CN110483654B - Preparation method and application of modified huperzia serrata polysaccharide - Google Patents
Preparation method and application of modified huperzia serrata polysaccharide Download PDFInfo
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Abstract
The invention discloses a preparation method and application of modified huperzia serrata polysaccharide, and relates to the technical field of high-molecular polysaccharide. The preparation method of the modified huperzia serrata polysaccharide comprises the following steps: dissolving huperzia serrata polysaccharide in a sodium hydroxide solution to obtain a huperzia serrata polysaccharide solution; adding a modifier into the huperzia serrata polysaccharide solution, and stirring for reaction to obtain a reaction solution; adjusting the reaction solution to be neutral, and dialyzing to obtain a concentrated solution; carrying out alcohol precipitation treatment on the concentrated solution, carrying out solid-liquid separation, collecting precipitate, and drying to obtain modified huperzia serrata polysaccharide; wherein the modifying agent is a phosphorylating agent or a carboxymethylating agent. The modified huperzia serrata polysaccharide prepared by modifying the huperzia serrata polysaccharide has good water solubility, and the market competitiveness of the huperzia serrata polysaccharide serving as an anti-tumor medicament is improved.
Description
Technical Field
The invention relates to the technical field of high molecular polysaccharides, in particular to a preparation method and application of modified huperzia serrata polysaccharide.
Background
Huperzia serrata is a Tibetan plant of huperzia genus of huperziaceae family, also called huperzia serrata, ophiorrhiza serrulata and the like, and is commonly used for treating traumatic injury, schizophrenia, inflammation and detoxification as a traditional Chinese herbal medicine. Research proves that huperzia serrata contains huperzine, terpenes and flavonoids, and because the huperzia serrata polysaccharide is poor in water solubility, the huperzia serrata has poor market competitiveness as an anti-tumor medicament.
Disclosure of Invention
The invention mainly aims to provide a preparation method and application of modified huperzia serrata polysaccharide, and aims to solve the problem of poor water solubility of the huperzia serrata polysaccharide.
In order to achieve the above object, the present invention provides a method for preparing modified huperzia serrata polysaccharide, comprising the steps of:
dissolving huperzia serrata polysaccharide in a sodium hydroxide solution to obtain a huperzia serrata polysaccharide solution;
adding a modifier into the huperzia serrata polysaccharide solution, and stirring for reaction to obtain a reaction solution;
adjusting the reaction solution to be neutral, and dialyzing to obtain a concentrated solution;
carrying out alcohol precipitation treatment on the concentrated solution, carrying out solid-liquid separation, collecting precipitate, and drying to obtain modified huperzia serrata polysaccharide;
wherein the modifier is a phosphorylating agent or a carboxymethylating agent.
Optionally, in the step of dissolving the huperzia serrata polysaccharide in the sodium hydroxide solution to obtain the huperzia serrata polysaccharide solution, the mass ratio of the huperzia serrata polysaccharide to the sodium hydroxide contained in the sodium hydroxide solution is 1: (8-15).
Optionally, the phosphorylating agent comprises a mixed agent of sodium tripolyphosphate and sodium trimetaphosphate.
Optionally, in the mixed reagent of sodium tripolyphosphate and sodium trimetaphosphate, the mass ratio of the sodium tripolyphosphate to the sodium trimetaphosphate is (5-7): 1.
optionally, the step of adding a modifier to the huperzia serrata polysaccharide solution and stirring for reaction to obtain a reaction solution comprises:
adding a phosphorylation reagent into the huperzia serrata polysaccharide solution, and stirring for reaction to obtain a reaction solution;
wherein the mass ratio of the phosphorylation reagent to the sodium hydroxide contained in the sodium hydroxide solution is 1: (1-2); and/or the presence of a gas in the gas,
the reaction temperature is 70-100 ℃; and/or the presence of a gas in the gas,
the reaction time is 3-7 h.
Optionally, the carboxymethylating agent comprises monochloroacetic acid.
Optionally, the step of adding a modifier to the huperzia serrata polysaccharide solution and stirring for reaction to obtain a reaction solution comprises:
adding a carboxymethylation reagent into the huperzia serrata polysaccharide solution, and stirring for reaction to obtain a reaction solution;
wherein the mass ratio of the carboxymethylation reagent to the sodium hydroxide contained in the sodium hydroxide solution is 1: (1-2); and/or the presence of a gas in the gas,
the reaction temperature is 40-60 ℃; and/or the presence of a gas in the gas,
the reaction time is 2-4 h.
Optionally, after adjusting the reaction solution to neutral, the step of dialyzing to obtain a concentrated solution comprises:
and (3) adjusting the reaction liquid to be neutral, dialyzing by adopting a dialysis bag with the molecular weight cutoff of 3000-4000, stopping dialysis and collecting concentrated liquid when no white precipitate is generated after silver nitrate solution is added into the dialyzate.
Optionally, in the step of performing alcohol precipitation treatment on the concentrated solution, performing solid-liquid separation, collecting the precipitate, and drying to obtain the modified huperzia serrata polysaccharide, the volume ratio of the added ethanol to the concentrated solution in the alcohol precipitation treatment is (3-5): 1.
the invention also provides application of the modified huperzia serrata polysaccharide prepared by the preparation method of the modified huperzia serrata polysaccharide in preparing an antioxidant or an anti-tumor medicament.
In the technical scheme provided by the invention, the huperzia serrata polysaccharide is dissolved in a sodium hydroxide solution to obtain a huperzia serrata polysaccharide solution; adding a modifier into the huperzia serrata polysaccharide solution, and stirring for reaction to obtain a reaction solution; adjusting the reaction solution to be neutral, and dialyzing to obtain a concentrated solution; carrying out alcohol precipitation treatment on the concentrated solution, carrying out solid-liquid separation, collecting precipitate, and drying to obtain modified huperzia serrata polysaccharide; wherein the modifier is a phosphorylating agent or a carboxymethylating agent. Compared with the serrate club moss polysaccharide in the prior art, the modified serrate club moss polysaccharide has high degree of substitution, better water solubility than the serrate club moss polysaccharide, and obviously improved oxidation resistance, and the antioxidant or the antitumor drug prepared from the modified serrate club moss polysaccharide has better efficacy and market competitiveness.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic flow diagram of one embodiment of a process for preparing phosphorylated huperzine serrate polysaccharides of the present invention;
FIG. 2 is a graph showing the trend of the removal rate of metal radicals by phosphorylated huperzine serrate polysaccharide prepared in example 3 in the measurement of antioxidant activity;
FIG. 3 is a graph showing the trend of the scavenging rate of ABTS free radicals of phosphorylated huperzia serrata polysaccharides prepared in example 3 in the determination of antioxidant activity;
FIG. 4 is a graph showing the trend of the clearance of metal radicals by carboxymethylated huperzine serrate polysaccharide prepared in example 4 in the measurement of antioxidant activity;
FIG. 5 is a graph showing the trend of the scavenging rate of the carboxymethylated huperzine serrate polysaccharide ABTS free radicals prepared in example 4 in the determination of antioxidant activity;
the implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It is to be understood that the described embodiments are merely a few embodiments of the invention, and not all embodiments.
It should be noted that those whose specific conditions are not specified in the examples were performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially. In addition, the meaning of "and/or" appearing throughout includes three juxtapositions, exemplified by "A and/or B" including either A or B or both A and B. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Huperzia serrata is used as a traditional Chinese herbal medicine for treating traumatic injury, schizophrenia and diminishing inflammation and detoxifying, but the water solubility of the Huperzia serrata polysaccharide is poor, so the market competitiveness of the Huperzia serrata polysaccharide as an anti-tumor medicine is poor. In view of this, the present invention provides a method for preparing modified huperzia serrata polysaccharide, which has good water solubility and improves the market competitiveness of huperzia serrata polysaccharide as an antitumor drug.
The preparation method of the modified huperzia serrata polysaccharide provided by the invention can be used for preparing the modified huperzia serrata polysaccharide. Referring to fig. 1, the preparation method of modified huperzia serrata polysaccharide comprises the following steps:
s10, dissolving huperzia serrata polysaccharide in a sodium hydroxide solution to obtain a huperzia serrata polysaccharide solution;
s20, adding a modifier into the huperzia serrata polysaccharide solution, and stirring to react to obtain a reaction solution;
s30, adjusting the reaction solution to be neutral, and dialyzing to obtain a concentrated solution;
s40, carrying out alcohol precipitation treatment on the concentrated solution to separate solid from liquid, collecting the precipitate and drying to obtain modified huperzia serrata polysaccharide;
wherein the modifier is a phosphorylating agent or a carboxymethylating agent.
In this embodiment, in step S10, an alkaline solution is added to the huperzia serrata polysaccharide to dissolve the huperzia serrata polysaccharide in the alkaline solution, so that on one hand, the dissolving speed of the huperzia serrata polysaccharide is increased, on the other hand, necessary reaction conditions are provided for subsequent phosphorylation or carboxymethylation modification, and compared with an alcohol solvent, the aqueous solution of sodium hydroxide has better solubility and faster dissolving speed, and the discharged waste liquid has no organic residue.
Specifically, the step S10 may include the following steps:
step S110, the huperzia serrata polysaccharide is crushed and sieved to obtain huperzia serrata polysaccharide powder.
And S120, adding a sodium hydroxide solution into the huperzia serrata polysaccharide powder, alkalizing for 0.5-1 h until huperzia serrata polysaccharide is fully dissolved, and thus obtaining a huperzia serrata polysaccharide solution.
Wherein the Huperzia serrata polysaccharide can be extracted from Huperzia serrata.
Further, the problem that the modified huperzia serrata polysaccharide structure is damaged, so that the number of by-products is increased and the reaction yield is reduced is avoided. The invention controls the alkalization degree of the huperzia serrata polysaccharide, and specifically controls the mass ratio of the huperzia serrata polysaccharide to sodium hydroxide contained in a sodium hydroxide solution to be 1: (8-15). The huperzia serrata polysaccharide is dissolved in the sodium hydroxide solution, and the sodium hydroxide solution may have any concentration, such as 0.1mol/L, 0.2mol/L, 1mol/L, 10mol/L, etc., as long as the mass ratio of the huperzia serrata polysaccharide to the sodium hydroxide solution containing sodium hydroxide is 1: (8-15). In addition, the sodium hydroxide solution can be continuously stirred when being added, and the dripping speed and the stirring speed are controlled to avoid the phenomenon that the by-product is generated due to overlarge local alkalinity; the sodium hydroxide solution can also be added once or for multiple times to control the reaction to be stably carried out and avoid side reactions.
The preparation method of the modified huperzia serrata polysaccharide can prepare and form phosphorylated huperzia serrata polysaccharide or carboxymethylated huperzia serrata polysaccharide according to different modifying agents. Obviously, if the added modifying agent is a phosphorylation reagent, the obtained huperzia serrata polysaccharide is phosphorylated huperzia serrata polysaccharide; if the modifier is carboxymethylation reagent, the obtained Huperzia serrata polysaccharide is carboxymethylation Huperzia serrata polysaccharide.
In order to make the prepared phosphorylated huperzine serrate polysaccharide have better water-solubility, the phosphorylation reagent comprises a mixed reagent of sodium tripolyphosphate and sodium trimetaphosphate. More specifically, the mass ratio of the sodium tripolyphosphate to the sodium trimetaphosphate is (5-7): 1. thus, if the modified huperzia serrata polysaccharide is a phosphorylated huperzia serrata polysaccharide, step S20 is specifically: and adding a phosphorylation reagent into the huperzia serrata polysaccharide solution, and stirring for reaction to obtain a reaction solution. Under alkaline conditions, the phosphorylating agent can phosphorylate the huperzia serrata polysaccharide structure. The phosphorylating agent is not limited to a mixed agent of sodium tripolyphosphate and sodium trimetaphosphate, and any agent that can phosphorylate a structure of huperzia serrata can be the phosphorylating agent of the present invention. Obviously, the amount of the phosphorylating agent, the reaction time and the reaction temperature during the reaction are large in response to the progress of the reaction. Wherein the mass ratio of the phosphorylation reagent to the sodium hydroxide contained in the sodium hydroxide solution is 1: (1-2); optionally, the reaction temperature is 70-100 ℃, and the reaction time is 3-7 h.
In addition, alternatives of the reaction temperature and the reaction time in the step S20 may be present at the same time or alternatively, depending on the actual needs.
If the modified huperzia serrata polysaccharide to be produced is the carboxymethylated huperzia serrata polysaccharide, step S20 specifically comprises: and adding a carboxymethylation reagent into the huperzia serrata polysaccharide solution, and stirring for reaction to obtain a reaction solution. In this example, the carboxymethylating agent is monochloroacetic acid. Of course, the carboxymethylating agent is not limited to monochloroacetic acid, and all agents that can carboxymethylate a huperzine serrate polysaccharide structure may be carboxymethylating agents of the present invention. Under alkaline conditions, the monochloroacetic acid can carboxymethylate the huperzia serrata polysaccharide structure. During the reaction, the amount of monochloroacetic acid, the reaction time and the reaction temperature have a great influence on the progress of the reaction. Wherein the mass ratio of the monochloroacetic acid to the sodium hydroxide contained in the sodium hydroxide solution is 1: (1-2); optionally, the reaction time of the reaction is 2-4 h, and the reaction temperature is 40-60 ℃. In addition, alternatives of the reaction temperature and the reaction time in the step S20 may be present at the same time or alternatively, depending on the actual needs.
And step S30, adjusting the reaction solution to be neutral, and dialyzing to obtain concentrated solution. Specifically, after the reaction solution is adjusted to be neutral, dialyzing by using a dialysis bag with the molecular weight cutoff of 3000-4000, stopping dialysis and collecting concentrated solution when no white precipitate is generated after a silver nitrate solution is added into the dialyzate.
And step S40, carrying out alcohol precipitation treatment on the concentrated solution, carrying out solid-liquid separation, collecting the precipitate, and drying to obtain the modified huperzia serrata polysaccharide. In the embodiment, when the alcohol precipitation treatment is performed, the volume ratio of the added ethanol to the concentrated solution is (3-5): 1. the concentration of the ethanol adopted is preferably 95% ethanol, and in order to further improve the purity of the modified huperzia serrata polysaccharide in the product, multiple alcohol precipitation treatments can be carried out.
In addition, when the precipitate is dried, the drying temperature is not higher than 30 ℃, and the drying mode can be vacuum drying, forced air drying and the like; in the case of solid-liquid separation, filtration, centrifugation, suction filtration or the like may be employed.
The modified huperzia serrata polysaccharide has better water solubility than huperzia serrata polysaccharide, and the metal free radicals, ABTS free radicals and the like show obvious scavenging action. Meanwhile, by optimizing each reaction condition, the preparation method is simple to operate, short in time consumption, low in reagent consumption and low in cost, and the prepared finished product has no monochloroacetic acid or other organic solvent residues and has a medium substitution degree.
In addition, for the sake of cost saving and environmental protection, after the above steps are finished, the following steps can be further included:
and step S50, collecting the liquid phase after solid-liquid separation, recovering ethanol, and using the recovered ethanol for alcohol precipitation treatment.
In addition, the invention also provides application of the modified huperzia serrata polysaccharide prepared by the preparation method of the modified huperzia serrata polysaccharide in preparing an antioxidant or an antitumor drug.
The modified huperzia serrata polysaccharide has good solubility and obvious antioxidant activity, and can be used for preparing antioxidant medicines and health products by using the modified huperzia serrata polysaccharide as an active ingredient. Specifically, the modified huperzia serrata polysaccharide can be used as one of the components, and is mixed with other active ingredients or auxiliary materials to prepare a composition; or combining modified Huperzia serrata polysaccharide as medicinal component with carrier to obtain carrier drug.
The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention.
Example 1
Crushing the huperzia serrata polysaccharide into huperzia serrata polysaccharide powder, taking 0.1g of huperzia serrata polysaccharide powder, adding a sodium hydroxide solution (containing 0.8g of sodium hydroxide), and alkalizing for 1h until the huperzia serrata polysaccharide is fully dissolved to obtain the huperzia serrata polysaccharide solution. Adding 0.4g of phosphorylation reagent (the mass ratio of sodium tripolyphosphate to sodium trimetaphosphate is 7: 1) into the huperzia serrata polysaccharide solution under stirring at 100 ℃, adjusting the pH of the reaction solution to 7, and reacting for 7.0h to obtain the reaction solution. And (3) putting the reaction solution into a dialysis bag with the molecular weight cutoff of 3500 for dialysis, stopping dialysis and collecting concentrated solution when no white precipitate is generated after a silver nitrate solution is added into the dialysis solution. Ethanol was added to the concentrate in an amount of 4 times the volume of the concentrate, and the mixture was allowed to stand overnight and centrifuged. Vacuum drying the precipitate at 25 deg.C to obtain phosphorylated huperzia serrata polysaccharide; the supernatant was distilled to recover ethanol.
Example 2
Crushing the huperzia serrata polysaccharide into huperzia serrata polysaccharide powder, taking 0.1g of huperzia serrata polysaccharide powder, adding a sodium hydroxide solution (containing 1g of sodium hydroxide), and alkalizing for 0.7h until the huperzia serrata polysaccharide is fully dissolved to obtain the huperzia serrata polysaccharide solution. Adding 1g of phosphorylation reagent (the mass ratio of sodium tripolyphosphate to sodium trimetaphosphate is 6: 1) into the huperzia serrata polysaccharide solution under stirring at 70 ℃, adjusting the pH of the reaction solution to 7, and reacting for 5.0h to obtain the reaction solution. And (3) putting the reaction solution into a dialysis bag with the molecular weight cutoff of 3500 for dialysis, stopping dialysis and collecting concentrated solution when no white precipitate is generated after a silver nitrate solution is added into the dialysis solution. Ethanol with the volume 3 times that of the concentrated solution is added into the concentrated solution, and after standing overnight, the concentrated solution is centrifuged. Vacuum drying the precipitate at 25 deg.C to obtain phosphorylated huperzia serrata polysaccharide; the supernatant was distilled to recover ethanol.
Example 3
The method comprises the steps of crushing the huperzia serrata polysaccharide into huperzia serrata polysaccharide powder, taking 0.1g of the huperzia serrata polysaccharide powder, adding a sodium hydroxide solution (containing 1.5g of sodium hydroxide), and alkalizing for 0.5h until the huperzia serrata polysaccharide is fully dissolved to obtain the huperzia serrata polysaccharide solution. Adding 1g of phosphorylation reagent (the mass ratio of sodium tripolyphosphate to sodium trimetaphosphate is 5: 1) into the huperzia serrata polysaccharide solution under stirring at 80 ℃, adjusting the pH of the reaction solution to 7, and reacting for 3.0h to obtain the reaction solution. And putting the reaction solution into a dialysis bag with the molecular weight cutoff of 3000 for dialysis, stopping dialysis and collecting concentrated solution when no white precipitate is generated after a silver nitrate solution is added into the dialysis solution. Ethanol of 5 times the volume of the concentrate was added to the concentrate, and after standing overnight, the mixture was centrifuged. Vacuum drying the precipitate at 25 deg.C to obtain phosphorylated huperzia serrata polysaccharide; the supernatant was distilled to recover ethanol.
Example 4
The method comprises the steps of crushing the huperzia serrata polysaccharide into huperzia serrata polysaccharide powder, taking 0.1g of the huperzia serrata polysaccharide powder, adding a sodium hydroxide solution (containing 0.8g of sodium hydroxide), and alkalizing for 0.5h until the huperzia serrata polysaccharide is fully dissolved to obtain the huperzia serrata polysaccharide solution. Adding 0.4g of monochloroacetic acid into the huperzia serrata polysaccharide solution under stirring at 40 ℃, and reacting for 2 hours to obtain reaction liquid. Adjusting the pH of the reaction solution to 7, placing the reaction solution in a dialysis bag with the molecular weight cutoff of 3500 for dialysis, and stopping dialysis and collecting the concentrated solution when no white precipitate is generated after adding silver nitrate solution into the dialysis solution. Ethanol was added to the concentrate in an amount of 4 times the volume of the concentrate, and the mixture was allowed to stand overnight and centrifuged. Vacuum drying the precipitate at 30 deg.C to obtain carboxymethyl huperzia serrata polysaccharide; the supernatant was distilled to recover ethanol.
Example 5
The method comprises the steps of crushing the huperzia serrata polysaccharide into huperzia serrata polysaccharide powder, taking 0.1g of the huperzia serrata polysaccharide powder, adding a sodium hydroxide solution (containing 1.5g of sodium hydroxide), and alkalizing for 0.5h until the huperzia serrata polysaccharide is fully dissolved to obtain the huperzia serrata polysaccharide solution. Under the condition of stirring and 45 ℃, 1g of monochloroacetic acid is added into the huperzia serrata polysaccharide solution, and the reaction is carried out for 3 hours to obtain reaction liquid. Adjusting the pH of the reaction solution to 7, placing the reaction solution in a dialysis bag with the molecular weight cutoff of 3500 for dialysis, and stopping dialysis and collecting the concentrated solution when no white precipitate is generated after adding silver nitrate solution into the dialysis solution. Ethanol with the volume 3 times that of the concentrated solution is added into the concentrated solution, and after standing overnight, the concentrated solution is centrifuged. Vacuum drying the precipitate at 27 deg.C to obtain carboxymethyl huperzia serrata polysaccharide; the supernatant was distilled to recover ethanol.
Example 6
Crushing the huperzia serrata polysaccharide into huperzia serrata polysaccharide powder, taking 0.1g of huperzia serrata polysaccharide powder, adding a sodium hydroxide solution (containing 1g of sodium hydroxide), and alkalizing for 0.5h until the huperzia serrata polysaccharide is fully dissolved to obtain the huperzia serrata polysaccharide solution. Under the condition of stirring and 60 ℃, 1g of monochloroacetic acid is added into the huperzia serrata polysaccharide solution, and the reaction is carried out for 4 hours to obtain reaction liquid. Adjusting the pH of the reaction solution to 7, putting the reaction solution into a dialysis bag with the molecular weight cutoff of 4000 for dialysis, and stopping dialysis and collecting concentrated solution when no white precipitate is generated after adding silver nitrate solution into the dialysis solution. Ethanol of 5 times the volume of the concentrate was added to the concentrate, and after standing overnight, the mixture was centrifuged. Vacuum drying the precipitate at 30 deg.C to obtain carboxymethyl huperzia serrata polysaccharide; the supernatant was distilled to recover ethanol.
Measurement of degree of substitution
1. Phosphorylated huperzia serrata polysaccharide prepared in the above examples was assayed as follows
The degree of substitution was determined as follows, using the phosphorylated huperzia serrata polysaccharides prepared in examples 1 to 3:
and (2) putting 10mg of sample into a beaker, respectively adding 1mL of concentrated sulfuric acid and concentrated nitric acid, heating on an electric furnace until smoke is generated, then cooling, adding 1mL of 30% hydrogen peroxide solution, slowly heating, and repeating the steps until no smoke is generated in the bottle, wherein the solution is colorless and transparent or light yellow. After cooling, 1mL of 6mol/L hydrochloric acid was added, and the mixture was heated on an electric furnace to completely decompose the acid, and transferred to a 50mL volumetric flask to fix the volume. Placing 5mL of the solution in a colorimetric tube, sequentially adding 2.0mL of 5% ammonium molybdate solution, shaking up, standing for a few seconds, respectively adding 1.0mL of 2% sodium sulfite solution and 1.0mL of 0.5% hydroquinone solution, shaking up, adding water to a scale, standing for 30min, zeroing with a reference, measuring absorbance at a wavelength of 660nm, and drawing a standard curve by taking the mass concentration of the phosphate radical standard solution (in terms of P) as a horizontal coordinate and the corresponding absorbance as a vertical coordinate to obtain a standard curve equation.
The calculation was performed according to the following formula, and the results are shown in Table 1.
Establishing a standard curve equation as follows:
y＝0.0004x+0.0001，R2＝0.9997
wherein y is absorbance, and x is phosphate radical standard solution mass concentration
Table 1 comparison of substitution ratios for phosphorylated huperzine serrate polysaccharide examples
| Detecting items | Example 1 | Example 2 | Example 3 |
| Degree of substitution/%) | 7.036 | 9.743 | 8.203 |
As can be seen from Table 1 above, the degree of substitution of phosphorylated huperzia serrata polysaccharides obtained in the examples is generally greater than 7%, while the degree of substitution of phosphorylated huperzia serrata polysaccharides obtained in comparative examples 2 and 3, which have excessive alkalinity, low temperature and excessive phosphorylating agent, is high.
2. Carboxymethylated huperzia serrata polysaccharide prepared in the above example was tested as follows
The degree of substitution was determined as follows, taking the carboxymethyl huperzia serrata polysaccharides prepared in examples 4 to 6:
10mg of sample was accurately weighed, 3mL of ethanol with a volume fraction of 70% was added, and the mixture was left for 5min after mixing. 10mL of distilled water and 50mL of 0.5mol/L sodium hydroxide solution were added in this order, mixed and stirred until the sample was dissolved. Then titrating with 0.1mol/LHCl solution, and taking phenolphthalein as a color developing agent. The calculation was performed according to the following formula and the results were recorded as in table 1.
The formula: a ═ V0M0-(V2-V1)M)/m
DS＝0.162A/(1-0.058A)
In the formula: v0Is the volume of sodium hydroxide solution added (mL); v1Determine the volume of HCl solution consumed (mL) for the blank; v2Determining the volume of HCl solution consumed (mL) for the sample; m0Is the concentration of the added sodium hydroxide solution; m is the concentration of the HCl solution used for the determination; m is the mass (g) of the sample used for the determination.
TABLE 2 comparison of degree of substitution for carboxymethylated Huperzia serrata polysaccharide examples
| Detecting items | Example 4 | Example 5 | Example 6 |
| Degree of substitution/%) | 0.259 | 0.238 | 0.158 |
As can be seen from Table 2, the degree of substitution of the carboxymethyl huperzine serrate polysaccharides obtained in examples 4 and 5 is above 0.2, while the degree of substitution of the carboxymethyl huperzine serrate polysaccharide obtained in example 6, which has excessive alkalinity, excessive temperature and excessive monochloroacetic acid, is significantly lower, indicating that the carboxymethyl huperzine serrate polysaccharide prepared by the preparation method of the present invention has a medium degree of substitution.
(II) measurement of antioxidant Activity
(1) The scavenging properties of phosphorylated huperzia serrata polysaccharide prepared in example 3 and carboxymethylated huperzia serrata polysaccharide prepared in example 4 were measured as follows:
sample solutions (1mg/mL, 2mg/mL, 4mg/mL, 8mg/mL, 10mg/mL) of different concentrations were taken and 400. mu.L of each sample solution was added with 40. mu.L of 5mmol/L FeCl2Measuring absorbance A of the solution, 80 mu L of phenanthroline solution and 1080 mu L of distilled water at 562nm1. Distilled water was used as a blank. Clearance was calculated according to the following formula and the change in clearance was plotted as shown in fig. 2.
The formula: clearance (%) - (1- (A)1-A2)/A0]×100％
In the formula: a. the0Absorbance of control group; a. the1Is the absorbance of the sample; a. the2Is the background absorbance value (FeCl) of the sample2The solution was changed to distilled water).
(2) The scavenging properties of the huperzia serrata polysaccharide prepared in example 3 and the carboxymethylated huperzia serrata polysaccharide prepared in example 4 for ABTS free radicals were determined as follows:
sample solutions (1mg/mL, 2mg/mL, 4mg/mL, 8mg/mL, 10mg/mL) of different concentrations, 400. mu.L each, were added to 1600. mu.L of the ABTS + stock solution, reacted at room temperature for 10min, and the absorbance measured at 734nm was A1. Distilled water was used as a blank. Clearance was calculated according to the following formula and the change in clearance was plotted as shown in fig. 3.
The formula: clearance (%) - (1- (A)1-A2)/A0]×100％
In the formula: a. the0Absorbance of control group; a. the1Is the absorbance of the sample; a. the2The background absorbance value of the sample (distilled water instead of ABTS + stock solution).
As can be seen from fig. 2 to fig. 3, the phosphorylated huperzia serrata polysaccharide prepared in example 3 has a scavenging effect on metal ion free radicals and ABTS free radicals, and particularly has a significant antioxidant effect on metal ion free radicals at a concentration of 8mg/mL, and has a significant antioxidant effect on ABTS free radicals at a concentration of 4 mg/mL. As can be seen from fig. 4 to fig. 5, the carboxymethyl huperzia serrata polysaccharide prepared in example 4 has a scavenging effect on metal ion free radicals and ABTS free radicals, and particularly has a significant antioxidant property at a concentration of 8mg/mL, which indicates that the carboxymethyl huperzia serrata polysaccharide prepared in the present invention has a good antioxidant activity and can be used for preparing an antioxidant. In conclusion, the modified huperzia serrata polysaccharide has better antioxidant activity and can be used for preparing antioxidants.
The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.
Claims (10)
1. A method for preparing modified huperzia serrata polysaccharide, which is characterized by comprising the following steps:
dissolving huperzia serrata polysaccharide in a sodium hydroxide solution to obtain a huperzia serrata polysaccharide solution;
adding a modifier into the huperzia serrata polysaccharide solution, and stirring for reaction to obtain a reaction solution;
adjusting the reaction solution to be neutral, and dialyzing to obtain a concentrated solution;
carrying out alcohol precipitation treatment on the concentrated solution, carrying out solid-liquid separation, collecting precipitate, and drying to obtain modified huperzia serrata polysaccharide;
wherein the modifier is a phosphorylating agent or a carboxymethylating agent.
2. The method of claim 1, wherein the step of dissolving the serrate club moss in the sodium hydroxide solution to obtain the serrate club moss solution comprises the step of mixing the serrate club moss with the sodium hydroxide contained in the sodium hydroxide solution in a mass ratio of 1: (8-15).
3. The method of claim 1, wherein the phosphorylating agent comprises a mixture of sodium tripolyphosphate and sodium trimetaphosphate.
4. The method for preparing modified huperzia serrata polysaccharide of claim 3, wherein the mass ratio of the sodium tripolyphosphate to the sodium trimetaphosphate in the mixed reagent of sodium tripolyphosphate and sodium trimetaphosphate is (5-7): 1.
5. the method of claim 1, wherein the step of adding a modifying agent to the solution of huperzia serrata polysaccharide and stirring the mixture to obtain a reaction solution comprises:
adding a phosphorylation reagent into the huperzia serrata polysaccharide solution, and stirring for reaction to obtain a reaction solution;
wherein the mass ratio of the phosphorylation reagent to the sodium hydroxide contained in the sodium hydroxide solution is 1: (1-2); and/or the presence of a gas in the gas,
the reaction temperature is 70-100 ℃; and/or the presence of a gas in the gas,
the reaction time is 3-7 h.
6. The method of claim 1, wherein the carboxymethylating agent comprises monochloroacetic acid.
7. The method of claim 1, wherein the step of adding a modifying agent to the solution of huperzia serrata polysaccharide and stirring the mixture to obtain a reaction solution comprises:
adding a carboxymethylation reagent into the huperzia serrata polysaccharide solution, and stirring for reaction to obtain a reaction solution;
wherein the mass ratio of the carboxymethylation reagent to the sodium hydroxide contained in the sodium hydroxide solution is 1: (1-2); and/or the presence of a gas in the gas,
the reaction temperature is 40-60 ℃; and/or the presence of a gas in the gas,
the reaction time is 2-4 h.
8. The method of claim 1, wherein the step of adjusting the reaction solution to neutral and dialyzing to obtain a concentrated solution comprises:
and (3) adjusting the reaction liquid to be neutral, dialyzing by adopting a dialysis bag with the molecular weight cutoff of 3000-4000, stopping dialysis and collecting concentrated liquid when no white precipitate is generated after silver nitrate solution is added into the dialyzate.
9. The method according to claim 1, wherein the modified huperzia serrata polysaccharide is obtained by subjecting the concentrated solution to alcohol precipitation, separating solid from liquid, collecting the precipitate, and drying, wherein the volume ratio of ethanol to the concentrated solution in the alcohol precipitation is (3-5): 1.
10. use of the modified huperzia serrata polysaccharide obtained by the method of any one of claims 1 to 9 in the preparation of an antioxidant or anti-tumor medicament.
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