CN117986395A - Ginseng fungus polysaccharide and preparation method and application thereof - Google Patents
Ginseng fungus polysaccharide and preparation method and application thereof Download PDFInfo
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Abstract
The invention particularly discloses a white ginseng fungus polysaccharide (SC-P) and a preparation method and application thereof. The composition of the SC-P comprises alpha-D-glucose, the chemical structure of the SC-P comprises 1, 4-connected alpha-D-glucose residues, 1,4, 6-connected alpha-D-glucose residues and 1, 6-connected alpha-D-glucose residues, and the molar ratio of residues of 1- > 4-Glu, 1- > 4,6-Glu and 1- > 6-Glu is 3:1:1:4. The SC-P is obtained by hot water extraction, ethanol precipitation, deproteinization, ion exchange column chromatography, dialysis and concentration. The SC-P has remarkable immunoregulatory activity and antitumor activity, and can be applied to medicines, health products or foods.
Description
Technical Field
The invention relates to the technical field of application of fungus polysaccharide, in particular to white ginseng fungus polysaccharide, and a preparation method and application thereof.
Background
Edible fungi commonly called mushrooms are large fungi, and the fruiting bodies of the edible fungi are rich in nutrients such as proteins, vitamins, mineral elements, amino acids, polysaccharides and the like. The edible fungus polysaccharide has the characteristics of resisting virus, resisting oxidation, resisting tumor, reducing blood fat, promoting proliferation and differentiation of immune cells, secretion of lymphokines, activating immune regulation of complement and the like, is safe and nontoxic, and has received wide attention in the fields of health food, biological medicine and the like. The edible fungus polysaccharide is a nonspecific immunopotentiator, can improve the immunity of the organism through various ways, and has no side effect on the organism.
The white ginseng fungus (Schizophyllum commune) is also called schizophyllum commune, sarcandra, and saussurea, and is a rare mushroom fungus used as both food and medicine of the phylum of fungi, basidiomycotina, agaricales, schizophyllaceae, and schizophyllum. The fruiting body of the white ginseng fungus is smaller, clustered or clustered, is similar to chrysanthemum, has a fan-shaped or kidney-shaped fungus cover and has a diameter of 1-5 cm, and the weight of a fresh product single plant cultivated artificially reaches 50-100 g. The fungus meat is thin, tough, white to off-white or brown. The white ginseng fungus has a plurality of split leaves, the edge is always palm-shaped longitudinal split inner coils, and the surface of the white ginseng fungus is long with fluff; basal stenosis, radial growth of bacterial folds from the basal, short or no stipe; tough, white to off-white, fluffy, fan-shaped or kidney-shaped, with multiple split lobes; the folds are narrow and radiate from the base; the handle is short or absent.
Zhoulin et al report on purification and characterization of high molecular weight schizophyllan polysaccharide (J, food and fermentation industries, 2008, 34 (12)) that crude polysaccharide products are obtained by centrifugation of fermentation liquor of white ginseng fungus, decolorization in activated carbon water bath, vacuum concentration of concentrated solution at 60 ℃ and deproteinization by Sevag method and precipitation with 95% ethanol for 24 hours; purifying with SEPHACRYL S-400HR filler, eluting with 0.05mol/LNaCl solution, concentrating, dialyzing, and freeze drying to obtain refined white ginseng fungus polysaccharide SPG. SPG was detected as a homogeneous component by gel column chromatography and HPLC. The weight average molecular mass (Mw) and the number average molecular mass (Mn) were measured by size exclusion chromatography and were 2.5X10 7 Da and 1.2X10 7 Da, respectively. It can be determined by HPLC, ultraviolet spectrum, infrared spectrum analysis to be beta-glucan. The structure was confirmed by amylase, cellulase degradation assays, and GC-MS and 13 C NMR data analysis to be 1 β - (1.fwdarw.6) branch per 3 glucose units on the β - (1.fwdarw.3) backbone. The prepared high molecular weight schizophyllan and bacterial cellulose are quite similar in surface morphology through scanning electron microscopy observation, and the high molecular weight schizophyllan is estimated to be applied to the fields of foods, biological materials and the like.
Yu et al at Structure and bioactivity of polysaccharide from a subseafloor strain of Schizophyllum commune 20R-7-F01([J],International Journal of Biological Macromolecules,2022,610–619.) report that the monosaccharide composition of the polysaccharide EPS obtained in the fruiting body of the white ginseng fungus is glucose, the molecular weight of the polysaccharide EPS is 6.088 multiplied by 10 5 Da, methylation and nuclear magnetic resonance analysis show that the main trunk of the EPS is (1- & gt 3) -beta-D-glucan, each third residue is connected with a side chain (1- & gt 6) -beta-D-glucan, and the biological activity analysis result shows that the EPS has stronger antioxidant activity and can strengthen the cell viability and phagocytosis of RAW264.7 cells.
Therefore, the research on the application of the white ginseng fungus polysaccharide in the immunoregulatory activity is lacking in the prior art.
Disclosure of Invention
The invention overcomes the defects existing in the prior art and provides a white ginseng fungus polysaccharide and a preparation method and application thereof.
In a first aspect the invention provides a white ginseng fungus polysaccharide (SC-P) which is a single polysaccharide consisting of alpha-D-glucose.
Further, the polysaccharide comprises 1, 4-linked alpha-D-glucose residues, 1,4, 6-linked alpha-D-glucose residues and 1, 6-linked alpha-D-glucose residues in a molar ratio of about 3:1:1:4.
Further, the chemical structure of the polysaccharide comprises a main chain composed of 1, 6-linked alpha-D-glucose and 1, 4-linked alpha-D-glucose residues, and a side chain composed of 1,4, 6-linked alpha-D-glucose residues and 1-linked alpha-D-glucose residues.
Further, the polysaccharide has a weight average molecular weight of 10000-100000Da (such as 10000Da、15000Da、20000Da、25000Da、30000Da、35000Da、40000Da、45000Da、50000Da、55000Da、60000Da、65000Da、70000Da、75000Da、80000Da、85000Da、90000Da、100000Da), preferably 10000-80000Da, more preferably 20000-70000 Da).
In one embodiment of the invention, the polysaccharide has a weight average molecular weight of 62032Da.
Further, the polysaccharide comprises the following structural formula:
where n is an integer from 20 to 50 (e.g., 20, 25, 30, 35, 40, 45, 50), preferably an integer from 30 to 40.
Wherein Glu is glucose.
In a second aspect the invention provides a composition comprising a polysaccharide according to the first aspect.
In a third aspect, the present invention provides a method for preparing a white ginseng fungus polysaccharide according to the first aspect, the method comprising the step of extracting white ginseng fungus fruiting bodies as raw materials.
Further preferably, the preparation method comprises the step of extracting the crude polysaccharide by a water extraction and alcohol precipitation method.
Further preferably, the preparation method further comprises a step of purifying the crude polysaccharide (e.g. by ion exchange column chromatography).
In one embodiment of the invention, the preparation method comprises the following steps:
(1) Extracting fruiting body powder of Ginseng radix alba with hot water, concentrating the water extract, precipitating with ethanol, and oven drying to obtain crude polysaccharide;
(2) Subjecting the crude polysaccharide obtained in the step (1) to ion exchange column chromatography, eluting, and collecting eluent;
(3) And (3) dialyzing and concentrating the eluent obtained in the step (2) by using a dialysis bag.
Further, the preparation method also comprises a step (4), specifically, freeze-drying the liquid in the dialysis bag after the step (3) is completed.
Further, in step (1), the temperature of the leaching may be 80-100 ℃ (e.g. 80, 85, 90, 95, 100 ℃); in one embodiment of the invention, the leaching temperature is 98 ℃.
Further, in the step (1), the mass ratio (W/V, mg/mL) of the white ginseng fungus fruiting body powder to water is 1:1-10 (such as 1:1, 1:2, 1:3, 1:5, 1:8, 1:10); in one embodiment of the invention, the mass ratio is 1:3.
Further, in the step (1), the leaching is performed 1 to 5 times (such as 1,2,3, 4, 5 times); in one embodiment of the invention, the number of leaches is 3.
Further, in the step (1), each leaching time is 1-10 hours (such as 1,3, 6, 8, 10 hours); in one embodiment of the invention, the time for each leaching is 6 hours.
In one embodiment of the invention, the leaching step in step (1) may comprise: mixing the powder of fruiting body of Ginseng radix alba with water, and boiling in water bath.
Further, in the step (1), in the alcohol precipitation step, the volume ratio of the alcohol to the aqueous extract concentrate is 1-10:1 (such as 1:1, 3:1, 4:1, 5:1, 10:1); in one embodiment of the invention, the volume ratio is 3:1;
in an embodiment of the present invention, in the alcohol precipitation step, the alcohol is ethanol.
In one embodiment of the present invention, step (1) comprises: extracting fruiting body powder of Ginseng radix alba with hot water, collecting supernatant, concentrating, adding anhydrous ethanol, collecting precipitate, oven drying, and removing protein to obtain crude polysaccharide.
Further, in the step (2), the ion exchange column may be a cellulose column, and a filler of the cellulose column is, for example, DEAE cellulose.
Further, in the step (2), the eluent used for elution may be a NaCl solution; specifically, the concentration of the NaCl solution is 0-0.3mol/L (such as 0, 0.05, 0.1, 0.15, 0.2, 0.25 and 0.3).
Further, in the step (2), the elution may be gradient elution, and the concentration of the eluent may be 0-0.3mol/L (e.g., 0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3) mol/L.
In one embodiment of the present invention, step (2) includes: and (3) passing the aqueous solution of the crude polysaccharide obtained in the step (1) through a cellulose column, carrying out gradient elution, collecting eluent, and concentrating.
Further, in the step (3), the molecular weight cut-off of the dialysis bag is 5000-10000Da (such as 5000, 6000, 7000, 8000, 9000, 10000 Da); in one embodiment of the invention, the molecular weight cut-off is 7000Da.
In one embodiment of the present invention, step (3) includes: and (3) placing the eluent obtained in the step (2) in a dialysis bag for dialysis for two days.
According to a fourth aspect of the present invention there is provided the use of a white ginseng fungus polysaccharide according to the first aspect or a composition according to the second aspect or a crude polysaccharide prepared by a process according to the third aspect, said use comprising:
(1) Use in the preparation of a product capable of enhancing immunity;
(2) The application in preparing products with anti-tumor activity.
Further, the product is food, health care product or medicine.
Further, in such applications, the polysaccharide may be used alone or in combination with other active ingredients.
Further, the concentration of the white ginseng fungus polysaccharide in the product is 0.5-25. Mu.g/mL (e.g., 0.5. Mu.g/mL, 0.625. Mu.g/mL, 1.25. Mu.g/mL, 2.5. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL, 25. Mu.g/mL), preferably 0.6-20. Mu.g/mL.
Further, in order to enhance the proliferation effect of RAW 264.7 cells, the concentration of the white ginseng fungus polysaccharide is preferably 5-20. Mu.g/mL (e.g., 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL), more preferably 20. Mu.g/mL.
Further, in order to enhance the inhibitory effect of MFC cells, the concentration of the white ginseng fungus polysaccharide is 5-20. Mu.g/mL (e.g., 5. Mu.g/mL, 10. Mu.g/mL, 20. Mu.g/mL), more preferably 10. Mu.g/mL.
The beneficial effects of the invention are as follows:
The polysaccharide SC-P is obtained by separating and purifying the white ginseng fungus, and the molecular weight, the monosaccharide composition, the chemical structure and the like of the polysaccharide SC-P are analyzed and identified, so that the weight average molecular weight and the structural composition of the polysaccharide SC-P are determined. Cell experiments show that the polysaccharide has remarkable immunoregulatory activity, and particularly has the highest proliferation promoting rate of RAW264.7 cells at the concentration of 20 mug/mL; the polysaccharide also has remarkable anti-tumor activity, and especially has highest MFC cell inhibition rate at the concentration of 10 mug/mL.
Drawings
FIG. 1 shows the HPGPC chart of FIG. 1 showing SC-P;
FIG. 2 shows an infrared spectrum of SC-P;
FIG. 3 shows a HLPC spectrum of SC-P;
FIG. 4 shows 1 H NMR spectra of SC-P;
FIG. 5 shows 13 C NMR of SC-P;
FIG. 6 shows a 1H-1 H-COSY spectrum of SC-P;
FIG. 7 shows the HMQC spectrum of SC-P;
FIG. 8 shows the HMQC spectrum of SC-P;
FIG. 9 shows experimental results of the effect of SC-P on RAW264.7 cell proliferation (1: blank; 2-4: final concentration 5, 10, 20. Mu.g/mL of SC-P;5: final concentration 10. Mu.g/mL of LPS);
FIG. 10 shows the experimental results of the effect of SC-P on MFC cell proliferation (1: blank; 2-4: final concentration of 5, 10, 20. Mu.g/mL of SC-P;5: final concentration of 10. Mu.g/mL of MAN).
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates.
In the present invention, the term "white ginseng fungus (Schizophyllum commune)" means a fungus of the genus schizophyllum of the family schizophyllaceae of the order agaricles of the phylum mycota, basidiomycotina, phylum agarices, comprising sporophores and mycelia.
The term "LPS" refers to lipopolysaccharide, the major component of the cell wall of gram-negative bacteria. LPS solution used in this experiment was purchased from Biosharp company in China.
The term "MAN" refers to mannatide. The MAN solution used in this experiment was purchased from the pharmaceutical industry of the lozenges, sichuan.
The term "CK" refers to Control check, blank.
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but 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 isolation and extraction of the white ginseng polysaccharide SC-P and Structure identification
1. Separation and extraction of white ginseng fungus polysaccharide SC-P
1.1 Extracting crude polysaccharide of white ginseng fungus by water extraction and alcohol precipitation method
200G of dried white ginseng fungus fruiting body is weighed and crushed, the crushed white ginseng fungus fruiting body and distilled water are added into a beaker according to the ratio of 1:3, water bath is carried out for 6 hours at 98 ℃, supernatant is collected and concentrated, the steps are repeated for 3 times, and finally, all supernatant is concentrated to 200mL. Adding three times of anhydrous ethanol to precipitate, collecting precipitate, drying, and removing protein from the extractive solution to obtain crude polysaccharide of Ginseng radix alba.
1.2 Separating and purifying crude polysaccharide of white ginseng fungus by DEAE-cellulose column chromatography
The cellulose 50gDEAE was weighed precisely and dissolved in 1L of ultrapure water and stirred thoroughly, and if no macroscopic cellulose particles were present, the stirring was stopped. Standing for 24h, and discarding supernatant for later use. Preparing 0.5mol/LNaOH, soaking cellulose for 6 hours, washing with ultrapure water to be neutral, discarding the supernatant, adding 0.5mol/L HCl for soaking for 6 hours, washing with distilled water to be neutral, discarding the supernatant; adding 0.5mol/LNaOH, soaking for 6 hours again, washing with distilled water to be neutral, and standing for standby.
And (3) loading the activated cellulose into a column, and balancing for 24 hours through a distilled water pressure column to separate and purify the crude polysaccharide. The supernatant (5 mL) after dilution of the crude polysaccharide was applied to a DEAE-cellulose column and eluted with different concentrations of NaCl (0, 0.05, 0.1 mol/L). The polysaccharide was measured by the sulfuric acid-phenol method. The distilled water eluate was concentrated to 5mL and the sample was purified on a cellulose column. Dialyzing with dialysis bag (Mw is greater than or equal to 7 kDa) for 48 hr, and lyophilizing to obtain Ginseng radix alba polysaccharide named SC-P.
2. Structure identification of white ginseng fungus polysaccharide SC-P
The structure analysis of the white ginseng fungus polysaccharide (SC-P) is carried out by using acid hydrolysis, methylation analysis, high-performance gel permeation chromatography, high-performance liquid chromatography, gas chromatography and mass spectrum combination technology, infrared spectrum technology and nuclear magnetic resonance technology.
2.1 Determination of molecular weight
10Mg of the polysaccharide SC-P sample of the white ginseng fungus was dissolved with 1mLddH 2 O and sonicated for 5min, and HPGPC analysis was performed.
2.2 Infrared Spectrometry of white ginseng fungus polysaccharide SC-P
2MgSC-P was mixed with KBr and tableted and scanned by an infrared spectrophotometer over the 4000cm -1-400cm-1 range.
2.3 Analysis of monosaccharide composition of Ginseng radix alba polysaccharide SC-P
Seven standards and SC-P samples after TFA acid hydrolysis were dissolved in mobile phase (85% acetonitrile) and subjected to HLPC analysis.
2.4 Nuclear magnetic resonance analysis of Ginseng radix alba polysaccharide SC-P
A50 mg sample of SC-P was dissolved in 0.6mL of heavy water (D 2 O), and the mixture was placed in a nuclear magnetic resonance tube and detected on a nuclear magnetic resonance apparatus.
2.5 Methylation and silanization derivatization of the white ginseng polysaccharide SC-P, and then GC-MS analysis
A20 mg sample of SC-P was weighed, the beaker was sealed, 2mL of DMSO (dimethyl sulfoxide) was added to the sealed beaker, and the beaker was gently shaken to dissolve the SC-P sufficiently. 200mg of NaOH are then added until just the NaOH is insoluble and left to stand in a shaker for 1h at room temperature. After the completion of the shaking, 1.5mL of methyl iodide was added, the reaction was carried out in a dark place for 1 hour, and after the reaction, water was added to terminate the reaction. Extracting the product with chloroform, and drying to obtain methylated polysaccharide. And (3) after the methylated polysaccharide is subjected to TFA complete acid hydrolysis, washing with water for three times to obtain a methylated complete acid hydrolysis product.
The sample was reacted with 2mL hexamethyldisilazane, 1mL trimethylchlorosilane, and 2mL anhydrous pyridine, and the mixture was subjected to water bath at 50℃for 20min, centrifuged at 12000rpm/min with a low temperature high speed centrifuge at 4℃for 10min, the precipitate was discarded, and the filtrate was filtered with a 0.22 μm filter, and the supernatant was used for GC-MS analysis.
3. Results
3.1 Basic Property results of white ginseng polysaccharide SC-P
The HPGPC chart of SC-P is shown in FIG. 1, which shows that SC-P has a weight average molecular weight of 62032Da.
3.2 FTIR Spectroscopy analysis of the white ginseng polysaccharide SC-P
The primary structure characterization of the SC-P is carried out by adopting a Fourier infrared spectrum, and the result is shown in figure 2, and the wave number is shown as a typical polysaccharide absorption peak in 3393.94cm -1,2922.02cm-1, 1400-1200 cm -1 and the like, and has no other miscellaneous peaks, which indicates that the separated and purified SC-P is a polysaccharide substance. The stretching vibration peak of the wide absorption peak O-H with the wave number of 3393.94cm -1, the stretching vibration peak of-CH 2 as the absorption peak within the range of 2922.02cm -1, the C=O stretching vibration peak as 1.80 cm -1, the C-H in-plane bending vibration peak as 1365.26cm -1 as-CHO, and the C-O stretching vibration peak as 1043.05cm -1. The absorption peak in the range of 1200-1000cm -1 is that produced by absorption of pyranose ring lactone and hydroxyl groups, indicating that SC-P has a pyran ring. The appearance of an absorption peak at 670.41cm -1 indicates that SC-P contains the alpha-configuration. In addition, there was no absorption peak around 1730cm -1, indicating that SC-P contained no uronic acid.
3.3 Analysis of monosaccharide composition of Ginseng radix alba polysaccharide SC-P
After SC-P was completely hydrolyzed, it was analyzed for monosaccharide composition by HPLC, the peak time of the standard and target samples are shown in table 4, the results of the target samples are shown in fig. 3, wherein the single symmetrical peak is glucose (Glu), and the retention time is 10.039min.
TABLE 4 Table 4
3.4 NMR Spectroscopy analysis of white ginseng polysaccharide SC-P
The 1 H NMR results of SC-P are shown in FIG. 4. The results showed that SC-P had four anomeric hydrogen signals, respectively: δ5.27ppm, δ5.03ppm, δ4.89ppm and δ4.42ppm, and the integrated area ratio was 0.74:0.19:0.23:1.03. signals between delta 3.1 and 4.2ppm are attributed to the hydrogen signals of C2-C6 in the sugar residues.
As shown in FIG. 5, the 13 C NMR results of SC-P show that SC-P has four anomeric carbon signals at delta 103.02ppm, delta 99.39ppm, delta 98.73ppm, delta 97.89 ppm. Signals between 60 and 82ppm are attributed to the C2-C6 carbon signals in the sugar residues.
The 1H-1 H-COSY spectrum of SC-P is shown in FIG. 6, from which the coupling relationship between adjacent hydrogen nuclei can be identified. The signals of the A part H1/H2 are delta 5.27/3.59, the signals of the B part H1/H2 are delta 5.03/3.92, the signals of the C part H1/H2 are delta 4.89/3.79, and the signals of the D part H1/H2 are delta 4.42/3.28.
All chemical shifts of hydrogen are summarized in table 1.
The HMQC spectrum of SC-P is shown in FIG. 7, from which the coupling relationship between the short-range-related 1 H and 13 C can be identified. The signals of the A part H1/C1 are delta 5.27/99.39, the signals of the B part H1/C1 are delta 5.03/98.73, the signals of the C part H1/C1 are delta 4.89/97.89, and the signals of the D part H1/C1 are delta 4.42/103.02.
The HMBC spectral plot for SC-P is shown in fig. 8, from which the coupling relationship between the remotely related 1 H and 13 C can be identified. The signals of H2/C4 of the A residue are delta 3.59/75.64, H6/C4 of the B residue are delta 3.41/73.09, H6/C4 of the C residue are delta 3.41/69.54, H1/C3 of the D residue are delta 4.42/73.09 and H2/C4 are 3.28/75.57.
All chemical shifts of carbon are summarized in table 2.
Table 1 chemical shifts of 1 H of SC-P
TABLE 2 chemical shift of 13 C in SC-P
3.5 Gas chromatography and Mass Spectrometry analysis of Ginseng radix alba polysaccharide SC-P
The methylation results are shown in Table 3, and indicate that the main repeating structural unit of SC-P is a main chain composed of 1, 6-linked alpha-D-glucose and 1, 4-linked alpha-D-glucose residues, and a side chain composed of 1,4, 6-linked alpha-D-glucose residues and 1-linked alpha-D-glucose residues. The structure of BA-T can be deduced preliminarily as shown in FIG. 9.
TABLE 3 analysis of SC-P methylation results
Example 2: immunomodulation and anti-tumor activity research of white ginseng fungus polysaccharide SC-P
In vitro, the immunoregulation and antitumor activities of the polysaccharide SC-P of the white ginseng fungus were measured by using the CCK-8 method.
1. Reagent(s)
CCK-8 kit, RPIM1640, FBS, DMSO, diabody and the like are all commercial products.
2. Instrument for measuring and controlling the intensity of light
An enzyme-labeled instrument; cell incubator.
3. Method of
3.1 Proliferation Effect of SC-P on RAW264.7 cells
The effect of white ginseng fungus polysaccharide (SC-P) on RAW264.7 cell proliferation was determined by a cell counting kit (CCK-8) method. RAW264.7 cells were cultured in vitro to logarithmic growth phase, after counting by a cell counting plate, the cell suspension was diluted to 1×10 5 cells/mL with a new culture solution, the cell suspension was added to a 96-well plate of 100 μl per well, and the 96-well plate was placed in a CO 2 incubator for 24 hours. After 24 hours, the experimental group (SC-P group) was added with SC-P solutions of different mass concentrations (final concentrations of 5, 10, 20. Mu.g/mL), the positive control group (MAN group) was added with 100. Mu.LLPS solution (final concentration of 10. Mu.g/mL), and the blank group (CK group) was added with 100. Mu.L of cell culture solution. After incubation for 24h in a CO 2 incubator, cell photographs were taken under an inverted microscope, 10. Mu.l of CCK-8 was added to each well, incubated in a CO 2 incubator for 3h, and absorbance at 450nm was measured on a microplate reader.
3.2 Influence of SC-P on proliferation of MFC cells
The effect of white ginseng fungus polysaccharide (SC-P) on MFC cell proliferation was determined by the cell counting kit (CCK-8) method. MFC cells were cultured in vitro to logarithmic phase, after counting by cell counting plate, the cell suspension was diluted to 1X 10 5 cells/mL with new culture solution, the cell suspension was added to 96-well plates, 100. Mu.L per well, and the 96-well plates were placed in CO 2 incubator for 24 hours. After 24 hours, the experimental group (SC-P group) was added with SC-P solutions of different mass concentrations (final concentrations of 5, 10, 20. Mu.g/mL), the positive control group (MAN group) was added with 100. Mu.LMAN solution (final concentration of 10. Mu.g/mL), and the blank group (CK group) was added with 100. Mu.L of cell culture solution. After incubation for 24h in a CO 2 incubator, cell photographs were taken under an inverted microscope, 10. Mu.l of CCK-8 was added to each well, incubated in a CO 2 incubator for 3h, and absorbance at 450nm was measured on a microplate reader.
4. Results
4.1 Proliferation Effect of SC-P on RAW264.7 cells
As shown in fig. 9, compared with the blank group, the LPS group can significantly (P < 0.01) promote proliferation of RAW264.7 cells with a proliferation rate of 107.43%; when the final concentration of SC-P is 5, 10 and 20 mug/mL, the proliferation of RAW264.7 cells can be obviously promoted (P < 0.05); when the final concentration of the SC-P is 20 mug/mL, the proliferation effect of the SC-P on RAW264.7 cells is most obvious, and the maximum proliferation rate reaches 90.98%. Therefore, the white ginseng fungus polysaccharide can promote the proliferation of immune cells and has an immunoregulatory effect.
4.2 Influence of SC-P on proliferation of MFC cells
As shown in fig. 10, the positive control group (MAN group) was able to significantly (P < 0.01) inhibit MFC cell proliferation with an inhibition rate of 32.02% compared to the blank group; when the final concentration of SC-P is 5, 10 and 20 mug/mL, the proliferation of MFC cells can be extremely obviously inhibited (P < 0.01); when the final concentration of the SC-P is 10 mug/mL, the inhibition effect of the SC-P on the MFC cells is most obvious, and the maximum inhibition rate reaches 48.11%. Therefore, the white ginseng fungus polysaccharide can inhibit cancer cell proliferation and has anti-tumor activity.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is to be construed as including any modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
The foregoing embodiments and methods described in this invention may vary based on the capabilities, experience, and preferences of those skilled in the art.
The listing of the steps of a method in a certain order in the present invention does not constitute any limitation on the order of the steps of the method.
Claims (10)
1. A ginseng fungus polysaccharide, characterized in that the polysaccharide is a single polysaccharide consisting of alpha-D-glucose, the chemical structure of the polysaccharide comprises 1, 4-linked alpha-D-glucose residues, 1,4, 6-linked alpha-D-glucose residues and 1, 6-linked alpha-D-glucose residues, and the molar ratio of the 1, 4-linked alpha-D-glucose residues, 1,4, 6-linked alpha-D-glucose residues and 1, 6-linked alpha-D-glucose residues is about 3:1:1:4;
The polysaccharide comprises the following structure:
Wherein n is an integer of 20 to 50.
2. Polysaccharide according to claim 1, wherein the weight average molecular weight of the polysaccharide is 10000-100000Da, preferably 10000-80000Da, further preferably 20000-70000Da, particularly preferably 62032Da.
3. The polysaccharide according to claim 1, wherein n is an integer from 30 to 40.
4. A composition comprising the polysaccharide of any one of claims 1-3.
5. The method for preparing polysaccharide according to claim 1, comprising the step of extracting the fruiting body of white ginseng fungus as a raw material:
Preferably, the preparation method comprises the step of extracting crude polysaccharide by a water extraction and alcohol precipitation method;
Further preferably, the preparation method further comprises a step of purifying the crude polysaccharide.
6. The preparation method according to claim 5, wherein the preparation method comprises the steps of:
(1) Extracting fruiting body powder of Ginseng radix alba with hot water, concentrating the water extract, precipitating with ethanol, and oven drying to obtain crude polysaccharide;
(2) Subjecting the crude polysaccharide obtained in the step (1) to ion exchange column chromatography, eluting, and collecting eluent;
(3) And (3) dialyzing and concentrating the eluent obtained in the step (2) by using a dialysis bag.
7. The method of claim 6, wherein in step (1), the leaching temperature is 80-100 ℃;
the mass ratio of the white ginseng fungus sporophore powder to the water is 1:1-10;
The volume ratio of the alcohol to the water extract concentrated solution in the alcohol precipitation is 1-10:1;
Preferably, the alcohol in the alcohol precipitation is ethanol.
8. The process of claim 6, wherein in step (2), the ion exchange column is a cellulose column, and the filler is DEAE cellulose;
the eluent used for elution is NaCl solution;
The elution is gradient elution.
9. Use of a polysaccharide according to any one of claims 1-3 or a composition according to claim 4 or a polysaccharide prepared by a method of preparation according to any one of claims 5-8, characterized in that the use comprises:
(1) Use in the preparation of a product capable of enhancing immunity;
(2) The application in preparing products with anti-tumor activity.
10. The use according to claim 9, characterized in that the concentration of white ginseng fungus polysaccharide in the product is 0.5-25 μg/mL.
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