CN111944860A - Marasmius androsaceus exopolysaccharide and application thereof - Google Patents
Marasmius androsaceus exopolysaccharide and application thereof Download PDFInfo
- Publication number
- CN111944860A CN111944860A CN201910411418.4A CN201910411418A CN111944860A CN 111944860 A CN111944860 A CN 111944860A CN 201910411418 A CN201910411418 A CN 201910411418A CN 111944860 A CN111944860 A CN 111944860A
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- Prior art keywords
- marasmius androsaceus
- exopolysaccharide
- polysaccharide
- fermentation
- androsaceus
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
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Abstract
The invention provides an Marasmius androsaceus extracellular polysaccharide and application thereof, belonging to the technical field of biological extraction. The molecular weight of the exocellular polysaccharide of Marasmius androsaceus is 10.47 kDa; wherein, the molar ratio of monosaccharide composition is mannose: glucose: galactose ═ 0.08: 0.34: 1.46. the marasmius androsaceus exopolysaccharide is extracted from marasmius androsaceus fermentation liquor for the first time, has a novel structure, adopts animal experiments to test that MEPS3 polysaccharide has a good anti-fatigue effect, and is mainly used for preparing anti-fatigue medicines, health-care products and the like.
Description
Technical Field
The invention belongs to the technical field of biological extraction, and particularly relates to Marasmius androsaceus extracellular polysaccharide and application thereof.
Background
Marasmius androsaceus (Marasmius androsaceus) is a medicinal fungus which generally grows in dense forest lands, and is called as Bidens pilosa, Aureobasidium fuscus and the like due to black color and the aweto-like rhizomorph with concentrated growth. Studies have shown that Marasmius androsaceus contains many active substances, such as cholesterol acetate, amino acids, ergosterol, cinnamic acid, beta-sitosterol, alkaloids, proteins, mannitol, adenosine, and polysaccharides.
The polysaccharide has various biological activities and functions, such as antioxidation, blood sugar reduction, immunity enhancement and the like, and the pharmacological activity and the like of the polysaccharide are closely related to the structure of the polysaccharide. At present, pharmacological research on marasmius androsaceus polysaccharide mainly focuses on the effects of immunoregulation, antioxidation, analgesia and the like.
For example, Chinese patent 201310150040.X discloses the use of enteric maramins androsaceus glycoprotein microspheres containing an absorption enhancer in the preparation of analgesic and anti-inflammatory drugs. The study on the in vitro antioxidant properties of extracellular and intracellular polysaccharides of Marasmius androsaceus was disclosed in the sixth academic report of Life sciences, Jilin, et al.
However, at present, research mainly aims at the crude polysaccharide component of marasmius androsaceus, and the component complexity of the crude polysaccharide hinders the improvement of the medicinal level of marasmius androsaceus, and no report is found on the research on the aspects of purification and separation of marasmius androsaceus to obtain a single polysaccharide component and related activity thereof.
Disclosure of Invention
The invention provides an marasmius androsaceus exopolysaccharide and application thereof, the polysaccharide is extracted from marasmius androsaceus for the first time, and the marasmius androsaceus exopolysaccharide has uniform components, novel structure and good anti-fatigue effect.
An marasmius androsaceus exopolysaccharide is prepared by the following steps:
(1) inoculating Marasmius androsaceus to potato culture medium, and activating to obtain seed solution;
(2) inoculating the seed liquid into a fermentation tank, and performing fermentation culture to obtain fermentation liquid;
(3) filter-pressing the fermentation liquor to obtain fermentation filtrate, concentrating the fermentation filtrate under reduced pressure, adding absolute ethyl alcohol, stirring, standing, centrifuging, collecting precipitate, and freeze-drying to obtain crude polysaccharide;
(4) and (3) performing ion exchange column chromatography on the crude polysaccharide to obtain three components, wherein the component with the lowest content is the obtained marasmius androsaceus exopolysaccharide.
Further, in the step (1), the preparation method of the potato culture medium comprises the following steps: peeling 200 and 300g of potatoes, dicing, boiling in boiling water for 30min, filtering, adding 15-20g of glucose, and metering to 1L;
the conditions for activation include: the activation is carried out in a shaking table, and the rotation speed of the shaking table is 180rpm-250 rpm;
the activation temperature is 26-30 ℃, and the activation time is 5-8 days.
Further, in the step (2), the culture medium for fermentation culture is: sucrose and/or glucose 10-20g/L, yeast extract powder and/or yeast extract and/or peptone 20-35g/L, KH2PO4 2.0-3.0g/L，MgSO4·7H2O2.0-3.5 g/L, pH 7.0;
the conditions of the fermentation culture comprise: the rotation speed is 220rpm-300rpm, the fermentation temperature is 26-30 ℃, the fermentation time is 5-8 days, and the ventilation volume is 150L/h-300L/h.
Further, in the step (3), the fermentation filtrate is decompressed and concentrated to a relative density of 1.15-1.25 at 80 ℃;
standing at 4 deg.C for 12 h;
the volume ratio of the absolute ethyl alcohol to the concentrated fermentation filtrate is 4: 1;
the centrifugation condition is 5000-.
Further, the step (4) is specifically as follows: dissolving the crude polysaccharide to prepare a crude polysaccharide solution, transferring the crude polysaccharide solution into a chromatographic column, performing gradient elution by using distilled water and a sodium chloride solution, and collecting the crude polysaccharide solution into an elution pipe to determine the content of the polysaccharide; drawing an ion exchange chromatography elution curve, combining eluents in the same absorption peak, concentrating, dialyzing and freeze-drying.
Further, the molecular weight of the exocellular polysaccharide of marasmius androsaceus is 10.47 kDa; wherein, the molar ratio of monosaccharide composition is mannose: glucose: galactose ═ 0.08: 0.34: 1.46.
furthermore, the exocellular polysaccharide of Marasmius androsaceus is pyran type polysaccharide.
The invention also provides application of the marasmius androsaceus exopolysaccharide in preparing anti-fatigue medicaments.
The invention also provides application of the Marasmius androsaceus extracellular polysaccharide in preparation of anti-fatigue health-care products.
Compared with the prior art, the marasmius androsaceus exopolysaccharide and the application thereof provided by the invention have the following advantages:
the exocellular polysaccharide of marasmius androsaceus is extracted from marasmius androsaceus fermentation liquor for the first time, has a novel structure and uniform components, the molecular weight of the exocellular polysaccharide is 10.47kDa, monosaccharide consists of mannose, glucose and galactose, and the molar ratio is 0.08: 0.34: 1.46. FT-IR and NMR are adopted to analyze the structure of the polysaccharide MEPS3 to obtain pyran type polysaccharide; the polysaccharide MEPS3 has heterogeneous morphology and various particle forms by SEM analysis. Animal experiments prove that the MEPS3 polysaccharide has good anti-fatigue effect. The purity of the exocellular polysaccharide of the marasmius androsaceus provided by the invention is up to more than 96%.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is the elution curves of ectopolysaccharides MEPS1, MEPS2 and MEPS3 from Marasmius androsaceus in the example of the present invention;
FIG. 2 is a graph of an infrared (FT-IR) spectrum of Marasmius androsaceus exopolysaccharide MEPS3 in example of the present invention;
FIG. 3 is a nuclear magnetic hydrogen spectrum (H-NMR) chart of Marasmius androsaceus exopolysaccharide MEPS3 in the example of the present invention;
FIG. 4 is the nuclear magnetic carbon spectrum (C) of ectochilus formosanus exopolysaccharide MEPS3 in the embodiment of the invention13-NMR) map;
FIG. 5 is a Scanning Electron Microscope (SEM) image of the extracellular polysaccharide MEPS3 of Marasmius androsaceus in the example of the present invention;
FIG. 6 shows the effect of extracellular polysaccharide MEPS1, MEPS2 and MEPS3 of Marasmius androsaceus on the swimming time of a mouse under load in the example of the present invention;
FIG. 7 shows the effect of extracellular polysaccharide MEPS1, MEPS2 and MEPS3 of Marasmius androsaceus on the balance time of the mouse fatigue rolling bar experiment in the example of the present invention;
FIG. 8 shows the effect of extracellular polysaccharide MEPS1, MEPS2 and MEPS3 of Marasmius androsaceus obtained in the present invention on the number of mouse shock experiments.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
Marasmius androsaceus (Marasmius androsaceus) is a medicinal fungus which contains many active substances, such as amino acids, polysaccharides and other macromolecular components. At present, the research on marasmius androsaceus polysaccharide mainly focuses on crude polysaccharide, but the components are complex, the medicinal mechanism is not clear, and the improvement of the medicinal level of marasmius androsaceus is hindered.
The embodiment of the invention provides an marasmius androsaceus exopolysaccharide which is a uniform component and has the molecular weight of 10.47 kDa; wherein, the molar ratio of monosaccharide composition is mannose (Man): glucose (Glc): galactose (Gal) ═ 0.08: 0.34: 1.46.
specifically, the Marasmius androsaceus exopolysaccharide is a pyran polysaccharide.
The invention further separates and purifies the crude polysaccharide outside the marasmius androsaceus cell to obtain uniform components of marasmius androsaceus extracellular polysaccharide, and analyzes monosaccharide composition, molecular weight, structure, medicinal activity and the like of the marasmius androsaceus extracellular polysaccharide to obtain a polysaccharide component with an anti-fatigue effect, thereby providing important parameter basis and novel thinking for the development of future marasmius androsaceus high added-value products.
The embodiment of the invention provides an marasmius androsaceus exopolysaccharide, which is prepared by the following steps:
(1) activating strains: inoculating Marasmius androsaceus to potato culture medium, and activating to obtain seed solution;
(2) and (3) strain culture: inoculating the seed liquid obtained in the step (1) into a fermentation tank, and culturing to obtain fermentation liquor;
(3) polysaccharide extraction: filter-pressing the fermentation liquor obtained in the step (2) to obtain fermentation filtrate, concentrating the fermentation filtrate under reduced pressure, adding absolute ethyl alcohol, stirring, standing, centrifuging, precipitating, removing protein, and freeze-drying to obtain crude polysaccharide;
(4) polysaccharide separation: and (3) performing ion exchange column chromatography to obtain three components, wherein the component with the lowest content is the obtained marasmius androsaceus exopolysaccharide, and is marked as MEPS 3.
In the invention, crude polysaccharide outside Marasmius androsaceus is extracted by an alcohol precipitation method, protein is removed by a Sevag method, and the polysaccharide is separated by an anion exchange chromatography method, so that uniform Marasmius androsaceus extracellular polysaccharide MEPS3 with high purity is extracted and separated.
In an embodiment of the present invention, in step (1), the preparation method of the potato culture medium includes: peeling 200 and 300g of potatoes, dicing, boiling in boiling water for 30min, filtering, adding 15-20g of glucose, and metering to 1L.
Further, in the step (1), the activation conditions include: the activation is carried out in a shaking table, and the rotation speed of the shaking table is 180rpm-250 rpm; the activation temperature is 26-30 ℃, and the activation time is 5-8 days.
In one embodiment of the present invention, in step (2), the culture medium for fermentation culture is: sucrose and/or glucose 10-20g/L, yeast extract powder and/or yeast extract and/or peptone 20-35g/L, KH2PO4 2.0-3.0g/L，MgSO4·7H2O2.0-3.5 g/L and pH 7.0. The sucrose and/or glucose is at least one of sucrose or glucose. At least one of yeast extract powder and/or yeast extract and/or peptone.
Further, in the step (2), the conditions of the fermentation culture comprise: the rotation speed is 220rpm-300rpm, the fermentation temperature is 26-30 ℃, the fermentation time is 5-8 days, and the ventilation volume is 150L/h-300L/h.
In one embodiment of the invention, in the step (3), the filter pressing is performed by using a plate-and-frame filter press.
Further, in the step (3), the temperature of the standing is 4 ℃, and the time of the standing is 12 hours.
Further, in the step (3), the fermentation filtrate is concentrated under reduced pressure to a relative density of 1.15 to 1.25.
Further, in the step (3), the volume ratio of the absolute ethyl alcohol to the concentrated fermentation filtrate is 4: 1.
Further, in the step (3), the centrifugation condition is that the rotation speed is 5000-.
In an embodiment of the present invention, the step (4) specifically includes: dissolving the crude polysaccharide to prepare a crude polysaccharide solution, transferring the crude polysaccharide solution into a chromatographic column, performing gradient elution by using distilled water and a sodium chloride solution, and collecting the crude polysaccharide solution into an elution pipe to determine the content of the polysaccharide; drawing an ion exchange chromatography elution curve, combining eluents in the same absorption peak, concentrating, dialyzing and freeze-drying.
The method specifically comprises the following steps: dissolving the crude polysaccharide to prepare a crude polysaccharide solution, transferring the crude polysaccharide solution into a chromatographic column, performing gradient elution by using distilled water and sodium chloride solutions of 0.1M (mol/L), 0.2M (mol/L), 0.3M (mol/L) and 0.4M (mol/L), collecting the crude polysaccharide solution into elution tubes with different serial numbers to measure the content of the polysaccharide, drawing an ion exchange chromatography elution curve, combining eluents in the same absorption peak, concentrating, dialyzing and freeze-drying to obtain each polysaccharide component.
Specifically, the concentration of the crude polysaccharide solution is 10-12 mg/mL.
Furthermore, DEAE gel column is selected as the chromatographic column for the ion exchange column chromatography; DEAE-52 was used as a column.
Furthermore, in the elution process, the flow rate of the eluent is 1.5-2mL/min, the dosage of the eluent is 500-800mL, and the automatic receiver is set to be 1-2 min/tube.
Further, the polysaccharide content in each tube was measured by the phenol-sulfuric acid method.
The invention also provides application of the marasmius androsaceus exopolysaccharide MEPS3 in preparation of anti-fatigue drugs.
The invention also provides application of the Marasmius androsaceus exopolysaccharide MEPS3 in preparation of anti-fatigue health care products.
In the embodiment of the invention, the fatigue resistance of the test animal taking the MEPS3 medicine is better through a weight swimming experiment, a fatigue rod rotating experiment and an electric shock experiment.
The present invention will be described in detail below with reference to the embodiments with reference to the attached drawings.
Example 1Extraction and separation of Marasmius androsaceus polysaccharide MEPS3 component
(1) Activating strains: inoculating Marasmius androsaceus to potato culture medium, and activating to obtain seed solution;
the preparation method of the potato culture medium comprises the following steps: peeling 250g of potato, dicing, boiling in boiling water for 30min, filtering, adding 18g of glucose, and metering to 1L; the activation conditions were: the rotating speed of the shaking table is 200rpm, the fermentation temperature is 28 ℃, and the fermentation time is 7 days.
(2) And (3) strain culture: inoculating the seed liquid obtained in the step (1) into a fermentation tank, and culturing to obtain fermentation liquor;
wherein the fermentation medium is: 15g/L of sucrose and/or glucose, 28g/L of yeast extract powder and/or yeast extract and/or peptone, KH2PO4 2.5g/L，MgSO4·7H2O3 g/L, pH 7.0; the fermentation culture conditions are as follows: the rotation speed is 260rpm, the fermentation temperature is 28 ℃, the fermentation time is 7 days, and the ventilation volume is 200L/h.
(3) Polysaccharide extraction: filtering the fermentation liquor obtained in the step (2) by adopting a plate-frame filter pressing method to obtain fermentation filtrate; concentrating the filtrate under reduced pressure to relative density of 1.2(80 deg.C) with rotary evaporator, adding four times volume of anhydrous ethanol, precipitating with ethanol at 4 deg.C for 12h, centrifuging at 5000r/min for 20min to obtain precipitate, adding water to the precipitate for redissolving, adsorbing pigment and Sevag reagent with activated carbon for repeatedly removing protein, centrifuging, concentrating under reduced pressure, and freeze drying to obtain crude polysaccharide.
(4) And (3) separating crude polysaccharide: dissolving the crude polysaccharide to prepare a crude polysaccharide solution, transferring the crude polysaccharide solution into a chromatographic column, performing gradient elution by using distilled water and sodium chloride solutions of 0.1M, 0.2M, 0.3M and 0.4M, and collecting the crude polysaccharide solution to an elution tube to determine the content of the polysaccharide; and then drawing an ion exchange chromatography elution curve by taking the serial number of each tube as an abscissa and the absorbance value of each tube as an ordinate. Combining eluates in the same absorption peak, concentrating under reduced pressure, dialyzing, lyophilizing, collecting corresponding components, respectively marking as MEPS1, MEPS2 and MEPS3, and calculating the content of each polysaccharide component, wherein the result shows that the content ratio of MEPS1, MEPS2 and MEPS3 is 6:2: 1. Wherein the component with the lowest content is the obtained target Marasmius androsaceus exopolysaccharide MEPS 3.
The elution curve of the Marasmius androsaceus exopolysaccharide (MEPS) sample by ion exchange column chromatography is shown in figure 1.
Example 2Extraction and separation of Marasmius androsaceus polysaccharide MEPS3 component
(1) Activating strains: inoculating Marasmius androsaceus to potato culture medium, and activating to obtain seed solution;
the preparation method of the potato culture medium comprises the following steps: peeling 200g of potato, dicing, boiling in boiling water for 30min, filtering, adding 15g of glucose, and metering to 1L; the activation conditions were: the rotating speed of the shaking table is 180rpm, the fermentation temperature is 26 ℃, and the fermentation time is 5 days.
(2) And (3) strain culture: inoculating the seed liquid obtained in the step (1) into a fermentation tank, and culturing to obtain fermentation liquor;
wherein the fermentation medium is: 10g/L of sucrose, 20g/L of yeast extract and KH2PO4 2.0g/L，MgSO4·7H2O2.0 g/L, pH 7.0; the fermentation culture conditions are as follows: the rotation speed is 220rpm, the fermentation temperature is 26 ℃, the fermentation time is 5 days, and the ventilation volume is 150L/h.
(3) Polysaccharide extraction: filtering the fermentation liquor obtained in the step (2) by adopting a plate-frame filter pressing method to obtain fermentation filtrate; concentrating the filtrate under reduced pressure to relative density of 1.15(80 deg.C) with rotary evaporator, adding four times volume of anhydrous ethanol, precipitating with ethanol at 4 deg.C for 12h, centrifuging at 5000r/min for 20min to obtain precipitate, adding water to the precipitate for redissolving, adsorbing pigment and Sevag reagent with activated carbon for repeatedly removing protein, centrifuging, concentrating under reduced pressure, and freeze drying to obtain crude polysaccharide.
(4) And (3) separating crude polysaccharide: dissolving the crude polysaccharide to prepare a crude polysaccharide solution, transferring the crude polysaccharide solution into a chromatographic column, performing gradient elution by using distilled water and sodium chloride solutions of 0.1M, 0.2M, 0.3M and 0.4M, and collecting the crude polysaccharide solution to an elution tube to determine the content of the polysaccharide; and then drawing an ion exchange chromatography elution curve by taking the serial number of each tube as an abscissa and the absorbance value of each tube as an ordinate. Combining eluates in the same absorption peak, concentrating under reduced pressure, dialyzing, lyophilizing, collecting corresponding components, respectively marking as MEPS1, MEPS2 and MEPS3, and calculating the content of each polysaccharide component, wherein the result shows that the content ratio of MEPS1, MEPS2 and MEPS3 is 6:2: 1. Wherein the component with the lowest content is the obtained target Marasmius androsaceus exopolysaccharide MEPS 3.
Example 3Extraction and separation of Marasmius androsaceus polysaccharide MEPS3 component
(1) Activating strains: inoculating Marasmius androsaceus to potato culture medium, and activating to obtain seed solution;
the preparation method of the potato culture medium comprises the following steps: peeling 300g of potato, dicing, boiling in boiling water for 30min, filtering, adding 20g of glucose, and metering to 1L; the activation conditions were: the rotating speed of the shaking table is 250rpm, the fermentation temperature is 30 ℃, and the fermentation time is 8 days.
(2) And (3) strain culture: inoculating the seed liquid obtained in the step (1) into a fermentation tank, and culturing to obtain fermentation liquor;
wherein the fermentation medium is: 20g/L glucose, 35g/L yeast extract powder and KH2PO4 3.0g/L，MgSO4·7H2O3.5 g/L, pH 7.0; the fermentation culture conditions are as follows: the rotation speed is 300rpm, the fermentation temperature is 30 ℃, the fermentation time is 8 days, and the ventilation volume is 300L/h.
(3) Polysaccharide extraction: filtering the fermentation liquor obtained in the step (2) by adopting a plate-frame filter pressing method to obtain fermentation filtrate; concentrating the filtrate under reduced pressure to relative density of 1.25(80 deg.C) with rotary evaporator, adding four times volume of anhydrous ethanol, precipitating with ethanol at 4 deg.C for 12h, centrifuging at 5000r/min for 20min to obtain precipitate, adding water to the precipitate for redissolving, adsorbing pigment and Sevag reagent with activated carbon for repeatedly removing protein, centrifuging, concentrating under reduced pressure, and freeze drying to obtain crude polysaccharide.
(4) And (3) separating crude polysaccharide: dissolving the crude polysaccharide to prepare a crude polysaccharide solution, transferring the crude polysaccharide solution into a chromatographic column, performing gradient elution by using distilled water and sodium chloride solutions of 0.1M, 0.2M, 0.3M and 0.4M, and collecting the crude polysaccharide solution to an elution tube to determine the content of the polysaccharide; and then drawing an ion exchange chromatography elution curve by taking the serial number of each tube as an abscissa and the absorbance value of each tube as an ordinate. Combining eluates in the same absorption peak, concentrating under reduced pressure, dialyzing, lyophilizing, collecting corresponding components, respectively marking as MEPS1, MEPS2 and MEPS3, and calculating the content of each polysaccharide component, wherein the result shows that the content ratio of MEPS1, MEPS2 and MEPS3 is 6:2: 1. Wherein the component with the lowest content is the obtained target Marasmius androsaceus exopolysaccharide MEPS 3.
Example 4Purity and molecular weight analysis of Marasmius androsaceus exopolysaccharide MEPS3
The polysaccharide MEPS3 from example 1 was formulated in a 1mg/mL solution with deionized water and filtered through a 0.22 μm filter.
Chromatographic conditions are as follows: an Agilent series high performance liquid chromatograph system is used; the chromatographic column is a TSK G5000PWXL column (7.8mm multiplied by 300 mm); the detector is an RI differential detector; the mobile phase is ultrapure water; the flow rate is 0.6 mL/min; the column temperature is 25 ℃; the temperature of the detector is 35 ℃; the sample volume of the sample and the glucan standard is 20 mu L;
log M of various dextran standards (2000KDa, 500KDa, 70KDa, 40KDa and 10KDa) is plotted on the ordinate versus the time to peak (t) on the abscissa, and the monosaccharide standard curve is-0.186 x + 7.997; r2＝0.9994；
The results showed that MEPS3 had a purity of 96.73% and a molecular weight of 10.47 kDa.
Example 5Monosaccharide composition analysis of Marasmius androsaceus exopolysaccharide MEPS3
The MEPS3 obtained in example 1 was analyzed for monosaccharide composition by High Performance Liquid Chromatography (HPLC).
Collecting polysaccharide MEPS3 sample 2mg, adding 2M TFA (trifluoroacetic acid) 2mL, sealing, hydrolyzing at 120 deg.C for 3h, cooling to room temperature, centrifuging to collect supernatant, adding 5mL methanol, concentrating under reduced pressure, and blowing nitrogen to dry to obtain polysaccharide hydrolysate.
PMP (1-phenyl-3-methyl-5 pyrazolone) is adopted for derivatization, and the specific operation is as follows: dissolving in water to obtain sample hydrolysate, mixing with 100 μ L of 0.3M NaOH solution and 100 μ L of 2M PMP-methanol solution, and mixing. Sealing in water bath at 70 deg.C for 30min, cooling to room temperature, centrifuging, collecting supernatant, adding 100 μ L0.3M HCl, mixing, adding 900 μ L chloroform to remove excessive PMP, centrifuging to remove lower chloroform layer, collecting aqueous layer polysaccharide derivative, diluting with 500 μ L water, passing through 0.22 μ M membrane, and performing HPLC analysis;
the HPLC detection conditions were as follows: c18 column chromatography (4.6X 150 mm); mobile phase 0.1M pH 6.7 phosphate buffer salt: acetonitrile (v/v) ═ 83: 17; the flow rate is 1 mL/min; the column temperature is 30 ℃; the detection wavelength is 250 nm; the loading amount is 20 mu L;
performing HPLC analysis under the above conditions with mannose, ribose, rhamnose, glucuronic acid, glucose, galactose (Gal), xylose, arabinose and fucose as standard substances;
the results show that MEPS3 is composed of three monosaccharides, mannose, glucose and galactose, in a molar ratio of 0.08: 0.34: 1.46, it can be seen that MEPS3 is a heteropolysaccharide.
Example 6FT-IR analysis of Marasmius androsaceus exopolysaccharide MEPS3
Weighing 1mg of the MEPS3 sample obtained in example 1, uniformly mixing with 150mg of KBr component, quickly and fully grinding and pressing a sheet, and scanning by using a Fourier transform infrared spectrometer, wherein the specific wavelength scanning range is 4000-400 cm-1The infrared spectrum of MEPS3 is shown in fig. 2. 3423cm, as can be seen in FIG. 2-1The peak is a stretching vibration absorption peak of hydroxyl (-OH) at 2926cm-1then-CH stretching vibration absorption peak is 1725cm-1Where is carbonyl (C ═ O) absorption peak, 1643cm-1The peak is bending vibration absorption peak of hydroxyl (-OH), 1412cm-1Is then-CH21384cm-1Is of the formula-CH2Deformation absorption peak at 1315cm-1The peak value is-CH bending vibration absorption peak, 1128cm-1The absorption peak is the absorption peak of carbon-oxygen (C-O) on the ring, 1065cm-1In which is an alcoholic hydroxyl group (R-CH)2OH) variable angle vibration absorption peak, 875cm-1And 806cm-1Is a characteristic absorption peak for mannose, which is consistent with the monosaccharide composition determination results described above.
The infrared spectrum measurement result shows that the MEPS3 polysaccharide component is pyran polysaccharide.
Example 7NMR analysis of Marasmius androsaceus exopolysaccharide MEPS3
A sample of MEPS3 obtained in example 1, 5mg, was accurately weighed and dissolved in 1.0mL of heavy water (D)2O), fully dissolved, placed in a nuclear magnetic tube, and measured for each of the hydrogen and carbon spectra of the sample by a nuclear magnetic resonance spectrometer, the results are shown in fig. 3 and 4.
The MEPS3 hydrogen spectrum showed that most of the signal was concentrated in 3.6-4.2ppm, 4.787ppm was characteristic peak for mannose, α -Gal signal appeared in 5.062 and 5.107, 5.159, and α -D-Glc anomeric hydrogen signal appeared in 5.313. The polysaccharide carbon spectrum also showed a beta-anomeric carbon signal at 102.23 ppm. The peak at 23.32ppm is the acetylmethyl carbon signal peak.
Example 8SEM analysis of Marasmius androsaceus exopolysaccharide MEPS3
A pipette gun is used for dropping a MEPS3 polysaccharide solution obtained in example 1 on a 1cm × 1cm mica sheet, the mica sheet is kept still for 5min, excess liquid is sucked off by filter paper, gold is sprayed, and SEM observation shows that the result is shown in FIG. 5, and SEM results show that MEPS3 has high heterogeneity, various particle shapes and rod-shaped structures with different sizes.
Example 9Fatigue resistance tests of marasmius androsaceus exopolysaccharide MEPS1, MEPS2 and MEPS3
Animal experiments are carried out on mice through a load swimming experiment, a fatigue rod rotating experiment and an electric shock experiment, the fatigue resistance performance of the MEPS1, the MEPS2 and the MEPS3 is tested, and the performance can be proved only when the following experiments show better fatigue resistance performance.
(1) Weight bearing swimming experiment
Experiment three experimental groups of MEPS1, MEPS2 and MEPS3 obtained in example 1 and a blank control group were set, and 10 of the experimental groups were used. Experimental groups are subjected to purification by ion exchange column chromatography, and preliminary studies show that the crude extracellular polysaccharide of Marasmius androsaceus has definite biological activity (data are not shown) under the condition that the administration dose is 250mg/kg, and the administration doses of each experimental group of MEPS1, MEPS2 and MEPS3 are calculated as follows: 180mg/kg, 60mg/kg and 30 mg/kg.
Continuously performing intragastric administration for 14 days, suspending lead granules at the root of the rat tail to bear a load of 5% of the weight of the mouse 30min after the last administration, placing in a bucket with the same specification and water temperature of 25 deg.C and water depth of 35cm, and placing one in each time. The Time from the start of swimming to exhaustion death was recorded with a stopwatch as mouse weight-bearing swimming Time (Time/s), criteria for exhaustion: the head of the mouse can not float out of the water surface after sinking into the water for 5s, so that the physical strength is exhausted. The experimental results are shown in fig. 6, compared with the blank control group, the weight swimming time of the mice in the MEPS1 test group is obviously prolonged to 146 ± 17s (p <0.05), the weight swimming time of the mice in the MEPS3 group is obviously prolonged to 157 ± 1s (p <0.01), and the weight swimming time of the mice in the MEPS2 test group is 58 ± 8s, which has no significant difference with the blank group. Therefore, both MEPS1 and MEPS3 can significantly increase the time for the mice to swim under load, and have certain anti-fatigue effect, while MEPS3 has the longest time for the mice to swim under load, and has the best anti-fatigue effect.
(2) Fatigue rod rotation test
The animals were divided into groups and administered with the same weight.
Three acclimation exercises are performed before the last administration, and each group of mice is placed on an organic glass rod respectively to make the muscles in a static tension state. The set rotation speed is 10 r/min. After training, the stomach was perfused, 30min after the last administration, each group of mice was placed on an organic glass rod, the rotation speed was set at 20r/min, the Time for the mice to keep in balance on the instrument (Rotarod Time/s) was recorded, and the experiments were performed in triplicate. As shown in fig. 7, the mice in MEPS1 and MEPS3 groups had equilibrium times of 61 ± 5s and 64 ± 4s, respectively, which were significantly different from those in the blank group (. P <0.05), while the mice in MEPS2 group had equilibrium times of 52 ± 6s, which were significantly different from those in the blank group. Therefore, MEPS1 and MEPS3 can prolong the time that the mice keep balance on the rotating rod, and have good anti-fatigue effect. While MEPS3 maximizes the time to maintain equilibrium on the rotor bar and optimizes the fatigue resistance.
(3) Electric shock test
The animal groups and administration methods were the same as those of the swimming test.
Before the last administration, the mice are put in a treadmill respectively, the rotating speed is set to be 20r/min, and the current is adjusted to be the highest so as to enable the mice to know the escaping from the charged area. After training, the mice are placed in a treadmill for 30min, the rotating speed is set to be 30r/min, the number of times of electric shock of the mice within 5min is recorded, and the three times are performed. The results are shown in fig. 8, the mice in MEPS1 and MEPS2 were shocked 27 ± 4 and 23 ± 4 respectively, and there was no significant difference from the blank group, and the mice in MEPS3 were shocked 15 ± 4 (. P < 0.05). Therefore, only MEPS3 can significantly reduce the number of times of electric shock to mice, and has good anti-fatigue effect.
In conclusion, the MEPS3 can simultaneously prolong the balance time of the mouse in the load swimming and balance rotating rod experiments, obviously reduce the number of electric shocks in the mouse endurance running experiments, and show good anti-fatigue effect.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (9)
1. An marasmius androsaceus exopolysaccharide is characterized by being prepared by the following steps:
(1) inoculating Marasmius androsaceus to potato culture medium, and activating to obtain seed solution;
(2) inoculating the seed liquid into a fermentation tank, and performing fermentation culture to obtain fermentation liquid;
(3) filter-pressing the fermentation liquor to obtain fermentation filtrate, concentrating the fermentation filtrate under reduced pressure, adding absolute ethyl alcohol, stirring, standing, centrifuging, collecting precipitate, and freeze-drying to obtain crude polysaccharide;
(4) and (3) performing ion exchange column chromatography on the crude polysaccharide to obtain three components, wherein the component with the lowest content is the obtained marasmius androsaceus exopolysaccharide.
2. The marasmius androsaceus exopolysaccharide of claim 1, wherein:
the molecular weight of the marasmius androsaceus exopolysaccharide is 10.47 kDa; wherein, the molar ratio of monosaccharide composition is mannose: glucose: galactose ═ 0.08: 0.34: 1.46.
3. the marasmius androsaceus exopolysaccharide of claim 1, wherein:
the Marasmius androsaceus extracellular polysaccharide is a pyran polysaccharide.
4. The marasmius androsaceus exopolysaccharide of claim 1, wherein:
in the step (1), the preparation method of the potato culture medium comprises the following steps: peeling 200 and 300g of potatoes, dicing, boiling in boiling water for 30min, filtering, adding 15-20g of glucose, and metering to 1L;
the conditions for the activation include: the activation is carried out in a shaking table, and the rotating speed of the shaking table is 180rpm-250 rpm;
the activation temperature is 26-30 ℃, and the activation time is 5-8 days.
5. The marasmius androsaceus exopolysaccharide of claim 1, wherein:
in the step (2), the culture medium for fermentation culture is as follows: sucrose and/or glucose 10-20g/L, yeast extract powder and/or yeast extract and/or peptone 20-35g/L, KH2PO4 2.0-3.0g/L，MgSO4·7H2O2.0-3.5 g/L, pH 7.0;
the conditions of the fermentation culture comprise: the rotation speed is 220rpm-300rpm, the fermentation temperature is 26-30 ℃, the fermentation time is 5-8 days, and the ventilation volume is 150L/h-300L/h.
6. The marasmius androsaceus exopolysaccharide of claim 1, wherein:
in the step (3), the fermentation filtrate is decompressed and concentrated to have the relative density of 1.15-1.25 at the temperature of 80 ℃;
standing at 4 deg.C for 12 h;
the volume ratio of the absolute ethyl alcohol to the concentrated fermentation filtrate is 4: 1;
the centrifugation condition is 5000-.
7. The marasmius androsaceus exopolysaccharide of claim 1, wherein:
the step (4) is specifically as follows: dissolving the crude polysaccharide to prepare a crude polysaccharide solution, transferring the crude polysaccharide solution into a chromatographic column, performing gradient elution by using distilled water and a sodium chloride solution, and collecting the crude polysaccharide solution into an elution pipe to determine the content of the polysaccharide; drawing an ion exchange chromatography elution curve, combining eluents in the same absorption peak, concentrating, dialyzing and freeze-drying.
8. Use of an marasmius androsaceus exopolysaccharide according to any one of claims 1 to 7 for the preparation of an antifatigue medicament.
9. Use of the marasmius androsaceus exopolysaccharide of any one of claims 1 to 7 for the preparation of an anti-fatigue health product.
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| CN112505222A (en) * | 2020-11-25 | 2021-03-16 | 南京中医药大学 | Method for determining monosaccharide composition and content in astragalus mongholicus medicinal material |
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| CN104073531A (en) * | 2014-07-15 | 2014-10-01 | 江苏神华药业有限公司 | Method for producing marasmius androsaceus (L. ex Fr.) Fr. extracellular polysaccharide through submerged liquid state fermentation |
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