CN116622000A - Preparation process for extracting active soluble polysaccharide by using agrocybe cylindracea - Google Patents
Preparation process for extracting active soluble polysaccharide by using agrocybe cylindracea Download PDFInfo
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- CN116622000A CN116622000A CN202310537954.5A CN202310537954A CN116622000A CN 116622000 A CN116622000 A CN 116622000A CN 202310537954 A CN202310537954 A CN 202310537954A CN 116622000 A CN116622000 A CN 116622000A
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- agrocybe cylindracea
- supernatant
- enzymolysis
- active soluble
- soluble polysaccharide
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- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 241000908270 Agrocybe cylindracea Species 0.000 title claims abstract 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 54
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- 238000000034 method Methods 0.000 claims abstract description 28
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- 206010022489 Insulin Resistance Diseases 0.000 description 3
- RYMZZMVNJRMUDD-UHFFFAOYSA-N SJ000286063 Natural products C12C(OC(=O)C(C)(C)CC)CC(C)C=C2C=CC(C)C1CCC1CC(O)CC(=O)O1 RYMZZMVNJRMUDD-UHFFFAOYSA-N 0.000 description 3
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- 229960002855 simvastatin Drugs 0.000 description 3
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- XUFXOAAUWZOOIT-SXARVLRPSA-N (2R,3R,4R,5S,6R)-5-[[(2R,3R,4R,5S,6R)-5-[[(2R,3R,4S,5S,6R)-3,4-dihydroxy-6-methyl-5-[[(1S,4R,5S,6S)-4,5,6-trihydroxy-3-(hydroxymethyl)-1-cyclohex-2-enyl]amino]-2-oxanyl]oxy]-3,4-dihydroxy-6-(hydroxymethyl)-2-oxanyl]oxy]-6-(hydroxymethyl)oxane-2,3,4-triol Chemical compound O([C@H]1O[C@H](CO)[C@H]([C@@H]([C@H]1O)O)O[C@H]1O[C@@H]([C@H]([C@H](O)[C@H]1O)N[C@@H]1[C@@H]([C@@H](O)[C@H](O)C(CO)=C1)O)C)[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O XUFXOAAUWZOOIT-SXARVLRPSA-N 0.000 description 2
- 235000008121 Agrocybe aegerita Nutrition 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
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- 229960002632 acarbose Drugs 0.000 description 2
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- 230000004060 metabolic process Effects 0.000 description 2
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- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 241000219161 Theobroma Species 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 229920001284 acidic polysaccharide Polymers 0.000 description 1
- 150000004805 acidic polysaccharides Chemical class 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000003178 anti-diabetic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
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- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
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- 230000005779 cell damage Effects 0.000 description 1
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- 238000011156 evaluation Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000012792 lyophilization process Methods 0.000 description 1
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- 229940111202 pepsin Drugs 0.000 description 1
- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Diabetes (AREA)
- Veterinary Medicine (AREA)
- Engineering & Computer Science (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Biochemistry (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Botany (AREA)
- Medical Informatics (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Hematology (AREA)
- Obesity (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention discloses a preparation process for extracting active soluble polysaccharide by using agrocybe cylindracea, which comprises the following steps: grinding agrocybe cylindracea into powder serving as a raw material, uniformly mixing the agrocybe cylindracea with distilled water, performing primary hot water leaching, and centrifuging to obtain filter residues and first supernatant; simultaneously, carrying out secondary hot water leaching and centrifugation on filter residues to obtain a second supernatant; mixing the first supernatant and the second supernatant, and concentrating in vacuum to obtain concentrated solution; carrying out enzymolysis on the concentrated solution to obtain enzymolysis solution, cooling the enzymolysis solution to room temperature, centrifuging the enzymolysis solution, and collecting enzymolysis supernatant; adding absolute ethyl alcohol into the enzymolysis supernatant, and then sequentially carrying out ethanol precipitation and centrifugation to obtain a precipitate; and re-dissolving the obtained precipitate, vacuum concentrating to remove ethanol, and freeze-drying. The polysaccharide obtained by the method has high purity. In vivo and in vitro experiments prove that the active soluble polysaccharide obtained by the method has better hypoglycemic effect.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a preparation process for extracting active soluble polysaccharide by using agrocybe cylindracea.
Background
The mushroom polysaccharide has good hypoglycemic and antidiabetic effects, and mainly influences sugar metabolism through the ways of promoting liver glycogen synthesis, promoting glycolysis or inhibiting the activity of alpha-glycosidase, and the like, can also directly act on islet beta cells, promote insulin secretion, increase insulin sensitivity and enhance antioxidant stress to play a hypoglycemic function. At present, a water extraction and alcohol precipitation method, an alkali extraction and alcohol precipitation method and an enzymolysis method are main extraction methods of polysaccharide, and ultrasonic and microwave auxiliary extraction methods are used for improving the yield. The water extraction and alcohol precipitation method is the most common and basic method for extracting the polysaccharide, has the advantages of simple operation, convenient extraction, low equipment requirement and the like, but the method has lower polysaccharide extraction yield and high time cost. The alkaline extraction and alcohol precipitation method is mainly suitable for extracting acidic polysaccharide, but part of impurities are soluble in polysaccharide leaching liquid in alkaline environment, so that the subsequent polysaccharide refining process is more complicated. The enzymolysis method can improve the yield when extracting polysaccharide, the reaction temperature is lower, and the extraction time is short; however, the disadvantages are that the price of the enzyme is high, the temperature control requirement is strict during the experiment, and the higher structure of the polysaccharide may be changed due to the action of the enzyme.
Disclosure of Invention
The invention aims to solve the problems of overcoming the defects of the prior art and providing a preparation process for extracting active soluble polysaccharide by using agrocybe cylindracea. The invention adopts the water extraction and alcohol precipitation method to assist the enzymolysis method to extract the agrocybe cylindracea soluble polysaccharide, thereby improving the purity of the polysaccharide.
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
The invention provides a preparation process for extracting active soluble polysaccharide by using agrocybe cylindracea, which comprises the following steps of:
s1: grinding agrocybe cylindracea into powder serving as a raw material, uniformly mixing the powder with distilled water to obtain a suspension, carrying out primary hot water extraction on the suspension, and centrifuging to obtain filter residues and first supernatant; simultaneously carrying out secondary hot water leaching and centrifugation on the filter residues to obtain a second supernatant; mixing the obtained first supernatant and the second supernatant, and concentrating to 15-30% of the original volume by vacuum to obtain a concentrated solution;
s2: carrying out enzymolysis on the obtained concentrated solution to obtain enzymolysis solution, cooling the enzymolysis solution to room temperature, centrifuging the enzymolysis solution, and collecting enzymolysis supernatant;
s3: adding absolute ethyl alcohol into the obtained enzymolysis supernatant to ensure that the concentration of the ethyl alcohol in the system reaches 70-90%, and then sequentially carrying out alcohol precipitation and centrifugation to obtain a precipitate;
s4: and re-dissolving the obtained precipitate, vacuum concentrating to remove ethanol, and freeze-drying to obtain the agrocybe cylindracea active soluble polysaccharide.
Preferably, the addition ratio (w: v) of agrocybe cylindracea powder to distilled water in the step S1 is 1:15-24.
Preferably, the primary hot water leaching conditions in step S1 are: the temperature is 80-95 ℃ and the time is 2-4 hours; the centrifugation conditions are as follows: the rotating speed is 3500-5600 rpm, and the time is 5-10min.
Preferably, the ratio of the amount of distilled water added to the amount of the residue (v: w) in the secondary hot water leaching process in step S1 is: 15-24:1; the secondary hot water leaching conditions are as follows: the temperature is 80-95 ℃ and the time is 2-4 hours; the centrifugation conditions are as follows: the rotating speed is 3500-5600 rpm, and the time is 5-10min.
Preferably, the enzyme used in the enzymolysis in the step S2 is papain, and the enzymolysis time is 2-4h.
Preferably, the centrifugation conditions in step S2 are: the rotating speed is 9500-12000 rpm/min, and the time is 15-25min.
Preferably, the alcohol precipitation time in step S3 is 8-12h, and the centrifugation conditions are: the rotating speed is 9500-12000 rpm, and the time is 10-20min.
Preferably, the reconstitution process in step S4 is: the extracted precipitate was completely dissolved using distilled water.
Preferably, the lyophilization process in step S4 is: pre-freezing for 1h at-80 ℃, and then putting the mixture into a freeze dryer for freeze drying for 3 days in a vacuum environment at-60 to-80 ℃.
By means of the technical scheme, the invention has at least the following advantages: the method can be used for extracting the water-soluble polysaccharide from agrocybe cylindracea serving as a raw material to obtain the polysaccharide with the purity of more than 80%. In vivo and in vitro experiments prove that the active soluble agrocybe cylindracea polysaccharide obtained by the method has better hypoglycemic effect.
The foregoing description is only an overview of the present invention, and is intended to provide a more thorough understanding of the present invention, and is to be accorded the full scope of the present invention.
Drawings
The present disclosure will become more readily understood with reference to the accompanying drawings. As will be readily appreciated by those skilled in the art: the drawings are only for illustrating the technical scheme of the present invention and are not intended to limit the scope of the present invention. In the figure:
FIG. 1 is a flow chart of the preparation process of the invention;
FIG. 2 is a graph showing the effect of agrocybe cylindracea active soluble polysaccharide on alpha-amylase activity;
FIG. 3 is a graph showing the effect of agrocybe cylindracea active soluble polysaccharide on the binding capacity of cholate;
FIG. 4 is a graph showing the destructive power of active soluble polysaccharides of agrocybe cylindracea on cholesterol;
FIG. 5 is a graph showing the effect of agrocybe cylindracea active soluble polysaccharide on blood glucose in mice;
FIG. 6 shows pancreatic pathology of different groups of mice;
FIG. 7 is an insulin resistance index of agrocybe cylindracea polysaccharide;
Detailed Description
In order to make the technical means, the creation features, the achievement of the purposes and the effects of the present invention easy to understand, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Agrocybe cylindracea is ground into powder and used as an extraction raw material of water-soluble polysaccharide, and distilled water with the ratio of 1:15 (w: v) is uniformly mixed. Extracting at 80deg.C for 4 hr, and centrifuging at 5600r for 10min to obtain supernatant. And leaching the filter residue with hot water for the second time, wherein the process comprises the following steps: mixing with distilled water in an amount of 1:15, extracting at 80deg.C for 4 hr, centrifuging at 5600r for 10min to obtain supernatant, mixing the two supernatants, and vacuum concentrating to 30% of the original volume. The concentrated supernatant was subjected to enzymolysis with papain at an optimum temperature for 4 hours, cooled to room temperature, and centrifuged at 9500rpm for 15 minutes, and the supernatant was collected. Adding absolute ethyl alcohol into the supernatant to ensure that the concentration of the ethyl alcohol in the system reaches 80%, precipitating the ethyl alcohol for 12h, centrifuging for 20min to obtain precipitate, re-dissolving the precipitate, vacuum concentrating to remove the ethyl alcohol, and freeze-drying (pre-freezing at-80 ℃ for 1h, and then freeze-drying in a vacuum environment at-60 to-80 ℃ for 3 days) to obtain the agrocybe cylindracea active soluble polysaccharide.
The content of active soluble polysaccharide of agrocybe cylindracea is determined as shown in table 1 below.
Example 2
Agrocybe cylindracea is ground into powder and used as an extraction raw material of water-soluble polysaccharide, and distilled water of 1:24 (w: v) is uniformly mixed. Extracting at 80deg.C for 4 hr, and centrifuging at 5600r for 10min to obtain supernatant. And leaching the filter residue with hot water for the second time, wherein the process comprises the following steps: mixing with distilled water at a ratio of 1:24, extracting at 80deg.C for 4 hr, centrifuging at 5600r for 10min to obtain supernatant, mixing the two supernatants, and vacuum concentrating to 30% of the original volume. The concentrated supernatant was subjected to enzymolysis with papain at an optimum temperature for 4 hours, cooled to room temperature, and centrifuged at 9500rpm for 15 minutes, and the supernatant was collected. Adding absolute ethyl alcohol into the supernatant to ensure that the concentration of the ethyl alcohol in the system reaches 80%, precipitating the ethyl alcohol for 12h, centrifuging for 20min to obtain precipitate, re-dissolving the precipitate, vacuum concentrating to remove the ethyl alcohol, and freeze-drying (pre-freezing at-80 ℃ for 1h, and then freeze-drying in a vacuum environment at-60 to-80 ℃ for 3 days) to obtain the agrocybe cylindracea active soluble polysaccharide.
Example 3
Agrocybe cylindracea is ground into powder and used as an extraction raw material of water-soluble polysaccharide, and distilled water with the ratio of 1:15 (w: v) is uniformly mixed. Extracting at 95deg.C for 4 hr, and centrifuging at 5600 r.p. for 10min to obtain supernatant. And leaching the filter residue with hot water for the second time, wherein the process comprises the following steps: mixing with distilled water in an amount of 1:15, extracting at 95deg.C for 4 hr, centrifuging at 5600r for 10min to obtain supernatant, mixing the two supernatants, and vacuum concentrating to 30% of the original volume. The concentrated supernatant was subjected to enzymolysis with papain at an optimum temperature for 4 hours, cooled to room temperature, and centrifuged at 9500rpm for 15 minutes, and the supernatant was collected. Adding absolute ethyl alcohol into the supernatant to ensure that the concentration of the ethyl alcohol in the system reaches 80%, precipitating the ethyl alcohol for 12h, centrifuging for 20min to obtain precipitate, re-dissolving the precipitate by using distilled water, vacuum concentrating to remove the ethyl alcohol, and freeze-drying (pre-freezing at-80 ℃ for 1h, and then freeze-drying in a vacuum environment at-60 to-80 ℃ for 3 days) to obtain the agrocybe cylindracea active soluble polysaccharide.
Comparative example 1
Weighing a certain amount of agrocybe cylindracea, grinding into powder, adding 15 times of distilled water, stirring at 80 ℃ for extraction for 4 hours, centrifuging at 5600r/min for 10min, and collecting filtrate. The filter residue is repeatedly extracted once, and the process comprises the following steps: mixing with distilled water at a ratio of 1:15, extracting at 80deg.C for 4 hr, centrifuging at 5600r for 10min to obtain supernatant, mixing the two extractive solutions, adjusting pH to neutrality, vacuum concentrating to 30% of original volume, performing enzymolysis with papain in 60 deg.C water bath for 2 hr, cooling to room temperature, centrifuging at 10000r/min for 15min, collecting supernatant, removing protein by Sevag method, and collecting supernatant. Absolute ethanol was slowly added to the concentrate until the ethanol concentration of the system reached 80% (v/v). After 12h of alcohol precipitation, centrifuging for 10min, and collecting the precipitate. Adding water into the precipitate for redissolution, vacuum concentrating to remove ethanol, and lyophilizing (pre-freezing at-80deg.C for 1 hr, and lyophilizing in vacuum environment at-60deg.C to-80deg.C for 3 days) to obtain Agrocybe aegerita water-extracted polysaccharide.
The content of active soluble polysaccharide of agrocybe cylindracea is determined by a phenol sulfuric acid method as shown in the table 1 below.
TABLE 1 Agrocybe cylindracea polysaccharide content extracted by different methods
As can be seen from the results of Table 1, agrocybe cylindracea active soluble polysaccharide, which contains more than 80% of polysaccharide purity, can be obtained using the method of example 1 of the present invention.
Application example 1 identification of Activity of soluble polysaccharide from agrocybe aegerita
1. Effect of Agrocybe cylindracea Activity soluble polysaccharide on alpha-amylase Activity
The experimental method comprises the following steps: acarbose was selected as positive Control (CON), 100 μl of the Sample (SAAP) extracted in example 1 or control was added to the centrifuge tube, 100 μl of α -amylase was added, incubation was performed in a thermostated incubator for about 10-20min, and then incubation was continued with 100 μl of starch substrate. Finally, 100. Mu.l of DNS was added to stop the reaction, the reaction was taken out and cooled to room temperature after 10 to 20 minutes of water bath in a boiling water bath, and the absorbance was measured at a wavelength of 540 nm. 3 samples were measured in parallel, and an alpha-amylase activity inhibition system table was set to calculate the inhibition ratio of alpha-amylase according to the formula:
IR＝[1-(A4-A3)/(A2-A1)]×100％；
wherein: a1 is the absorbance value of the blank group, A2 is the absorbance value of the blank group, A3 is the absorbance value of the sample control group, and A4 is the absorbance value of the sample group.
The results are shown in FIG. 2, and it can be seen from FIG. 2 that both polysaccharide group (SAAP) and positive control group (CON) acarbose have inhibitory effect on alpha-amylase activity. Wherein the inhibition ratio is more than 15% at a polysaccharide concentration of 0.15mg/mL (corresponding to SAAP-0.15), and the inhibition effect is more than that of the positive control group (CON-0.15). The alpha-amylase can promote the hydrolysis of starch components into glucose, and the more obvious the inhibition effect on the alpha-amylase is, the more obvious the blood sugar reducing effect is.
Application example 2 evaluation of hypoglycemic Effect of Agrocybe cylindracea active soluble polysaccharide
1. Binding ability of active soluble polysaccharide of agrocybe cylindracea to cholate
The experimental method comprises the following steps: firstly, respectively drawing taurocholate and glycocholate standard curves by taking the concentration of cholate as an abscissa and the absorbance as an ordinate. Simvastatin is adopted as a positive control group (CON), a proper amount of agrocybe cylindracea polysaccharide (SAAP) extracted in the example 1 with the volume and mass concentration of a series of gradients is respectively taken and added into test tubes, pepsin solution and hydrochloric acid solution are respectively added into each test tube, and the mixture is subjected to constant-temperature shaking digestion for 1-2 hours. Adjusting the pH value to 7.6 by using sodium hydroxide solution, adding trypsin solution, and oscillating at constant temperature for digestion for 1-2 h. Then glycocholate/taurocholate is added into each test tube, and the mixture is oscillated for 1 to 2 hours in a constant temperature shaking table. Centrifuging for 10-20min under proper conditions, collecting supernatant, and adding into H 2 SO 4 The solution is heated in a water bath for 10-20min, taken out and then is subjected to ice bath for 5-10min, and the absorbance value is measured at 387nm wavelength. All reagents were dissolved using PBS buffer. And calculating the content of the residual glycocholate and taurocholate according to the standard curve. The cholate combined reaction system is reasonably arranged, and the inhibition rate is calculated according to the following formula:
IR＝[1-(A4-A3)/(A2-A1)]×100％；
wherein: a1 is the absorbance value of the blank group, A2 is the absorbance value of the blank group, A3 is the absorbance value of the sample control group, and A4 is the absorbance value of the sample group.
The results are shown in FIG. 3, and it can be seen from FIG. 3 that the experiment of the binding capacity with cholate is divided into two parts of glycocholate and taurocholate. In glycocholate experiments, the maximum binding rate of agrocybe cylindracea polysaccharide and cholate is not greatly different; in the taurocholate experiments, the agrocybe cylindracea polysaccharide has outstanding effect. Diabetes and hyperlipidemia are often indiscernible, cholate is mainly related to metabolism of cholesterol in vivo, and after agrocybe cylindracea polysaccharide and cholate are combined, the content of bile acid in vivo can be effectively reduced, so that the purposes of reducing the blood lipid content and reducing the cholesterol content are achieved
2. Destructive effect of active soluble polysaccharide of agrocybe cylindracea on cholesterol micelle
The experimental method comprises the following steps: using simvastatin as a positive Control (CON), an appropriate volume of 3 different concentrations of the polysaccharide Solution (SAAP) extracted from example 1 was added to the cholesterol micelle solution and stirred slowly at constant temperature for a period of time. Centrifuging at 5000rpm/min for 10-15 min, and collecting supernatant. The cholesterol micelle concentration in the supernatant was measured using a kit to evaluate the degree of damage to the cholesterol micelle. The blank group was distilled water in the same amount; samples were assayed in 3 replicates.
As a result, as shown in FIG. 4, it can be seen from FIG. 4 that the effect of water-extractable polysaccharide has a certain effect in the experiment of the destructive effect on cholesterol, and the destructive effect is more than 20% at a concentration of 2.5mg/mL (SAAP-2.5). The polysaccharide has a destructive effect on cholesterol micelle in the body, so that the cholesterol can be effectively prevented from being absorbed and utilized by the human body.
Application example 3 influence of Theobroma cacumen Alni active soluble polysaccharide on physical and chemical indexes of mice
The experimental method comprises the following steps: the CON group is normal mice, and the normal feed is eaten. The remaining groups were modeled using STZ, so that mice all met the model of type two diabetes. The DM group was only allowed to consume high-fat feed, the MET group was infused daily with the stomach-positive drug simvastatin, SAAP was the agrocybe cylindracea polysaccharide group extracted in example 1. All mice were assayed for fasting blood glucose weekly by tail vein blood withdrawal using a glucometer.
The results are shown in FIGS. 5, 6 and 7.
As can be seen from fig. 5, the agrocybe cylindracea water-soluble active polysaccharide (SAAP group) has a lower blood sugar level and is more stable than the model group (DM); the SAAP group and the positive drug control group (MET) have lower blood sugar values, which indicates that the agrocybe cylindracea water-soluble active polysaccharide has the effect of reducing blood sugar.
As can be seen from fig. 6, the islet cells of the CON group mice were aligned and distributed regularly, and the DM group mice showed severe islet cell damage and vacuolation, and no intact islet cells were present in the visual field. The MET and SAAP groups had varying degrees of islet cell atrophy, with irregular islet shape, but improved conditions compared to the DM group.
As can be seen from fig. 7, the insulin concentration of the type II diabetes mice is significantly increased, the MET and SAAP groups are reduced, and the SAAP significantly reduces the insulin resistance index of the type II diabetes mice compared with the DM group.
While the invention has been described with respect to preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and that any such changes and modifications as described in the above embodiments are intended to be within the scope of the invention.
Claims (9)
1. A preparation process for extracting active soluble polysaccharide by using agrocybe cylindracea, which is characterized by comprising the following steps of:
s1: grinding agrocybe cylindracea into powder serving as a raw material, uniformly mixing the powder with distilled water to obtain a suspension, carrying out primary hot water extraction on the suspension, and centrifuging to obtain filter residues and first supernatant; simultaneously carrying out secondary hot water leaching and centrifugation on the filter residues to obtain a second supernatant; mixing the obtained first supernatant and the second supernatant, and concentrating to 15-30% of the original volume by vacuum to obtain a concentrated solution;
s2: carrying out enzymolysis on the obtained concentrated solution to obtain enzymolysis solution, cooling the enzymolysis solution to room temperature, centrifuging the enzymolysis solution, and collecting enzymolysis supernatant;
s3: adding absolute ethyl alcohol into the obtained enzymolysis supernatant to ensure that the concentration of the ethyl alcohol in the system reaches 70-90%, and then sequentially carrying out alcohol precipitation and centrifugation to obtain a precipitate;
s4: and re-dissolving the obtained precipitate, vacuum concentrating to remove ethanol, and freeze-drying to obtain the agrocybe cylindracea active soluble polysaccharide.
2. The process for preparing an active soluble polysaccharide extracted from agrocybe cylindracea according to claim 1, wherein the ratio of agrocybe cylindracea powder to distilled water (w: v) in the step S1 is 1:15-24.
3. The process for preparing active soluble polysaccharides extracted from agrocybe cylindracea according to claim 1, wherein the primary hot water extraction conditions in step S1 are: the temperature is 80-95 ℃ and the time is 2-4 hours; the centrifugation conditions are as follows: the rotating speed is 3500-5600 rpm, and the time is 5-10min.
4. The process for preparing an active soluble polysaccharide extracted from agrocybe cylindracea according to claim 1, wherein the ratio of the amount of distilled water added to the amount of filter residue (v: w) in the secondary hot water extraction process in step S1 is: 15-24: 1, a step of; the secondary hot water leaching conditions are as follows: the temperature is 80-95 ℃ and the time is 2-4 hours; the centrifugation conditions are as follows: the rotating speed is 3500-5600 rpm, and the time is 5-10min.
5. The process for preparing active soluble polysaccharide extracted from agrocybe cylindracea according to claim 1, wherein the enzyme used in the enzymolysis in the step S2 is papain, and the enzymolysis time is 2-4h.
6. The process for preparing an active soluble polysaccharide extracted from agrocybe cylindracea according to claim 1, wherein the centrifugation conditions in step S2 are: the rotating speed is 9500-12000 rpm, and the time is 15-25min.
7. The process for preparing active soluble polysaccharides extracted from agrocybe cylindracea according to claim 1, wherein the alcohol precipitation time in step S3 is 8-12h, and the centrifugation conditions are as follows: the rotating speed is 9500-12000 rpm, and the time is 10-20min.
8. The process for preparing active soluble polysaccharides extracted from agrocybe cylindracea according to claim 1, wherein the re-dissolving process in step S4 is as follows: the extracted precipitate was completely dissolved using distilled water.
9. The process for preparing an active soluble polysaccharide from agrocybe cylindracea according to claim 1, wherein the freeze-drying process in step S4 is: pre-freezing for 1h at-80 ℃, and then putting the mixture into a freeze dryer for freeze drying for 3 days in a vacuum environment at-60 to-80 ℃.
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| CN103694366A (en) * | 2013-12-12 | 2014-04-02 | 上海市农业科学院 | Preparation method of high-content lentinan |
| CN104387485A (en) * | 2014-11-12 | 2015-03-04 | 绥化学院 | Method for extracting polysaccharides in flammulina velutipes by synergism of complex enzymes and high-pressure hot water extraction process |
| CN106883304A (en) * | 2017-03-06 | 2017-06-23 | 南昌大学 | Heterogeneity polysaccharide is comprehensively prepared and purification process in a kind of Hericium erinaceus |
| CN109180832A (en) * | 2018-10-12 | 2019-01-11 | 绩溪县徽菜宝生物科技有限公司 | A kind of efficient Extraction Methods of Polysaccharides From Agrocybe aegerita |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103694366A (en) * | 2013-12-12 | 2014-04-02 | 上海市农业科学院 | Preparation method of high-content lentinan |
| CN104387485A (en) * | 2014-11-12 | 2015-03-04 | 绥化学院 | Method for extracting polysaccharides in flammulina velutipes by synergism of complex enzymes and high-pressure hot water extraction process |
| CN106883304A (en) * | 2017-03-06 | 2017-06-23 | 南昌大学 | Heterogeneity polysaccharide is comprehensively prepared and purification process in a kind of Hericium erinaceus |
| CN109180832A (en) * | 2018-10-12 | 2019-01-11 | 绩溪县徽菜宝生物科技有限公司 | A kind of efficient Extraction Methods of Polysaccharides From Agrocybe aegerita |
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