CN117801133A - Preparation method of momordica grosvenori pectic polysaccharide - Google Patents
Preparation method of momordica grosvenori pectic polysaccharide Download PDFInfo
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 83
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 83
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 83
- 241001409321 Siraitia grosvenorii Species 0.000 title claims abstract description 61
- 235000011171 Thladiantha grosvenorii Nutrition 0.000 title claims abstract description 61
- 229920001277 pectin Polymers 0.000 title claims abstract description 15
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims abstract description 41
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 claims abstract description 23
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- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 18
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- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 16
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Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The invention relates to the technical field of momordica grosvenori polysaccharide, in particular to a preparation method of momordica grosvenori pectic polysaccharide. According to the invention, pectin type momordica grosvenori polysaccharide SGP-C2 which is highly rich in D-galacturonic acid is extracted and separated from momordica grosvenori for the first time, SGP-D1 is further separated, and 2 polysaccharides show stronger immune regulation activity in vitro experiments.
Description
Technical Field
The invention relates to the technical field of momordica grosvenori polysaccharide, in particular to a preparation method of momordica grosvenori pectic polysaccharide.
Background
Polysaccharides (polysaccharides), also known as polysaccharides, have complex and diverse biological functions, most of which are associated with the immune system. The plant polysaccharide is one of the most widely available polysaccharides in the nature, and the polysaccharide component of many medicinal and edible Chinese herbal medicines has bidirectional immunoregulation effect, is called as a natural immunoregulator, and has important effects of maintaining the health of organisms and preventing or treating related diseases.
Fructus Siraitiae Grosvenorii is fruit of fructus Siraitiae Grosvenorii of Siraitia in Cucurbitaceae, is medicinal and edible Chinese medicinal material, is rich in triterpene saponin, polysaccharide, flavone, amino acid and lignan, has various pharmacological activities, and has great development potential. However, the grosvenor momordica fruit industry focuses on zero-calorie sweet glycosides for a long time, 60% of grosvenor momordica fruit in the market is only used for extracting sweet glycosides, and functional research and product development of other components are very little, so that the waste of grosvenor momordica fruit resources is caused.
Disclosure of Invention
In view of the above, the invention aims to provide a preparation method of momordica grosvenori pectic polysaccharide, which can successfully obtain momordica grosvenori pectic polysaccharide and improve the utilization of momordica grosvenori resources.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of momordica grosvenori pectic polysaccharide, which comprises the following steps:
pretreating fructus Siraitiae Grosvenorii to obtain fructus Siraitiae Grosvenorii crude polysaccharide;
separating the crude polysaccharide of the momordica grosvenori by a cellulose column, and eluting by adopting water, 0.1mol/L sodium chloride aqueous solution, 0.3mol/L sodium chloride aqueous solution and 0.5mol/L sodium chloride aqueous solution in sequence to respectively obtain water eluent, 0.1mol/L sodium chloride aqueous solution eluent, 0.3mol/L sodium chloride aqueous solution eluent and 0.5mol/L sodium chloride aqueous solution eluent;
dialyzing water eluent, 0.1mol/L sodium chloride aqueous solution eluent, 0.3mol/L sodium chloride aqueous solution eluent and 0.5mol/L sodium chloride aqueous solution eluent respectively to obtain components with the molecular weight of more than 1000, and obtaining SGP-A, SGP-B, SGP-C and SGP-D respectively;
separating SGP-C by gel column, eluting with water to obtain SGP-C2;
the SGP-C2 includes glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose; the molar ratio of glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose in the SGP-C2 is 0.78:1.84:26.57:1.31:0.49:0.06.
preferably, the method further comprises separating SGP-D by a gel column, eluting with water to obtain SGP-D1; the SGP-D1 comprises glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose; the molar ratio of glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose in SGP-D1 is 1.37:2.00:20.79:0.61:0.12:0.25.
preferably, the pretreatment comprises:
degreasing fructus Siraitiae Grosvenorii with ethanol water solution to obtain fructus Siraitiae Grosvenorii residue;
extracting the momordica grosvenori residues with water, removing compounds with molecular weight smaller than 300 from the obtained extracting solution through an ultrafiltration membrane, and sequentially concentrating and performing first alcohol precipitation on the obtained filtrate to obtain a first alcohol precipitation substance;
and dissolving the first alcohol precipitate in water, and sequentially removing proteins, performing second alcohol precipitation and decoloring to obtain the crude polysaccharide of the momordica grosvenori.
Preferably, the cellulose column is a DE-52 cellulose column; the gel column is a SephadexG-200 gel column.
Preferably, the flow rate of the eluent is 5mL/min when the cellulose column is separated.
Preferably, the SGP-C is separated by a gel column at a water flow rate of 0.2mL/min.
Preferably, the volume concentration of the ethanol aqueous solution is 70-90%.
Preferably, the usage ratio of the momordica grosvenori to the ethanol water solution is 1g:5-20mL.
Preferably, the degreasing is heating reflux degreasing, the degreasing temperature is 60-80 ℃, the degreasing times are 1-3 times, and each time is 1-3 hours.
Preferably, the volume concentration of the ethanol aqueous solution of the first alcohol precipitation is 75-100%.
The invention provides a preparation method of momordica grosvenori pectic polysaccharide, which comprises the steps of pre-treating momordica grosvenori to obtain momordica grosvenori crude polysaccharide; separating the crude polysaccharide of the momordica grosvenori by a cellulose column, and eluting by adopting water, 0.1mol/L sodium chloride aqueous solution, 0.3mol/L sodium chloride aqueous solution and 0.5mol/L sodium chloride aqueous solution in sequence to respectively obtain water eluent, 0.1mol/L sodium chloride aqueous solution eluent, 0.3mol/L sodium chloride aqueous solution eluent and 0.5mol/L sodium chloride aqueous solution eluent; dialyzing the water eluent, 0.1mol/L sodium chloride aqueous solution eluent, 0.3mol/L sodium chloride aqueous solution eluent and 0.5mol/L sodium chloride aqueous solution eluent respectively to obtain SGP-A, SGP-B, SGP-C and SGP-D respectively; separating SGP-C by gel column, eluting with water to obtain SGP-C2; the SGP-C2 includes glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose; the molar ratio of glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose in the SGP-C2 is 0.78:1.84:26.57:1.31:0.49:0.06. according to the invention, pectin type momordica grosvenori polysaccharide SGP-C2 which is highly rich in D-galacturonic acid is extracted and separated from momordica grosvenori for the first time, SGP-D1 is further separated, and 2 polysaccharides show stronger immune regulation activity in vitro experiments.
Drawings
FIG. 1 shows the DEAE-52 column chromatography elution profile of SGP;
FIG. 2 shows SephadexG-200 gel column elution curves for SGP-C and SGP-D;
FIG. 3 is a SGP-C2 and SGP-D1 ultraviolet full wavelength scan;
FIG. 4 is a graph showing SGP-C2 and SGP-D1 molecular weight distribution;
FIG. 5 is a diagram showing the composition of SGP-C2 and SGP-D1 monosaccharides;
FIG. 6 is a Fourier transform infrared spectrum of SGP-C2 and SGP-D1;
FIG. 7 is a SGP-C2 nuclear magnetic spectrum;
FIG. 8 is a SGP-D1 nuclear magnetic spectrum;
FIG. 9 is an SGP-C2 and SGP-D1 Congo red experiment;
FIG. 10 is a scanning electron microscope image of SGP, SGP-C2 and SGP-D1;
FIG. 11 shows proliferation and phagocytosis experiments of SGP, SGP-C2 and SGP-D1 on RAW264.7 cells;
FIG. 12 is a graph showing the effect of SGP, SGP-C2 and SGP-D1 on the secretion of NO, TNF- α and IL-6 in RAW264.7 cells.
Detailed Description
The invention provides a preparation method of momordica grosvenori pectic polysaccharide, which comprises the following steps:
pretreating fructus Siraitiae Grosvenorii to obtain fructus Siraitiae Grosvenorii crude polysaccharide;
separating the crude polysaccharide of the momordica grosvenori by a cellulose column, and eluting by adopting water, 0.1mol/L sodium chloride aqueous solution, 0.3mol/L sodium chloride aqueous solution and 0.5mol/L sodium chloride aqueous solution in sequence to respectively obtain water eluent, 0.1mol/L sodium chloride aqueous solution eluent, 0.3mol/L sodium chloride aqueous solution eluent and 0.5mol/L sodium chloride aqueous solution eluent;
dialyzing water eluent, 0.1mol/L sodium chloride aqueous solution eluent, 0.3mol/L sodium chloride aqueous solution eluent and 0.5mol/L sodium chloride aqueous solution eluent respectively to obtain components with the molecular weight of more than 1000, and obtaining SGP-A, SGP-B, SGP-C and SGP-D respectively;
separating SGP-C by gel column, eluting with water to obtain SGP-C2;
the SGP-C2 includes glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose; the molar ratio of glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose in the SGP-C2 is 0.78:1.84:26.57:1.31:0.49:0.06.
in the present invention, the raw materials used in the present invention are preferably commercially available products unless otherwise specified.
The invention carries out pretreatment on the momordica grosvenori to obtain the crude polysaccharide of the momordica grosvenori.
In the present invention, the pretreatment preferably includes the steps of:
degreasing fructus Siraitiae Grosvenorii with ethanol water solution to obtain fructus Siraitiae Grosvenorii residue;
extracting the momordica grosvenori residues with water, removing compounds with molecular weight smaller than 300 from the obtained extracting solution through an ultrafiltration membrane, and sequentially concentrating and precipitating with first alcohol from the obtained filtrate to obtain a first alcohol precipitate;
and dissolving the first alcohol precipitate in water, and sequentially removing proteins, performing second alcohol precipitation and decoloring to obtain the crude polysaccharide of the momordica grosvenori.
The invention uses ethanol water solution to degrease the momordica grosvenori to obtain momordica grosvenori residues.
In the invention, before degreasing, the method further comprises crushing, wherein the crushing is preferably performed until the particle size is 0.1-0.3 cm. In the present invention, the volume concentration of the aqueous ethanol solution is preferably 70 to 90%, more preferably 80%; the dosage ratio of the momordica grosvenori to the ethanol water solution is preferably 1g:5-20mL, preferably 1g:5-15mL, more preferably 1g:8mL.
In the present invention, the degreasing is preferably thermal reflow degreasing, the degreasing temperature is preferably 60-80 ℃, more preferably 78 ℃, the number of times of degreasing is preferably 1-3, more preferably 2, and the time is preferably 1-3h, more preferably 3h each time.
After the momordica grosvenori residues are obtained, the momordica grosvenori residues are subjected to water extraction, the obtained extracting solution is subjected to ultrafiltration membrane to remove compounds with molecular weight smaller than 300, and the obtained filtrate is sequentially subjected to concentration and first alcohol precipitation to obtain a first alcohol precipitate.
In the invention, the ratio of the residue of the momordica grosvenori to the water is preferably 1g:5-20mL, more preferably 1g:10mL; the temperature of the water extraction is preferably 60-80 ℃, more preferably 70 ℃, the number of times of water extraction is preferably 2, and the time of each time is preferably 2-5h, more preferably 4h.
In the present invention, the concentration is preferably vacuum concentration, and the temperature of the vacuum concentration is preferably 40-60 ℃, more preferably 55 ℃; the concentration is preferably 1/4 of the original volume of the system.
In the present invention, the volume concentration of the aqueous ethanol solution of the first alcohol precipitation is preferably 75 to 100%, more preferably 95%. In the present invention, in the first alcohol precipitation, the volume ratio of the concentrate obtained by concentration to the aqueous ethanol solution is preferably 1:10.
after the first alcohol sediment is obtained, the first alcohol sediment is dissolved in water, and protein removal, second alcohol sediment and decoloration are sequentially carried out, so that the grosvenor momordica crude polysaccharide is obtained.
In the present invention, after the first alcohol precipitation, centrifugation and washing with absolute ethanol are preferably further included before removing proteins. In the present invention, the rotational speed of the centrifugation is preferably 4000r/min, and the time is preferably 10min. In the present invention, the number of times of washing with absolute ethanol is preferably 3.
In the present invention, the protein removal method is preferably a sevage method. In the present invention, after removing the protein, it is preferable to further include centrifugation and spin-evaporation to remove the excess organic solvent in the supernatant.
In the present invention, the second precipitation is preferably performed by concentrating the supernatant after removing the protein to remove the excessive solvent, adding water, then adding absolute ethanol, and standing at 4 ℃ for 12 hours.
In the present invention, it is preferable that after the second precipitation, centrifugation and washing with absolute ethanol are further included. In the invention, the rotating speed of the centrifugation is preferably 4000r/min, and the time is preferably 10min; in the present invention, the number of times of washing with the absolute ethanol is preferably 3.
In the present invention, the resin used for the decoloring is preferably an AB-8 macroporous resin. In the present invention, the decoloring is preferably performed by dissolving the alcohol precipitate obtained by the second precipitation in water, and then loading the obtained solution onto AB-8 macroporous resin.
In the present invention, the method further comprises concentrating the water eluate obtained after the decolorization.
After the crude polysaccharide of the momordica grosvenori is obtained, the crude polysaccharide of the momordica grosvenori is separated by a cellulose column, and water, 0.1mol/L sodium chloride aqueous solution, 0.3mol/L sodium chloride aqueous solution and 0.5mol/L sodium chloride aqueous solution are sequentially adopted for eluting, so that water eluent, 0.1mol/L sodium chloride aqueous solution eluent, 0.3mol/L sodium chloride aqueous solution eluent and 0.5mol/L sodium chloride aqueous solution eluent are respectively obtained.
In the present invention, the cellulose column is preferably a DE-52 cellulose column; when the cellulose column is separated, the flow rate of the eluent is 5mL/min. In the present invention, the elution is preferably 2 column volumes per eluent.
The invention carries out dialysis on water eluent, 0.1mol/L sodium chloride aqueous solution eluent, 0.3mol/L sodium chloride aqueous solution eluent and 0.5mol/L sodium chloride aqueous solution eluent respectively, and components with the molecular weight cutoff more than 1000 are respectively obtained to obtain SGP-A, SGP-B, SGP-C and SGP-D;
in the present invention, the dialysis is preceded by concentration, preferably concentration under reduced pressure; the molecular weight cut-off of the dialysis is preferably 1000.
In the present invention, the post-dialysis treatment further comprises freeze-drying the dialyzed material, wherein the temperature of the freeze-drying is preferably-80 ℃ and the time is preferably 36 hours.
After SGP-C is obtained, the SGP-C is separated by a gel column and eluted by water to obtain SGP-C2.
In the present invention, the gel column is preferably a SephadexG-200 gel column; the flow rate of water at the time of the elution is preferably 0.2mL/min.
In the present invention, the SGP-C2 includes glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose; the molar ratio of glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose in SGP-C2 is preferably 0.78:1.84:26.57:1.31:0.49:0.06.
in the invention, the preparation method of the siraitia grosvenorii pectic polysaccharide further comprises the steps of separating SGP-D through a gel column and eluting with water to obtain SGP-D1. In the invention, the conditions for separating SGP-D by the gel column are the same as those for separating SGP-C by the gel column, and will not be described again.
In the present invention, the SGP-D1 preferably includes glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose; the molar ratio of glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose in the SGP-D1 is preferably 1.37:2.00:20.79:0.61:0.12:0.25.
the invention also provides application of the grosvenor momordica grosvenori crude polysaccharide SGP and refined pectic polysaccharides SGP-C2 and SGP-D1 in preparation of immunity enhancing products.
Experimental results show that the momordica grosvenori polysaccharide SGP, SGP-C2 and SGP-D1 can stimulate RAW264.7 cells to secrete NO, TNF-alpha, IL-6 and other factors, and enhance the phagocytic capacity of the RAW264.7 cells, so that the cellular immune function is enhanced, and the momordica grosvenori polysaccharide SGP, SGP-C2 and SGP-D1 can be applied to product development for enhancing the immunity.
Dried fructus Siraitiae Grosvenorii was purchased from Navy limited liability company in Gui Lin Shi Yongfu county forests. The mouse mononuclear macrophage cell line RAW264.7 is purchased from a cell bank of China center for type culture Collection. DEAE-52 cellulose and Sephadex G200 were purchased from Shanghai Seiyaka leaf Biotechnology Co. The monosaccharide controls DL-arabinose, D-mannose, D-ribose, D (+) -anhydrous glucose, D-glucuronic acid, D-galactose, rhamnose, D-galacturonic acid were purchased from Chengdoman Biotech Co. Dextran molecular weight standards were purchased from chinese food and drug assay institute. Cell proliferation assay (CCK 8) kit was purchased from the biotech company, inc. NO detection kits were purchased from Shanghai Biyun Tian Co. TNF- α and IL-6 detection kits were purchased from Elabscience; other reagents were all of analytically pure grade.
LC-2030C liquid chromatograph (shimadzu corporation, japan); nicolet fourier infrared spectrometer (Therom Fisher, usa); brucker Avance500MHz superconducting nuclear magnetic resonance spectrometer (Brucker Corp., germany); ALPHA1-2LD PLU freeze dryer (Beijing bo line Instrument Co., ltd.); a new century T6 ultraviolet visible spectrophotometer (beijing pro analysis general instruments, inc.); zeiss EVO18 scanning electron microscope (Zeiss Co., germany); spark microplate reader (Tecan, switzerland); BSP-150 biochemical incubator (Shanghai Bolus medical instruments Co., ltd.); 5% CO 2 Cell incubator (Thermo Forma company, usa); dmi1 inverted phase contrast microscope (Leka Co., USA).
The technical solution in the present invention will be clearly and completely described in the following in connection with the embodiments in the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the embodiments herein, which are obtained by persons of ordinary skill in the art without making any inventive effort, are within the scope of the present invention.
Example 1
Extraction, separation and purification of momordica grosvenori polysaccharide
(1) Weighing 2kg of dried fructus momordicae, crushing, adding 16L of 80vol% ethanol water solution, heating, refluxing and degreasing for 3 hours at 78 ℃, and repeating for 2 times to obtain fructus momordicae residues.
(2) Drying the fructus Siraitiae Grosvenorii residue in a blast drying oven at 55deg.C, adding water at a ratio of feed liquid to 1g:10mL, heating and extracting in water bath at 70deg.C for 2 times, each time for 4 hr; after the extract is filtered by an ultrafiltration membrane, the filtrate is concentrated to 1/4 of the total volume in vacuum at 55 ℃, an ethanol water solution with the volume percentage of 95 percent and 10 times of the volume of the concentrated solution is added, the mixture is stood overnight at 4 ℃, the ethanol sediment material is centrifuged for 10min under the condition of 4000r/min, and the sediment is leached by absolute ethanol for 3 times.
Dissolving the washed precipitate in water, removing protein for 1 time by using a sevage method, removing redundant chloroform by rotary evaporation of filtrate, dissolving the filtrate to 400mL by adding water, slowly adding 1400mL of absolute ethyl alcohol while stirring, standing overnight at 4 ℃, centrifuging the alcohol sediment for 10min under the condition of 4200r/min, collecting the precipitate, washing the precipitate with absolute ethyl alcohol for 3 times, dissolving the washed precipitate in water, decolorizing by using AB-8 macroporous, eluting by using pure water, concentrating the eluent, and freeze-drying (the temperature is-80 ℃ for 48 h) to obtain the grosvenor momordica fruit crude polysaccharide SGP with the yield of 1.58%.
(3) Dissolving the crude Siraitia grosvenorii polysaccharide SGP with water to obtain 50mg/mL solution, and loading with 30mL solution onto Cellulose DE-52 Cellulose column (4.5 cm. Times.40 cm, OH) - Type) was sequentially eluted with water, 0.1mol/L, 0.3mol/L and 0.5mol/L aqueous sodium chloride at a flow rate of 5mL/min, 2 column volumes per eluent, 15 mL/tube, the eluent was collected from each portion, concentrated under reduced pressure, dialyzed (molecular weight cut-off 1000), and freeze-dried to give water, 0.1M, 0.3M and 0.5M sodium chloride eluted samples SGP-A, SGP-B, SGP-C and SGP-D, as shown in the elution profile of FIG. 1.
(4) Dissolving SGP-C and SGP-D in water, purifying with SephadexG-200 gel column, eluting with distilled water at flow rate of 0.2mL/min, collecting eluate (figure 2), concentrating, and freeze drying to obtain refined polysaccharide of Siraitia grosvenorii into SGP-C2 and SGP-D1.
The yields of SGP-C2 and SGP-D1 were 22.2% and 38.3%, respectively.
The yield calculation is calculated according to formulas 1 to 3:
crude polysaccharide SGP yield (%) =m/m 0 *100 formula 1;
refined polysaccharide SGP-C2 yield (%) =m 1 M is 100 formula 2;
refined polysaccharide SGP-D1 yield (%) =m 2 And/m is 100 formula 3.
Wherein, m: the quality of the grosvenor momordica crude polysaccharide; m is m 0 : the quality of the fructus momordicae; m is m 1 SGP-C2 mass, m 2 SGP-D1 mass.
Example 2
Structural characterization of Siraitia grosvenorii refined polysaccharide SGP-C2 and SGP-D1
(1) Siraitia grosvenorii refined polysaccharide SGP-C2 and SGP-D1 purity
The purity of SGP-C2 and SGP-D1 prepared in example 1 was checked by using a method of ultraviolet full-wavelength scanning. The specific method comprises the following steps: 2mg of the dried sample is taken and dissolved in pure water to prepare 0.5mg/mL polysaccharide solution, pure water is used as a blank control, and full-wavelength scanning is carried out within the range of 190-400 nm by an ultraviolet scanning method. As can be seen from fig. 3: the SGP-C2 or SGP-D1 has no characteristic absorption peaks of nucleic acid and protein in the range of 260-280 nm, and the SGP-C2 and SGP-D1 are proved to contain no protein and nucleic acid impurities.
(2) Determination of molecular weight
The specific method comprises the following steps: and (3) taking dry SGP-C2 or SGP-D1 and dextran standards with different molecular weights, adding water for dissolving to prepare a solution with the concentration of 2mg/mL, filtering by a filter membrane, and performing high-efficiency gel chromatography analysis.
Conditions for high performance gel chromatography analysis: a TSKgel G4000PWXL (7.8 mm i.d.×30cm,10 μm) column; mobile phase: ultrapure water; column temperature: 30 ℃; flow rate: 0.5mL/min; a RID 10 type a moveout detector; sample injection amount: 20. Mu.L.
A standard curve was plotted with retention time on the abscissa and the logarithm of dextran molecular weight on the ordinate, and the standard curve regression equation was y= -0.2728 ×+9.5237 (r2= 0.9977). Then calculating SGP-C2 or S according to the standard curve and the peak time of SGP-C2 or SGP-D1GP-D1 has a molecular weight of 5.0X10, respectively 5 Da and 7.6X10 5 Da。
HPGPC analysis of FIG. 4 revealed that SGP-C2 or SGP-D1 exhibited a single chromatographic peak with good symmetry.
(3) SGP-C2 and SGP-D1 monosaccharide composition
The monosaccharide composition was determined by PMP-pre-column derivatization high performance liquid chromatography.
2mg of the SGP-C2 or SGP-D1 dry sample prepared in example 1 was taken, 3mL of trifluoroacetic acid was added for dissolution, shaking up, sealing, hydrolysis at 110℃for 3 hours, 2mL of methanol was added after cooling, and evaporation to dryness under reduced pressure was repeated 5 times to remove the excess trifluoroacetic acid, to obtain a residue.
The residue and monosaccharide standards D- (+) -glucose, D-glucuronic acid, D-galactose, D-galacturonic acid, D-mannose, DL-arabinose, rhamnose, D-ribose are respectively dissolved in 2mL of ultrapure water to respectively prepare polysaccharide solution and standard solution with the concentration of 1 mg/mL.
And (3) derivatization treatment: 600. Mu.L of polysaccharide solution was taken, 300. Mu.L of 0.3mol/LNaOH solution and 300. Mu.L of 0.5mol/L PMP-methanol solution were added, and after mixing, derivatization was carried out in an oven at 70℃for 100min under protection from light. After the reaction, cooling to room temperature, adding 400 mu L of 0.3mol/L hydrochloric acid solution, mixing, adding chloroform for extraction for 5 times to remove unreacted PMP, discarding the lower layer, centrifuging the extract for 3min at 13000r/min, filtering the supernatant with 0.22 mu m aqueous microporous membrane, and detecting by HPLC.
Derivatization treatment is carried out on monosaccharide standard solution according to the same method, and 200 mu L of monosaccharide standard solution is respectively sucked into liquid phase vials after the derivatization treatment is finished to be used as a mixed standard to be detected.
HPLC conditions: ZORBAX SB-C18 (4.6mm. Times.250 mm,5 μm) column; mobile phase: acetonitrile-phosphate buffer (ph 6.9) =15: 85; column temperature: 30 ℃; flow rate: 1mL/min; ultraviolet detection wavelength: 254nm; the sample injection amount was 30. Mu.L. The results of the HPLC detection are shown in FIG. 3.
As can be seen from FIG. 5, the molar ratio of the monosaccharides of SGP-C2 and SGP-D1 is 0.78, respectively: 1.84:26.57:1.31:0.49:0.06 and 1.37:2.00:20.79:0.61:0.12:0.25, indicating that SGP-C2 and SGP-D1 are HG pectic polysaccharides, with HG domains at 79.7% and 78.7%, respectively.
(4) SGP-C2 and SGP-D1 Fourier transform infrared chromatographic analysis
1.5mg of SGP-C2 or SGP-D1 dry sample prepared in example 1 and 150mg of dry potassium bromide are weighed, uniformly ground under an infrared lamp, tabletted and subjected to infrared scanning. The specific results are shown in FIG. 4.
As can be seen from FIG. 6, SGP-C2 and SGP-D1 are each at 3400cm -1 And 3420cm -1 The absorption peak is an O-H telescopic vibration peak of 2950cm -1 And 2940cm- 1 The absorption peak is C-H stretching vibration peak; 1620cm -1 The presence of a strong absorption peak indicates the presence of uronic acid; 1420cm -1 The characteristic absorption of the left and right parts is caused by C-H deformation vibration, and 1150 cm and 1100cm -1 And 1020cm -1 The continuous peak appearing at this point is caused by the absorption vibration of C-O-C in the pyran ring, indicating the presence of the pyran ring; 960cm -1 The characteristic absorption peak at the site is caused by asymmetric stretching vibration of the D-glucopyranose ring, and 895cm -1 The nearby weak absorption peak indicates the presence of β -type glycosidic linkages; 771cm -1 The absorption peak at the position is related to the stretching vibration of the pyranose symmetrical ring; while SGP-D1 is 835cm -1 Weak absorption at this point suggests the presence of an alpha glycosidic bond.
(5) SGP-C2 and SGP-D1 Nuclear magnetic resonance analysis
25mg of SGP-C2 or SGP-D1 dry sample prepared in example 1 was weighed and dissolved in 0.5mL of D 2 And O, loading the magnetic core tube, and detecting.
The nuclear magnetic analysis of SGP-C2 is shown in FIG. 7: SGP-C2 1 HNMR proton signals are concentrated in the δ1.20-5.36 interval, with severe stacking. There are two distinct hetero-head hydrogen proton signals at δ5.12 and δ4.66; there is a rhamnose residue signal peak at δ1.21. 13 The CNMR carbon signal is concentrated in the delta 16.94-176.13 interval and has low resolution in the 68.28-78.16 interval. Wherein, there are two anomeric carbon signal peaks at δ 99.07 and δ96.20, and a methoxy signal at δ 57.53, and it is presumed that methyl esterification of part of galacturonic acid occurs. Combined two-dimensional spectrum @ 1 H- 1 H COSY and HMQC), the cross peaks delta 5.12/99.07 of the anomeric hydrogen signal/anomeric carbon signal (H1/C1), the signal of methyl esterified galacturonic acid and the signal of non-methyl esterified galacturonic acid partially overlap peaks and delta 4.66/96.2 signal peaks, respectively, are labeled residues A, B and C, respectively, and the deduced residue structure is shown in table 1.
The nuclear magnetic analysis of SGP-D1 is shown in FIG. 8: SGP-D1 1 HNMR proton signals are concentrated in the interval δ1.31-5.12, with one end group proton signal at δ5.12. 13 The carbon signal of CNMR is concentrated in the interval δ 68.29-175.53, with one end carbon signal at δ 99.08. And there was a cross peak delta 5.12/99.08 on the HSQC spectrum, and the structure of the deduced residues is shown in Table 1.
TABLE 1 Nuclear magnetic resonance chemical shifts and residue Structure of SGP-C2 and SGP-D1
(6) Congo red experiment
Taking SGP-C2 or SGP-D1 dry samples prepared in example 1, adding water to prepare a polysaccharide solution of 0.5mg/mL, and uniformly mixing with a Congo red solution of 200 mu mol/L. And adding a proper amount of NaOH solution with the volume of 1mol/L to ensure that the final concentration of NaOH is 0mol/L, 0.1mol/L, 0.2mol/L, 0.3mol/L, 0.4mol/L and 0.5mol/L respectively. And (3) carrying out light-shielding reaction at room temperature for 10min, carrying out full-wavelength scanning by using an ultraviolet spectrophotometer, and drawing a line graph by taking NaOH concentration as an abscissa and the maximum absorption wavelength as an ordinate. If the polysaccharide has a triple helix structure, the polysaccharide will undergo a complex reaction with congo red reagent, and the maximum absorption wavelength (λmax) of the mixed solution will shift in the long wave direction, i.e. redshift. Whereas sodium hydroxide may disrupt the polysaccharide triple helix structure, so that the mixed solution λmax gradually decreases, i.e., blue shifts, as the concentration of sodium hydroxide increases.
As can be seen from fig. 9, after SGP-C2 or SGP-D1 was mixed with congo red, λmax did not undergo significant red shift, and as the concentration of sodium hydroxide increased, the mixed solution had a similar trend to that of congo red single-solution λmax, thus judging that the two polysaccharides did not contain triple helix structure.
(7) Scanning electron microscope analysis
1.5mg of the SGP-C2 or SGP-D1 sample prepared in example 1 was weighed and analyzed for microscopic morphology using a thermal field emission scanning electron microscope (ZEISS EVO 18). As can be seen from fig. 10, at 200X times, the SGP-C2 surface is rough, exhibiting irregular globular, rod-like and band-like structures; SGP-D1 exhibits irregular flakes of varying size and shape. Under the condition of 1.00KX times, the SGP-C2 can clearly observe a rod shape and a belt shape, and the surface has folds; SGP-D1 had smooth and fine fragments on the surface and had cracks.
Example 3
In vitro immune regulation activity detection of grosvenor momordica grosvenori crude polysaccharide SGP, refined polysaccharide SGP-C2 and SGP-D1
(1) Proliferation assay of RAW264.7 cells
Detecting influence of polysaccharide on cell proliferation by CCK-8 method, selecting cells in logarithmic growth phase, and adjusting to 1×10 5 Inoculating in 96-well plate at 37deg.C and 5% CO 2 Is cultured for 24 hours under the condition of (2). Polysaccharide solutions (SGP, SGP-C2 and SGP-D1 prepared in example 1) with different concentrations (6.25 mug/mL, 12.5 mug/mL and 25 mug/mL) are added to each well of the administration group, 1 mug/mL LPS solution is added to the positive control group, the same volume of complete medium is added to the blank group, three wells are added to each group, and the culture is carried out for 24 hours after administration. 10 mu L of CCK-8 solution is added into each hole, the mixture is incubated for 45min at 37 ℃, the absorbance value at 450nm is detected by an enzyme label instrument, and the cell proliferation rate is calculated according to the following test:
P(％)＝(A-A0)/(A1-A0)×100％
wherein: a is the average absorbance of the administration group; a0 is background group mean absorbance; a1 is the mean absorbance of the blank group.
As can be seen from FIG. 11A, the crude Siraitia grosvenorii polysaccharide SGP, the homogeneous polysaccharide SGP-C2 and SGP-D1 significantly promoted proliferation of RAW264.7 cells (P < 0.001) in the concentration range of 6.25-25. Mu.g/mL compared with the blank group, indicating that the concentration of less than 25. Mu.g/mL is not toxic to RAW264.7 cells.
(2) Phagocytic capacity of RAW264.7 cells
Neutral red assay for RAW264.7 cell phagocytic capacity: selecting cells in logarithmic growth phase, and adjusting to 1×10 5 Inoculating individual/mLIn 96-well plates at 37℃in 5% CO 2 Is cultured for 24 hours under the condition of (2). Polysaccharide solutions (SGP, SGP-C2 and SGP-D1 prepared in example 1) with different concentrations (6.25 mug/mL, 12.5 mug/mL and 25 mug/mL) are added to each well of the administration group, 1 mug/mLLPS solution is added to the positive control group, the same volume of complete medium is added to the blank group, three wells are added to each group, and the culture is performed for 24 hours after administration. The supernatant was discarded, washed 2 times with PBS, incubated for 1h with 100. Mu.L of 0.05% neutral red solution per well, discarded, washed twice with PBS, and incubated for 1h with 100. Mu.L of cell lysate (glacial acetic acid: absolute ethanol=1:1) at room temperature, and absorbance at 540nm was detected by an ELISA reader.
Phagocytic capacity index= (A1/A0) ×100%;
wherein: a1 is the mean absorbance of the dosing group; a0 is the mean absorbance of the blank group.
As shown in FIG. 11B, the crude Siraitia grosvenorii polysaccharide SGP, the refined polysaccharide SGP-C2 and the SGP-D1 can promote the phagocytosis of RAW264.7 cells, the SGP can obviously promote the phagocytosis of macrophages at the low concentration of 6.25 mu g/mL, the SGP-C2 can obviously promote the phagocytosis of macrophages (P is less than 0.05) at the high concentration of 12.5-25 mu g/mL, and the SGP-D1 can obviously promote the phagocytosis of macrophages (P is less than 0.001) at the range of 6.25-12.5 mu g/mL.
(3) RAW264.7 cell NO, TNF-alpha, IL-6 secretion amount
The effect of LPS and different Siraitia grosvenorii polysaccharides (SGP, SGP-C2 and SGP-D1) on the NO secretion of RAW264.7 cells was examined by Griess method. ELISA was used to examine the effect of Lipopolysaccharide (LPS) and different Siraitia grosvenorii polysaccharides (SGP, SGP-C2 and SGP-D1) on TNF-alpha and IL-6 secretion of RAW264.7 cells. Cell culture was carried out as in the case of administration, and the cells were cultured for 24 hours after administration. The supernatant was collected and subjected to the procedure described in the kit.
As shown in fig. 12A, the level of NO increased significantly after LPS stimulation of RAW264.7 cells. Compared with a blank control group, the grosvenor momordica grosvenori crude polysaccharide SGP, the refined polysaccharide SGP-C2 and the SGP-D1 can increase the level of NO in cells within the concentration range of 6.25-25 mug/mL, and are in concentration dependency relationship, which shows that the grosvenor momordica grosvenori polysaccharide can activate and improve the capability of releasing NO by macrophages RAW264.7 within a certain concentration range. FIG. 12B shows that Siraitia grosvenorii polysaccharide has an effect on TNF-alpha secretion of RAW264.7 cells, and SGP, SGP-C2 and SGP-D1 significantly promote TNF-alpha secretion in the concentration range of 6.25-25 μg/mL, and are concentration-dependent. FIG. 12C shows that SGP significantly promotes RAW264.7 cell IL-6 secretion in the concentration range of 12.5-25 μg/mL, SGP-C2 and SGP-D1 have no significant effect on IL-6 secretion in the concentration range of 6.25-12.5 μg/mL, and IL-6 secretion can be significantly promoted at high concentration of 25 μg/mL.
Comparative example 1
Extraction of grosvenor momordica fruit crude polysaccharide
(1) Weighing 2kg of dried fructus momordicae, crushing, adding 16L of 80vol% ethanol water solution, heating, refluxing and degreasing for 3 hours at 78 ℃, and repeating for 2 times to obtain fructus momordicae residues;
(2) Drying the fructus Siraitiae Grosvenorii residue in a 55 deg.C blast drying oven, adding water at a ratio of feed liquid to 1 g/5 mL, heating and extracting in water bath at 80deg.C for 2 times, each time for 3 hr; filtering the extract by ultrafiltration membrane, vacuum concentrating the filtrate to 1/4 of the total volume at 55deg.C, adding 10 times of 95% ethanol water solution, standing overnight at 4deg.C, centrifuging the ethanol sediment material at 4000r/min for 10min, and eluting the sediment material with anhydrous ethanol for 3 times. Dissolving the washed precipitate with water, removing protein for 1 time by using a sevage method, removing redundant chloroform by rotary evaporation of filtrate, dissolving to 400mL by adding water, slowly adding 1400mL of absolute ethyl alcohol while stirring, standing overnight at 4 ℃, centrifuging the alcohol sediment material for 10min under the condition of 4200r/min, collecting the precipitate, washing the precipitate with absolute ethyl alcohol for 3 times, dissolving the washed precipitate with water, decolorizing with AB-8 macroporous, eluting with pure water, concentrating the eluent, and freeze-drying (the temperature is-80 ℃ for 48 h) to obtain the momordica grosvenori crude polysaccharide with the yield of 0.99%.
Comparative example 2
Extraction of grosvenor momordica fruit crude polysaccharide
(1) Weighing 2kg of dried fructus momordicae, crushing, adding 16L of 80vol% ethanol water solution, heating, refluxing and degreasing for 3 hours at 78 ℃, and repeating for 2 times to obtain fructus momordicae residues;
(2) Drying the fructus Siraitiae Grosvenorii residue in a blast drying oven at 55deg.C, adding water at a ratio of feed liquid to 1g:20mL, heating and extracting in water bath at 70deg.C for 2 times, each time for 4 hr; filtering the extract by ultrafiltration membrane, vacuum concentrating the filtrate to 1/4 of the total volume at 55deg.C, adding 10 times of 95% ethanol water solution, standing overnight at 4deg.C, centrifuging the ethanol sediment material at 4000r/min for 10min, and eluting the sediment material with anhydrous ethanol for 3 times. Dissolving the washed precipitate with water, removing protein for 1 time by using a sevage method, removing redundant chloroform by rotary evaporation of filtrate, dissolving to 400mL by adding water, slowly adding 1400mL of absolute ethyl alcohol while stirring, standing overnight at 4 ℃, centrifuging the alcohol sediment material for 10min under the condition of 4200r/min, collecting the precipitate, washing the precipitate with absolute ethyl alcohol for 3 times, dissolving the washed precipitate with water, decolorizing with AB-8 macroporous, eluting with pure water, concentrating the eluent, and freeze-drying (the temperature is-80 ℃ for 48 h) to obtain the crude momordica grosvenori polysaccharide with the yield of 1.61%.
Comparative example 3
Extraction of grosvenor momordica fruit crude polysaccharide
(1) Weighing 2kg of dried fructus momordicae, crushing, adding 16L of 80vol% ethanol water solution, heating, refluxing and degreasing for 3 hours at 78 ℃, and repeating for 2 times to obtain fructus momordicae residues;
(2) Drying the fructus Siraitiae Grosvenorii residue in a blast drying oven at 55deg.C, adding water at a ratio of feed liquid to 1g:10mL, heating and extracting in water bath at 70deg.C for 2 times, each time for 4 hr; filtering the extract by using an ultrafiltration membrane, concentrating the filtrate at 55 ℃ in vacuum to 1/4 of the total volume, adding an ethanol water solution with the volume percentage of 95% and the volume percentage of 10 times of the volume of the concentrated solution, standing overnight at 4 ℃, centrifuging the alcohol sediment material for 10min under the condition of 4000r/min, leaching the sediment by using absolute ethanol for 3 times, dissolving the washed sediment by adding water, decolorizing by using AB-8 macroporous, eluting by using pure water, concentrating the eluent, and freeze-drying (the temperature is-80 ℃ and the time is 48 h) to obtain the momordica grosvenori crude polysaccharide with the yield of 1.02%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. The preparation method of the momordica grosvenori pectic polysaccharide is characterized by comprising the following steps of:
pretreating fructus Siraitiae Grosvenorii to obtain fructus Siraitiae Grosvenorii crude polysaccharide;
separating the crude polysaccharide of the momordica grosvenori by a cellulose column, and eluting by adopting water, 0.1mol/L sodium chloride aqueous solution, 0.3mol/L sodium chloride aqueous solution and 0.5mol/L sodium chloride aqueous solution in sequence to respectively obtain water eluent, 0.1mol/L sodium chloride aqueous solution eluent, 0.3mol/L sodium chloride aqueous solution eluent and 0.5mol/L sodium chloride aqueous solution eluent;
dialyzing water eluent, 0.1mol/L sodium chloride aqueous solution eluent, 0.3mol/L sodium chloride aqueous solution eluent and 0.5mol/L sodium chloride aqueous solution eluent respectively to obtain components with the molecular weight of more than 1000, and obtaining SGP-A, SGP-B, SGP-C and SGP-D respectively;
separating SGP-C by gel column, eluting with water to obtain SGP-C2;
the SGP-C2 includes glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose; the molar ratio of glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose in the SGP-C2 is 0.78:1.84:26.57:1.31:0.49:0.06.
2. the method of claim 1, further comprising separating SGP-D by a gel column, eluting with water to obtain SGP-D1; the SGP-D1 comprises glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose; the molar ratio of glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose in SGP-D1 is 1.37:2.00:20.79:0.61:0.12:0.25.
3. the method of claim 1, wherein the pretreatment comprises:
degreasing fructus Siraitiae Grosvenorii with ethanol water solution to obtain fructus Siraitiae Grosvenorii residue;
extracting the momordica grosvenori residues with water, removing compounds with molecular weight smaller than 300 from the obtained extracting solution through an ultrafiltration membrane, and sequentially concentrating and performing first alcohol precipitation on the obtained filtrate to obtain a first alcohol precipitation substance;
and dissolving the first alcohol precipitate in water, and sequentially removing proteins, performing second alcohol precipitation and decoloring to obtain the crude polysaccharide of the momordica grosvenori.
4. The method of claim 1, wherein the cellulose column is a DE-52 cellulose column; the gel column is a SephadexG-200 gel column.
5. The method according to claim 1 or 4, wherein the flow rate of the eluent is 5mL/min when the cellulose column is separated.
6. The method according to claim 1 or 4, wherein the SGP-C is separated by a gel column at a water flow rate of 0.2mL/min.
7. The method according to claim 2, wherein the aqueous ethanol solution has a volume concentration of 70-90%.
8. The preparation method according to claim 2, wherein the ratio of the amount of the momordica grosvenori to the aqueous ethanol solution is 1g:5-20mL.
9. The method according to claim 2, wherein the degreasing is thermal reflow degreasing, the degreasing temperature is 60-80 ℃, the number of times of degreasing is 1-3, and each time is 1-3 hours.
10. The method according to claim 2, wherein the aqueous ethanol solution of the first alcohol precipitation has a volume concentration of 80-100%.
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