CN113968916B - Extraction method and application of phlebopus portentosus polysaccharide - Google Patents
Extraction method and application of phlebopus portentosus polysaccharide Download PDFInfo
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Abstract
The invention relates to an extraction method and application of phlebopus portentosus polysaccharide, which comprises the following steps: s1, adding 95% ethanol into the powder of the Phlebopus portentosus fruiting body, performing ultrasonic dispersion, and standing at room temperature to leave a precipitate 1; s2, adding water, leaching in a water bath, and concentrating; s3, adding 95% ethanol for precipitation under magnetic stirring, standing at low temperature, and centrifuging to obtain a precipitate 2; s4, extracting by using a Sevage reagent, and taking supernatant; s5, adding SP-825 macroporous resin, and decoloring; s6, adding 95% ethanol for precipitation, standing at low temperature, and centrifuging to obtain a precipitate 3; s7, performing DEAE-52 fiber column chromatography, eluting with distilled water and NaCl solutions with different concentrations; s8, concentrating, dialyzing and drying; the boletus fuscogilus polysaccharide powder obtained by multiple water extraction and alcohol precipitation extraction combined with adsorption, elution and purification of a multi-gradient NaCl solution-DEAE-52 cellulose chromatographic column has obvious inhibition activity on alpha-glucosidase, and can be used for preparing hypoglycemic drugs.
Description
Technical Field
The invention relates to the technical field of fungal polysaccharide extraction, and particularly relates to an extraction method and application of phlebopus portentosus polysaccharide.
Background
Diabetes is a chronic metabolic disease characterized by hyperglycemia, and can be mainly classified into type 1, type 2, and gestational diabetes. Wherein, the number of the type 2 diabetes patients accounts for about 90 to 95 percent of the total number of the diabetes patients. Hyperglycemia causes dysfunction and chronic damage of tissues and organs such as eyes, kidneys, heart, nerves and blood vessels. Clinical drug treatment modes aiming at diabetes mainly comprise oral drug treatment and insulin injection treatment, wherein the oral drugs mainly comprise sulfonylurea drugs, biguanide hypoglycemic drugs, alpha-glucosidase inhibition and the like. Through modern pharmacological research, compared with chemical synthetic drugs, the hypoglycemic active substance extracted from natural products generally has the advantages of remarkable effect, high biological tolerance, small side effect and the like. Therefore, the development of the natural active substances not only can provide clinical medicines with better curative effect, but also can bring remarkable social and economic benefits.
Phlebopus portentosus also called Phlebopus portentosus, commonly known as "Hepatiella nigra", is a Phlebopus fungus belonging to the genus Phlebopus of the family Boletaceae. The species are mainly distributed in the areas of Hainan, yunnan and Guangxi provinces in China. The boletus fuscous is fat and beautiful, delicious in taste, rich in nutrition, rich in various mineral elements, high in protein, low in fat and popular with the public, and has a good medicinal value, for example, the polysaccharide-protein complex is obtained by Karnchanat and the like by using a water extraction and alcohol precipitation method, and experiments show that the boletus fuscous has antioxidant capacity.
However, currently, research on the in-vitro hypoglycemic activity of the Phlebopus portentosus polysaccharide is less, so that the invention provides an extraction method and application of the Phlebopus portentosus polysaccharide, and provides an important basis for the deep research and application of the Phlebopus portentosus.
Disclosure of Invention
In view of this, the invention provides an extraction method and application of phlebopus portentosus polysaccharide.
The technical scheme of the invention is realized as follows:
a method for extracting Phlebopus portentosus polysaccharide comprises the following steps:
s1, taking powder of a Phlebopus portentosus fruiting body, adding 95% ethanol, mixing, performing ultrasonic dispersion, standing at room temperature, removing a supernatant, and keeping a precipitate 1;
s2, adding water into the precipitate 1, leaching in a water bath, filtering, retaining filtrate, adding water into filter residues, repeatedly extracting for 3-5 times under the same condition, combining the filtrates, performing rotary evaporation, and concentrating to obtain a concentrated extract;
s3, adding 95% ethanol with 4 times volume of the concentrated extracting solution into the concentrated extracting solution under magnetic stirring for precipitation, standing at low temperature, centrifuging, and removing supernatant to obtain a precipitate 2;
s4, adding water to dissolve the precipitate 2, adding a Sevage reagent to extract, taking supernate, repeating the process for 2-4 times, and combining the supernate;
s5, adding the supernatant into SP-825 macroporous resin, and carrying out magnetic stirring for decoloration to obtain decolored liquid;
s6, adding the decolorized solution into 95% ethanol for precipitation, standing at a low temperature, removing supernatant, centrifuging to obtain a precipitate 3, pre-freezing, and freeze-drying to obtain polysaccharide crude extract powder;
s7, adding water to the polysaccharide crude extract powder for dissolving, centrifuging, taking supernatant, performing DEAE-52 fiber column chromatography, and sequentially eluting with distilled water and NaCl solutions with different concentrations to obtain eluent;
and S8, concentrating, dialyzing and freeze-drying the eluent to obtain phlebopus portentosus polysaccharide powder.
Preferably, in step S4, the Sevage reagent is prepared by mixing chloroform and n-butanol at a volume ratio of 4:1.
Preferably, in step S7, the NaCl solution elution is performed by sequentially eluting with 0.1mol/L, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L and 1.0mol/L NaCl solutions, and the eluates are combined.
Preferably, in step S1, the ultrasonic dispersion temperature is 40 ℃ and the ultrasonic time is 30min.
Preferably, in the step S2, the water bath leaching temperature is 80 ℃, the leaching time is 1-3 hours, and the rotary evaporation temperature is 50-70 ℃.
Preferably, in step S3, the centrifugation rotation speed is 4000rpm, and the centrifugation time is 5min.
Preferably, in steps S3 and S6, the low temperature rest is at 4 ℃ for 24h.
Preferably, in step S7, the concentrated solution is placed in an MD34 dialysis bag, placed on a constant temperature magnetic stirrer, dialyzed in tap water for 24 hours, during which tap water is changed every 4 hours, water is changed for three times, and left to stand overnight for 12 hours, and then dialyzed for 12 hours, during which distilled water is changed every 4 hours, water is changed for three times.
A Phlebopus portentosus polysaccharide obtained by the above extraction method, and its application in preparing medicines for reducing blood sugar are provided.
Preferably, the phlebopus portentosus polysaccharide is used for preparing an alpha-glucosidase inhibitor for a hypoglycemic medicament.
Compared with the prior art, the invention has the beneficial effects that:
the invention takes the Phlebopus portentosus sporocarp as a raw material, and the Phlebopus portentosus polysaccharide is obtained by multiple water extraction and alcohol precipitation extraction combined with adsorption, elution and purification of a multi-gradient NaCl solution-DEAE-52 cellulose chromatographic column.
The purified polysaccharide components contain PPP-0, PPP-1 and PPP-2, the content of the PPP-0 is at most and reaches 62.7mg, the PPP-1 and the PPP-2 in the purified polysaccharide components show a rising trend in the range of 0.25 mg/mL-2.5 mg/mL of polysaccharide concentration, the inhibition rate of the PPP-1 on alpha-glucosidase is 32.20-57.15 percent along with the increase of the concentration, the inhibition rate of the PPP-2 on the alpha-glucosidase is 39.04-70.55 percent, and the direct quantitative-effect relationship is formed; the result of enzyme inhibition kinetic analysis shows that the inhibition of the PPP-0 on the alpha-glucosidase is competitive reversible inhibition.
The invention provides experimental basis for the blood sugar reducing active ingredients of the Phlebopus portentosus, provides theoretical reference for the development and production of the Phlebopus portentosus, and provides basic data for the development of polysaccharide products of the Phlebopus portentosus.
Drawings
FIG. 1 is a graph showing the enzyme inhibition rate of Phlebopus portentosus fruiting body polysaccharide;
FIG. 2 is a graph showing the judgment of the mechanism of inhibition of alpha-glucosidase by PPP-0;
FIG. 3 is a graph showing the determination of the type of alpha-glucosidase inhibition by PPP-0;
FIG. 4 is a DEAE-52 cellulose chromatogram elution curve of Phlebopus portentosus fruiting body polysaccharide.
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
Example 1
(1) Crude extraction of boletus fuscogilus fruiting body crude polysaccharide: extracting the crude polysaccharide of the Phlebopus portentosus fruiting body by water extraction and alcohol precipitation, namely taking 50g of Phlebopus portentosus fruiting body powder, adding 1000mL of 95% ethanol, placing in an ultrasonic instrument at 35 ℃ for ultrasonic treatment for 20min, and properly stirring by using a glass rod in the ultrasonic treatment; standing at room temperature overnight, removing supernatant, recovering ethanol, and retaining precipitate; adding 1000mL of deionized water into the precipitate, leaching for 1h in a 70 ℃ water bath, filtering, retaining the filtrate, adding 1000mL of deionized water into the filter residue, continuing the extraction under the same conditions, repeating the extraction for 3 times, combining the filtrates, performing rotary evaporation at 50 ℃, and concentrating the extracting solution to about 250 mL; placing the beaker containing the extracted concentrated solution on a magnetic stirrer, slowly adding 95% ethanol with 4 times of volume of the concentrated solution while stirring for precipitation, and standing at 4 ℃ for 18 hours; centrifuging for 3min at 4000rpm by using a centrifuge, separating the precipitate from ethanol, discarding the supernatant to obtain a precipitate, adding 200mL of deionized water into the precipitate, and uniformly stirring to dissolve the precipitate; adding the solution obtained by dissolving the precipitate into a 500mL separating funnel, adding 200mL Sevage reagent for extraction, removing protein, reserving supernatant, and repeating the process for 2 times; putting the supernatant into a beaker, adding a proper amount of SP-825 macroporous resin, putting the beaker on a magnetic stirrer for stirring and decoloring, and performing suction filtration to recover the SP-825 macroporous resin; adding 95% ethanol with 4 times volume of the solution obtained after decolorization for precipitation, sealing, and standing overnight in a refrigerator at 4 ℃; removing supernatant, recovering ethanol, centrifuging lower layer, precipitating in culture dish, pre-freezing, freeze drying, and drying for 18 hr to obtain coarse polysaccharide powder of Phlebopus portentosus fruiting body;
(2) Separating and purifying components of the Phlebopus portentosus fruiting body polysaccharide: weighing 1.5g of boletus fuscogilus fruiting body crude polysaccharide powder, dissolving in 10mL of deionized water, subpackaging into 2mL of centrifuge tubes, centrifuging, and keeping supernatant; adding a crude polysaccharide solution by using DEAE-52 fiber column chromatography, eluting with distilled water and NaCl solutions with the concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mol/L respectively, controlling the elution speed (once every 9 minutes by using an automatic collection device and 10 mL/tube every time), setting the automatic collection device to collect eluent, and detecting the polysaccharide content by using a phenol-concentrated sulfuric acid method to obtain a gradient curve of the purified polysaccharide content of the boletus fuscogilus fruiting body; concentrating the eluent of each stage to 10mL; putting the concentrated solution into an MD34 dialysis bag, placing the bag on a constant-temperature magnetic stirrer, dialyzing the bag in tap water for 24 hours, changing tap water once every 4 hours during the period, changing water for three times, standing the bag overnight for 12 hours, then changing distilled water for dialysis for 12 hours, changing distilled water once every 4 hours during the period, changing water for three times, transferring the dialyzate into a culture dish after the dialysis is finished, and freeze-drying the dialyzate for 24 hours to obtain the purified polysaccharide powder of the Phlebopus portentosus fruiting body.
Example 2
(1) Crude extraction of boletus fuscogilus fruiting body crude polysaccharide: extracting the crude polysaccharide of the Phlebopus portentosus fruiting body by water extraction and alcohol precipitation, namely taking 50g of Phlebopus portentosus fruiting body powder, adding 1000mL of 95% ethanol, placing in an ultrasonic instrument at 45 ℃ for ultrasonic treatment for 40min, and properly stirring by using a glass rod in the ultrasonic treatment; standing at room temperature overnight, removing supernatant, recovering ethanol, and retaining precipitate; adding 1000mL of deionized water into the precipitate, leaching for 1-3h in a water bath kettle at 90 ℃, filtering, retaining the filtrate, adding 1000mL of deionized water into the filter residue, continuing extraction under the same conditions, repeating extraction for 5 times, combining the filtrates, performing rotary evaporation at 70 ℃, and concentrating the extract to about 250 mL; placing the beaker containing the extracted concentrated solution on a magnetic stirrer, slowly adding 95% ethanol with 4 times of volume of the concentrated solution while stirring for precipitation, and standing at 4 ℃ for 32 hours; centrifuging for 7min at 4000rpm by using a centrifuge, separating the precipitate from ethanol, discarding the supernatant to obtain a precipitate, adding 200mL of deionized water into the precipitate, and uniformly stirring to dissolve the precipitate; adding the solution obtained by dissolving the precipitate into a 500mL separating funnel, adding 200mL Sevage reagent for extraction, removing protein, reserving supernatant, and repeating the process for 4 times; putting the supernatant into a beaker, adding a proper amount of SP-825 macroporous resin, putting the beaker on a magnetic stirrer for stirring and decoloring, and performing suction filtration to recover the SP-825 macroporous resin; adding 4 times volume of 95% ethanol into the decolorized solution for precipitation, sealing, and standing overnight in a refrigerator at 4 deg.C; removing supernatant, recovering ethanol, centrifuging lower layer, precipitating in culture dish, pre-freezing, freeze drying, and drying for 32 hr to obtain coarse polysaccharide powder of Phlebopus portentosus fruiting body;
(2) Separating and purifying components of the Phlebopus portentosus fruiting body polysaccharide: weighing 1.5g of boletus fuscogilus fruiting body crude polysaccharide powder, dissolving in 10mL of deionized water, subpackaging into 2mL of centrifuge tubes, centrifuging, and keeping supernatant; adding a crude polysaccharide solution by using DEAE-52 fiber column chromatography, eluting with distilled water and NaCl solutions with the concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mol/L respectively, controlling the elution speed (once every 9 minutes by using an automatic collection device and 10 mL/tube every time), setting the automatic collection device to collect eluent, and detecting the polysaccharide content by using a phenol-concentrated sulfuric acid method to obtain a gradient curve of the purified polysaccharide content of the boletus fuscogilus fruiting body; concentrating each eluent to 20mL; putting the concentrated solution into an MD34 dialysis bag, placing the bag on a constant-temperature magnetic stirrer, dialyzing the bag in tap water for 24 hours, changing tap water once every 4 hours during the period, changing water for three times, standing the bag overnight for 12 hours, then changing distilled water for dialysis for 12 hours, changing distilled water once every 4 hours during the period, changing water for three times, transferring the dialyzate into a culture dish after the dialysis is finished, and freeze-drying the dialyzate for 24 hours to obtain the purified polysaccharide powder of the Phlebopus portentosus fruiting body.
Example 3
(1) Extracting crude polysaccharide of Phlebopus portentosus fruiting body: extracting the crude polysaccharide of Phlebopus portentosus fruiting body by water extraction and alcohol precipitation, namely taking 50g of Phlebopus portentosus fruiting body powder, adding 1000mL of 95% ethanol, placing in an ultrasonic instrument at 40 ℃ for ultrasonic treatment for 30min, and properly stirring by using a glass rod in the ultrasonic treatment process; standing at room temperature overnight, removing supernatant, recovering ethanol, and retaining precipitate; adding 1000mL of deionized water into the precipitate, leaching for 2h in a water bath at 80 ℃, filtering, retaining the filtrate, adding 1000mL of deionized water into the filter residue, continuing to extract under the same conditions, repeatedly extracting for 4 times, combining the filtrates, performing rotary evaporation at 60 ℃, and concentrating the extracting solution to about 250 mL; placing the beaker containing the extracted concentrated solution on a magnetic stirrer, slowly adding 95% ethanol with 4 times of volume of the concentrated solution while stirring for precipitation, and standing at 4 ℃ for 24 hours; centrifuging for 5min at 4000rpm by using a centrifuge, separating the precipitate from ethanol, discarding the supernatant to obtain a precipitate, adding 200mL of deionized water into the precipitate, and uniformly stirring to dissolve the precipitate; adding the solution obtained by dissolving the precipitate into a 500mL separating funnel, adding 200mL Sevage reagent for extraction, removing protein, retaining supernatant, and repeating the process for 3 times; putting the supernatant into a beaker, adding a proper amount of SP-825 macroporous resin, putting the beaker on a magnetic stirrer for stirring and decoloring, and performing suction filtration to recover the SP-825 macroporous resin; adding 95% ethanol with 4 times volume of the solution obtained after decolorization for precipitation, sealing, and standing overnight in a refrigerator at 4 ℃; removing supernatant, recovering ethanol, centrifuging the lower layer, precipitating, placing into a culture dish, pre-freezing, lyophilizing, and drying for 24 hr to obtain coarse polysaccharide powder of Phlebopus portentosus fruiting body;
(2) Separating and purifying components of the Phlebopus portentosus fruiting body polysaccharide: weighing 1.5g of boletus fuscogilus fruiting body crude polysaccharide powder, dissolving in 10mL of deionized water, subpackaging into 2mL of centrifuge tubes, centrifuging, and keeping supernatant; adding a crude polysaccharide solution by using DEAE-52 fiber column chromatography, eluting with distilled water and NaCl solutions with the concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mol/L respectively, controlling the elution speed (once every 9 minutes by using an automatic collection device and 10 mL/tube every time), setting the automatic collection device to collect eluent, and detecting the polysaccharide content by using a phenol-concentrated sulfuric acid method to obtain a gradient curve of the purified polysaccharide content of the boletus fuscogilus fruiting body; concentrating each eluent to 15mL; putting the concentrated solution into an MD34 dialysis bag, placing the bag on a constant-temperature magnetic stirrer, dialyzing the bag in tap water for 24 hours, changing tap water once every 4 hours during the period, changing water for three times, standing the bag overnight for 12 hours, then changing distilled water for dialysis for 12 hours, changing distilled water once every 4 hours during the period, changing water for three times, transferring the dialyzate into a culture dish after the dialysis is finished, and freeze-drying the dialyzate for 24 hours to obtain the purified polysaccharide powder of the Phlebopus portentosus fruiting body.
Example 4
The purified polysaccharide powder of the fruit body of Phlebopus portentosus obtained in example 3 was subjected to the measurement of α -glucosidase activity, the determination of the inhibition mechanism of glucosidase and the kinetic analysis of α -glucosidase inhibition.
(1) Preparing a polysaccharide sample of the Phlebopus portentosus fruiting body;
preparing a polysaccharide sample of the Phlebopus portentosus fruiting body: 10mg of a Phlebopus portentosus fruiting body polysaccharide sample is precisely weighed, dissolved by 4mL of 0.01mol/L PBS solution to obtain 2.5mg/mL of Phlebopus portentosus fruiting body polysaccharide solution, and the solution is respectively diluted into polysaccharide solutions with the concentration of 0.25-2.0mg/mL (0.25 mg/mL, 0.5mg/mL, 1.0mg/mL, 1.5mg/mL and 2.0 mg/mL), and each solution is 1mL.
(2) Measuring the activity of alpha-glucosidase;
A. and (3) a phlebopus portentosus fruiting body polysaccharide sample group: absorbing 40 mu L of the phlebopus portentosus sporocarp polysaccharide solution with different concentrations into a 96-pore plate, adding 30 mu L of alpha-glucosidase with the concentration of 0.2U/mL, mixing the solution evenly by gentle shaking, incubating the solution for 10min in a biochemical incubator at 37 ℃, then adding 30 mu L of PNPG solution with the concentration of 0.5mmol/L, incubating the solution for 20min in the biochemical incubator at 37 ℃, finally adding 50 mu L of sodium carbonate solution into each pore to terminate the reaction, wherein the concentration of the sodium carbonate solution is 0.5mol/L, and measuring the absorbance of the solution at 405nm by an enzyme reader after 5 min;
B. control group of Phlebopus portentosus fruiting body samples: changing the step of adding 30 mu L of alpha-glucosidase into the step of adding 30 mu L of PBS solution, and the rest steps are the same;
C. blank control group: replacing the polysaccharide of the Phlebopus portentosus fruiting body with an equal amount of PBS solution, and carrying out the same steps; using acarbose solution as a positive control, each sample was subjected to three parallel experiments, and the inhibitory activity of α -glucosidase was calculated as follows:
alpha-glucosidase inhibition (%) = [1- (A1-A2)/A0 ]. Times.100%
A0 is the absorbance value of the blank control group; a1 is the absorbance value of a polysaccharide sample group; a2 is the absorbance value of the sample control group, and all results are measured as inhibition.
(3) Determination of the inhibitory mechanism of glucosidase
Under the condition of fixing the concentration of a substrate, changing the dosage of enzyme, measuring the influence of polysaccharide components of the bolete portentosus sporocarp with different concentrations on the inhibition of alpha-glucosidase, taking 0.5mmol/L PNPG as the substrate, taking no inhibitor as a blank group, taking the bolete portentosus polysaccharide with different concentrations as a sample group, respectively adding 0.2U/mL of 30-120 mu L (30, 60, 90 and 120 mu L) of alpha-glucosidase solution, and plotting by using an enzyme reader at 405nm according to the alpha-glucosidase activity measuring method of (2) and taking the dosage of the enzyme as a horizontal coordinate and the corresponding reaction rate as a vertical coordinate, wherein the plot reflects the relationship between the residual enzyme activity of the alpha-glucosidase after the action of the bolete portentosus polysaccharose components of the bolete portentosae sporocarp and the dosage of the enzyme.
(4) Kinetic analysis of α -glucosidase inhibition:
preparation of a PNP standard curve: adding PNP solution (0.5 mmol/L) of 0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL into the test tube in sequence, adding sodium carbonate solution of 0.5mol/L to 2.3mL, using distilled water as blank control, and measuring the absorbance value at 405nm by using an enzyme-labeling instrument; and (3) taking the volume of the PNP as an abscissa and the absorbance value A as an ordinate to make a linear regression equation to obtain the PNP standard curve.
Determination of the type of alpha-glucosidase inhibition: under the condition that the concentration of the immobilized enzyme is 0.2U/mL, the concentration of the added substrate PNPG is changed, and the influence of the phlebopus portentosus fruiting body polysaccharide on the activity of the alpha-glucosidase is measured. The method is the same as the method (2), and the concentration of the substrate PNPG is changed to be 0.125, 0.25, 0.5, 1.0 and 2.0mmol/L. The initial rate of the enzyme reaction was plotted against the substrate concentration, plotted by the Lineweaver-Burk reciprocal double equation, and the type of inhibition was judged.
(5) And (3) analysis of experimental results:
A. as shown in fig. 4, the refined crude polysaccharide powder obtained by alcohol precipitation of the polysaccharide extract of the Phlebopus portentosus fruiting body is sequentially eluted by distilled water and NaCl solutions with the concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mol/L to obtain an elution curve; after DEAE-52 cellulose column chromatography, three obvious elution peaks with different contents are obtained. Wherein, the elution peak area of the distilled water elution peak and the elution peak area of NaCl with the concentration of 0.1mol/L are larger, the content is relatively more, the corresponding parts of the eluents are combined together, and after dialysis treatment and freeze drying, three kinds of purified polysaccharide component powder of phlebopus portentosus are obtained: PPP-0, PPP-1 and PPP-2 (front PP is the first letter of Phlebus portentosus of the Latin of Phlebus veitchii, and the latter P is the first letter of Polysaccharide English Polysaccharide, so called PPP for short, and the three distinct elution peaks are marked as PPP-0, PPP-1 and PPP-2 in the order of elution in sequence), wherein the content of PPP-0 is the maximum, and the content of PPP-2 is 62.7mg as minimum.
B. An alpha-glucosidase inhibition experiment is carried out to obtain a phlebopus portentosus fruiting body crude polysaccharide inhibition rate curve, and the inhibition rate curve of the positive control drug acarbose is compared under the same condition. Therefore, the crude polysaccharide of the Phlebopus portentosus fruiting body can be preliminarily judged to have a certain inhibition effect on the alpha-glucosidase.
In order to screen out the purified polysaccharide component with the best hypoglycemic activity, enzyme inhibition experiments are respectively carried out on the crude polysaccharide of the phlebopus portentosus sporocarp, PPP-0, PPP-1 and PPP-2, the inhibition rate curve is shown in figure 1, when the concentration of the polysaccharide is 0.25-2.5 mg/mL, the crude polysaccharide, PPP-0, PPP-1 and PPP-2 of the phlebopus portentosus polysaccharide have certain alpha-glucosidase inhibition activity, and the inhibition rates of the PPP-1 and PPP-2 and the concentration of the polysaccharide have positive correlation dose-effect relationship.
The PPP-1 and PPP-2 in the purified polysaccharide component show a rising trend in the range of 0.25 mg/mL-2.5 mg/mL of polysaccharide concentration, and the inhibition rate of the PPP-1 on the alpha-glucosidase is from 32.20% to 57.15% and the inhibition rate of the PPP-2 on the alpha-glucosidase is from 39.04% to 70.55% along with the increase of the concentration. At the same polysaccharide concentration (1.0 mg/mL), the inhibition ratios of different polysaccharide components to alpha-glucosidase are as follows: PPP-2 >; at the same polysaccharide concentration (2.0 mg/mL), the inhibition ratios of different polysaccharide components to alpha-glucosidase are as follows: PPP-2> crude polysaccharide > PPP-0> < PPP-1, wherein the inhibitory activity of PPP-2 is much higher than that of the other three components. Because the content of the polysaccharide component PPP-2 obtained by the extraction, separation and purification experiment is the least, and the content of the polysaccharide component PPP-0 is the greatest, the invention selects the PPP-0 component to carry out the enzyme inhibition kinetics experiment, and explores the inhibition mechanism and the type.
C. The absorbance values of all groups are measured at 405nm by an enzyme-labeling instrument, the absorbance values are substituted into a PNP standard curve regression equation, the reaction rates after different enzyme amounts are added are obtained through calculation, the reaction rates corresponding to the enzyme amounts are plotted, the result is shown in figure 2, the inhibition kinetic curve of PPP-0 with different concentrations on alpha-glucosidase is obtained, and the relation between the residual enzyme activity after the alpha-glucosidase is acted by PPP-0 polysaccharide components and the added different enzyme amounts is analyzed. After the alpha-glucosidase is acted by 1mg/mL PPP-0, the data analysis shows that the relationship between the enzyme activity and the enzyme quantity is a straight line, the equation is y =0.0174x-7E-05, R 2 =0.9999; as the concentration of PPP-0 is increased to 2mg/mL, the relationship between the enzyme activity and the enzyme amount is obliqueStraight line of rate decrease, equation y =0.0169x-9E-05 2 =0.9998. Since the vertical intercept of the two curves is small, approaching 0, it can be considered to cross the origin, indicating that PPP-0 inhibits α -glucosidase reversibly.
The PPP-0 and the alpha-glucosidase are combined reversibly, the activity of the enzyme is inhibited, and the catalytic efficiency of the enzyme is reduced, but the molecular conformation of the enzyme is changed permanently due to the increase of the concentration of the PPP-0, so that the effective enzyme amount is reduced, and the catalytic efficiency of the enzyme is reduced.
Definition of enzyme activity units: an amount of enzyme capable of converting PNPG into 1. Mu. Mol PNP within 1min at 37 ℃ and pH 6.8.
D. In an enzyme activity measuring system, the concentration of a substrate PNPG is changed, an alpha-glucosidase solution with the concentration of 0.2U/mL is added, the influence of PPP-0 with different concentrations on the enzyme activity is measured, and a group without adding enzyme solution PPP-0 is used as a control. The results of double reciprocal mapping by the Lineweaver-Burk method are shown in FIG. 3, and it can be seen that two straight lines of PPP-0 and non-inhibition intersect at the ordinate 1/[ V ], and when an inhibitor exists, the Km value of the Michaelis constant is gradually increased, the maximum reaction rate Vmax is kept constant, and the inhibition type of the Phlebopus portentosus PPP-0 component on the alpha-glucosidase can be presumed to be competitive inhibition.
Therefore, the inhibition of the alpha-glucosidase activity by the polysaccharide of the Phlebopus portentosus fruiting body is one of the reasons for the hypoglycemic activity of the Phlebopus portentosus fruiting body, the PPP-0 inhibition type is competitive reversible inhibition, and the generation of blood sugar after meal is reduced.
In conclusion, the invention takes the phlebopus portentosus sporocarp as a raw material, the phlebopus portentosus polysaccharide obtained by multiple water extraction and alcohol precipitation extraction, combined adsorption elution by a multi-gradient NaCl solution-DEAE-52 cellulose chromatographic column, separation and purification mainly comprises three polysaccharide components of PPP-0, PPP-1 and PPP-2, the content of the PPP-0 is the most, the PPP-1 and the PPP-2 are in a positive correlation dose-effect relationship in a concentration range of 0.25-2.5 mg/mL, and reversible inhibition of the PPP-0 polysaccharide component on alpha-glucosidase is known to be competitive inhibition in determination of an enzyme activity system and enzyme inhibition kinetics analysis, so that the phlebopus portus portentosus polysaccharide can be used for preparing a hypoglycemic medicament.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (6)
1. An application of the Phlebopus portentosus polysaccharide prepared by the extraction method of the Phlebopus portentosus polysaccharide is characterized in that the Phlebopus portentosus polysaccharide is applied to the preparation of a hypoglycemic medicament, and the extraction method of the Phlebopus portentosus polysaccharide comprises the following steps:
s1, taking powder of a Phlebopus portentosus fruiting body, adding 95% ethanol, mixing, performing ultrasonic dispersion, standing at room temperature, removing a supernatant, and keeping a precipitate 1; the ultrasonic dispersion temperature is 40 ℃, and the ultrasonic time is 30min;
s2, adding water into the precipitate 1, leaching in a water bath, filtering, retaining filtrate, adding water into filter residues, repeatedly extracting for 3-5 times under the same condition, combining the filtrates, performing rotary evaporation, and concentrating to obtain a concentrated extract; the water bath extraction temperature is 80 ℃, the extraction time is 1 to 3 hours, and the rotary evaporation temperature is 50 to 70 ℃;
s3, adding 95% ethanol with 4 times volume of the concentrated extracting solution into the concentrated extracting solution under magnetic stirring for precipitation, standing at low temperature, centrifuging, and removing supernatant to obtain a precipitate 2;
s4, adding water to dissolve the precipitate 2, adding a Sevage reagent to extract, taking supernate, repeating the process for 2-4 times, and combining the supernate;
s5, adding the supernatant into SP-825 macroporous resin, and carrying out magnetic stirring and decoloring to obtain a decoloring solution;
s6, adding the decolorized solution into 95% ethanol for precipitation, standing at a low temperature, removing supernatant, centrifuging to obtain a precipitate 3, pre-freezing, and freeze-drying to obtain polysaccharide crude extract powder;
s7, adding water to the crude polysaccharide powder for dissolving, centrifuging, taking supernatant, performing DEAE-52 fiber column chromatography, sequentially eluting with distilled water and NaCl solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0mol/L, detecting polysaccharide content by using a phenol-concentrated sulfuric acid method to obtain a gradient curve of purified polysaccharide content of the phlebopus portentosus fruiting body, obtaining three obvious elution peaks, and marking the three obvious elution peaks as PPP-0, PPP-1 and PPP-2 in a sequential elution order to obtain PPP-2 eluent;
s8, concentrating, dialyzing and freeze-drying the PPP-2 eluent to obtain the boletus fuscogilus polysaccharide powder.
2. The application of the phlebopus portentosus polysaccharide prepared by the extraction method of the phlebopus portentosus polysaccharide according to claim 1 is characterized in that in the step S4, the Sevage reagent is prepared by mixing chloroform and n-butanol in a volume ratio of 4:1.
3. The application of the phlebopus portentosus polysaccharide prepared by the extraction method of the phlebopus portentosus polysaccharide according to claim 1 is characterized in that in the step S3, the centrifugal rotating speed is 4000rpm, and the centrifugal time is 5min.
4. The use of a polysaccharide of phlebopus portentosus prepared by the method for extracting a polysaccharide of phlebopus portentosus according to claim 1, wherein in steps S3 and S6, the low temperature standing is 24h at 4 ℃.
5. The use of the polysaccharide of phlebopus portentosus as claimed in claim 1, wherein in step S8, said dialysis comprises placing the concentrated solution in MD34 dialysis bag, placing it on a constant temperature magnetic stirrer, dialyzing 24h in tap water, while changing tap water once every 4h, changing water three times, placing it overnight for 12h, then changing distilled water for dialysis for 12h, while changing distilled water once every 4h, and changing water three times.
6. The use of the Phlebopus portentosus polysaccharide obtained by the method for extracting Phlebopus portentosus polysaccharide according to claim 1, wherein the Phlebopus portentosus polysaccharide is used for preparing the Phlebopus portentosus polysaccharideα-glucosidase inhibitors for use in hypoglycemic agents.
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