cellulose DE-52 column by a wet method, sequentially carrying out gradient elution by using distilled water, 0.1M NaCl solution, 0.3M NaCl solution and 0.5M NaCl solution at the flow rate of 1.9-2.1 mL/h, respectively collecting each fraction, tracking and detecting the eluent by using a phenol-sulfuric acid method until sugar-free groups flow out, respectively dialyzing each fraction by using distilled water, concentrating the solution in a dialysis bag under reduced pressure, and respectively carrying out vacuum freeze drying to obtain the agrocybe cylindracea polysaccharide.
6. The method for enriching and purifying agrocybe aegerita polysaccharides with hypoglycemic function according to claim 2, characterized in that: in the step (1), the drying temperature is 50-53 ℃, the crushing time is 2-3 min, and the sieving is performed by a 50-70 mesh sieve.
7. The method for enriching and purifying agrocybe aegerita polysaccharides with hypoglycemic function according to claim 2, characterized in that: in the step (2), the material-liquid ratio of the agrocybe aegerita powder to the distilled water is 1: 19-21, the soaking time is 28-33 min, the extraction temperature is 98-103 ℃, and the extraction time is 115-125 min.
8. The method for enriching and purifying agrocybe aegerita polysaccharides with hypoglycemic function according to claim 2, characterized in that: in the step (3), the concentration of the absolute ethyl alcohol is 85%, the standing time is 11.5-13 h, and the temperature is 4 ℃.
9. The method for enriching and purifying agrocybe aegerita polysaccharides with hypoglycemic function according to claim 2, characterized in that: the temperature of the reduced pressure concentration is 48-53 ℃, the temperature of the freeze drying is-55 to-50 ℃, and the vacuum degree is 0-100 Pa.
10. The application of the agrocybe aegerita polysaccharide with the function of reducing blood sugar, which is prepared by the enrichment and purification method according to any one of claims 1 to 8, in preparing medicines, health-care products or foods for reducing blood sugar.
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