CN110423355B - Preparation method of carboxymethylated lotus root polysaccharide-trichostatin A conjugate - Google Patents
Preparation method of carboxymethylated lotus root polysaccharide-trichostatin A conjugate Download PDFInfo
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Abstract
The invention discloses a preparation method of carboxymethylated lotus root polysaccharide-trichostatin A conjugate, which comprises the following steps: grafting polyethyleneimine-polyethylene glycol copolymer on carboxymethylated lotus root polysaccharide to obtain carboxymethylated lotus root polysaccharide complex; grafting folic acid on the carboxymethylated lotus root polysaccharide complex to obtain a carboxymethylated lotus root polysaccharide-folic acid complex; and grafting the carboxymethylated lotus root polysaccharide-folic acid compound to the trichostatin A to obtain the carboxymethylated lotus root polysaccharide-trichostatin A conjugate. The end product of the lotus root polysaccharide-trichostatin A conjugate has strong biological activity, and the performance research shows that the conjugate has obvious effect on the aspect of anti-tumor cells and can be widely applied to the production of anti-tumor drugs.
Description
Technical Field
The invention belongs to the field of drug synthesis, and particularly relates to a preparation method of carboxymethylated lotus root polysaccharide-trichostatin A conjugate.
Background
Nelumbo nucifera is the rhizome of Nymphaeaceae, and is mainly distributed in Asian countries such as China and India. According to the related records in Ben Cao Jing Shu, Nelumbo Nucifera Gaertn has the effects of clearing heat, stopping bleeding, regulating endocrine, nourishing and tranquilizing. According to the modern pharmacopoeia, the unprocessed lotus roots can play the roles of clearing heat, dissipating fatigue, cooling blood, treating heat stranguria and the like, while the mature lotus roots have the efficacies of strengthening spleen and stomach, benefiting blood, promoting tissue regeneration, stopping diarrhea and the like. A large number of researches show that the polysaccharide is an important component in the active ingredients of the lotus roots, and the polysaccharide has various biological activities including antioxidant activity, antitumor activity, immunoregulation activity, blood sugar reduction activity, antiviral activity and the like. According to the current development level, the technology level of lotus root processing is relatively low, and the extraction and development of polysaccharide can be an innovative way for developing and utilizing the resources. Polysaccharides are closely related to the maintenance of life-related functions and are one of the basic substances constituting human life.
Researches show that the molecular structure of the polysaccharide has a certain relationship with the exertion of the biological activity of the polysaccharide, and the modification of the molecular structure can obviously enhance the biological activity of the polysaccharide or generate new activity. However, the application of the polysaccharide drugs is not wide at present, particularly, the research results in the aspect of treating cancers are few, and the requirement of anti-tumor drugs is very urgent.
Disclosure of Invention
The invention mainly aims to provide a preparation method of carboxymethylated lotus root polysaccharide-trichostatin A conjugate, aiming at synthesizing a polysaccharide drug with an anti-tumor effect.
To achieve the above object, the present invention provides a preparation method of carboxymethylated lotus root polysaccharide-trichostatin a conjugate, which comprises the following steps:
grafting polyethyleneimine-polyethylene glycol copolymer on carboxymethylated lotus root polysaccharide to obtain carboxymethylated lotus root polysaccharide complex;
grafting folic acid on the carboxymethylated lotus root polysaccharide complex to obtain a carboxymethylated lotus root polysaccharide-folic acid complex;
and grafting the carboxymethylated lotus root polysaccharide-folic acid compound to the trichostatin A to obtain the carboxymethylated lotus root polysaccharide-trichostatin A conjugate.
Optionally, the step of grafting the carboxymethylated lotus root polysaccharide with polyethyleneimine-polyethylene glycol copolymer to obtain the carboxymethylated lotus root polysaccharide complex comprises:
uniformly dispersing carboxymethylated lotus root polysaccharide in a phosphate buffer solution, then sequentially adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a polyethyleneimine-polyethylene glycol copolymer, uniformly mixing, and reacting in the dark to obtain a reaction solution;
and transferring the reaction solution to a dialysis bag, carrying out running water dialysis treatment, centrifuging the retention solution, discarding the supernatant, and drying the precipitate to obtain the carboxymethylated lotus root polysaccharide complex.
Optionally, the carboxymethylated lotus root polysaccharide is uniformly dispersed in a phosphate buffer solution, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the polyethyleneimine-polyethylene glycol copolymer are respectively added and uniformly mixed, and the reaction solution is obtained by reacting in the dark: the weight ratio of carboxymethylated lotus root polysaccharide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and polyethyleneimine-polyethylene glycol copolymer is (40-60): (6-10): (40-60).
Optionally, the carboxymethylated lotus root polysaccharide is uniformly dispersed in a phosphate buffer solution, then 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the polyethyleneimine-polyethylene glycol copolymer are respectively added and uniformly mixed, and the reaction solution is obtained by reacting in the dark: the reaction time is 24-48 h; and/or the presence of a gas in the gas,
transferring the reaction solution to a dialysis bag, and performing running water dialysis treatment, wherein the method comprises the following steps: the time of running water dialysis treatment is 24-48 h.
Optionally, the step of grafting folic acid to the carboxymethylated lotus root polysaccharide complex to obtain the carboxymethylated lotus root polysaccharide-folic acid complex comprises:
dissolving folic acid in a mixed solution of triethylamine and dimethyl sulfoxide to obtain a first mixed solution;
dissolving N, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide in dimethyl sulfoxide to obtain a second mixed solution;
adding the second mixed solution into the first mixed solution, and reacting in a dark place to obtain folic acid active ester;
and (3) dissolving the carboxymethylated lotus root polysaccharide complex and the folic acid active ester in dimethyl sulfoxide, and carrying out a dark reaction to obtain the carboxymethylated lotus root polysaccharide-folic acid complex.
Optionally, the weight ratio of folic acid, N-dicyclohexylcarbodiimide, N-hydroxysuccinimide is (30-50): (15-25): (8-12).
Optionally, the carboxymethylated lotus root polysaccharide complex and the folic acid active ester are dissolved in dimethyl sulfoxide and react away from light to obtain the carboxymethylated lotus root polysaccharide-folic acid complex, wherein the step of dissolving the carboxymethylated lotus root polysaccharide complex and the folic acid active ester in dimethyl sulfoxide comprises the following steps: the weight ratio of the carboxymethylated lotus root polysaccharide complex to the folic acid active ester is (18-22) to (6-9).
Optionally, adding the second mixed solution into the first mixed solution, and performing a reaction in the absence of light to obtain the active folic ester: the reaction time is 8-16 h; and/or the presence of a gas in the gas,
dissolving carboxymethylated lotus root polysaccharide complex and folic acid active ester in dimethyl sulfoxide, and reacting in a dark place to obtain carboxymethylated lotus root polysaccharide-folic acid complex: the reaction time is 8-16 h.
Optionally, the step of grafting the carboxymethylated lotus root polysaccharide-folic acid complex to trichostatin a to obtain the carboxymethylated lotus root polysaccharide-trichostatin a conjugate comprises:
dissolving carboxymethylated lotus root polysaccharide-folic acid compound in dimethyl sulfoxide to obtain a solution to be reacted;
and adding the dimethyl sulfoxide solution of the trichostatin A into the solution to be reacted, and reacting to obtain carboxymethylated lotus root polysaccharide-trichostatin A conjugate.
Optionally, in the step of adding a dimethyl sulfoxide solution of the trichostatin A into the solution to be reacted to obtain carboxymethylated lotus root polysaccharide-trichostatin A conjugate: the reaction time is 8-16 h.
According to the technical scheme provided by the invention, carboxymethylated lotus root polysaccharide is preferably selected, the carboxymethylated lotus root polysaccharide has better water solubility and multiple effects, and then polyethyleneimine-polyethylene glycol copolymer is taken as a framework to respectively graft carboxymethylated lotus root polysaccharide, folic acid and trichostatin A, so that the final product lotus root polysaccharide-trichostatin A conjugate has strong biological activity, and performance research shows that the conjugate has obvious effect on anti-tumor cells, and can be widely applied to production of anti-tumor drugs.
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 method for preparing carboxymethylated Nelumbo nucifera-Trigoncomycin A conjugates of the present invention;
FIG. 2 is an ultraviolet spectrum of carboxymethylated lotus root polysaccharides and carboxymethylated lotus root polysaccharide complexes in example 1 of the present invention;
FIG. 3 is the ultraviolet spectra of carboxymethylated lotus root polysaccharide complex and carboxymethylated lotus root polysaccharide-folic acid complex in example 1 of the present invention;
FIG. 4 is an ultraviolet spectrum of carboxymethylated Nelumbo nucifera-folate complex and carboxymethylated Nelumbo nucifera-trichostatin A conjugate in example 1 of the present invention;
FIG. 5 is an infrared spectrum of carboxymethylated lotus root polysaccharide and carboxymethylated lotus root polysaccharide complex in example 1 of the present invention;
FIG. 6 is a comparison of IR spectra of carboxymethylated Nelumbo nucifera-folate complex and carboxymethylated Nelumbo nucifera-Trigoncomycin A conjugate of example 1 according to the present invention;
FIG. 7 is a scanning electron microscope observation of carboxymethylated Nelumbo nucifera-folate complex and carboxymethylated Nelumbo nucifera-Trigoncomycin A conjugate in example 1 of the present invention;
FIG. 8 is a graph showing the cell activity of ovarian cancer cells A2780 treated with different concentrations of carboxymethylated Nelumbo Nucifera polysaccharide-Folic acid complex and carboxymethylated Nelumbo Nucifera polysaccharide-Trigoncomycin A conjugate in example 1 of the present invention.
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.
Modern science shows that polysaccharide drugs not only have the function of promoting and regulating the immunity of organisms, but also have the functions of resisting tumors, free radical, oxidation, aging, viruses, blood fat and blood sugar, radiation and the like, have the characteristics of low toxicity and few adverse reactions, attract the wide attention of scientists, and become a hotspot of the current life science research. The lotus root polysaccharide has various biological activities, and the lotus root polysaccharide-drug conjugate is constructed, so that the hydrophilicity of the drug can be improved, the bioavailability of the drug can be enhanced, and more possibilities are provided for the polysaccharide-drug conjugate. The invention provides a preparation method of carboxymethylated lotus root polysaccharide-trichostatin A conjugate, which combines polysaccharide and drug by covalent bond, and constructs polysaccharide-drug conjugate. Referring to the schematic flow diagram of one example of a method for preparing carboxymethylated lotus rhizome polysaccharide-trichostatin a conjugates presented in fig. 1, the method for preparing carboxymethylated lotus rhizome polysaccharide-trichostatin a conjugates comprises the following steps:
step S10, grafting the carboxymethylated lotus root polysaccharide with polyethyleneimine-polyethylene glycol copolymer to obtain the carboxymethylated lotus root polysaccharide compound.
Grafting refers to "graft copolymerization", i.e., a reaction in which a polyethyleneimine-polyethylene glycol copolymer is used as a backbone and appropriate functional side groups are bonded to the side chains via chemical bonds. In specific implementation, the method can be implemented according to the following steps:
step S11, weighing carboxymethylated lotus root polysaccharide, placing the carboxymethylated lotus root polysaccharide in a flask, adding phosphate buffer (pH is 6.0), stirring to uniformly disperse the carboxymethylated lotus root polysaccharide in the buffer, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction system to activate the carboxymethylated lotus root polysaccharide, adding polyethyleneimine-polyethylene glycol copolymer, uniformly mixing, and stirring to react under a dark condition to obtain a reaction solution;
and step S12, transferring the reaction solution into a dialysis bag, performing running water dialysis treatment, taking the retention solution, centrifuging, removing the supernatant, and performing freeze drying treatment to obtain the carboxymethylated lotus root polysaccharide complex.
Wherein the weight ratio of carboxymethylated lotus root polysaccharide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and polyethyleneimine-polyethylene glycol copolymer is (40-60): (6-10): (40-60); the reaction time of the light-shielding reaction in the step S11 is 24-48 h; the time of the running water dialysis treatment in the step S12 is 24-48 h.
Step S20, grafting folic acid to the carboxymethylated lotus root polysaccharide complex to obtain the carboxymethylated lotus root polysaccharide-folic acid complex.
In this example, folic acid was activated first, and then reacted with carboxymethylated lotus root polysaccharide complex to graft folic acid onto carboxymethylated lotus root polysaccharide complex, with the entire reaction process protected from light. In specific implementation, step S20 includes the following steps:
s21, placing folic acid in a flask, then uniformly mixing triethylamine and dimethyl sulfoxide to form a mixed solution, adding the mixed solution into the flask, and performing ultrasonic treatment to completely dissolve folic acid to obtain a first mixed solution;
and step S22, dissolving the N, N-dicyclohexylcarbodiimide and the N-hydroxysuccinimide in dimethyl sulfoxide to prepare a second mixed solution.
Wherein the weight ratio of folic acid, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide is (30-50): (15-25): (8-12).
And step S23, adding the second mixed solution into the first mixed solution under stirring, and reacting in a dark place to obtain the folic acid active ester.
In this example, the reaction time was 8 to 16 hours with exclusion of light.
In specific implementation, step S23 may further include:
adding the second mixed solution into the first mixed solution under stirring, and reacting in a dark place to obtain a reaction mixed solution; filtering to remove a reaction byproduct dicyclohexylurea, adding ultrapure water into the obtained filtrate under stirring to separate out a yellow precipitate, standing overnight, filtering, and freeze-drying to obtain a crude product of the folic acid active ester; grinding the crude product of the folic acid active ester into powder, repeatedly washing with hot water for many times, soaking with acetone and performing ultrasonic treatment to remove unreacted folic acid and dicyclohexylurea serving as a reaction byproduct, and filtering and freeze-drying to obtain the folic acid active ester.
And step S24, dissolving the carboxymethylated lotus root polysaccharide complex and the folic acid active ester in dimethyl sulfoxide, and carrying out a dark reaction to obtain the carboxymethylated lotus root polysaccharide-folic acid complex.
Wherein, carboxymethylation lotus root polysaccharide complex and folic acid active ester are weighed according to a certain weight ratio and dissolved in dimethyl sulfoxide for reaction, and the weight ratio of the carboxymethylation lotus root polysaccharide complex to the folic acid active ester is (18-22) to (6-9); the reaction time is 8-16h without light.
In specific implementation, in step S24, after obtaining the carboxymethylated lotus root polysaccharide-folic acid complex mixed solution by the dark reaction, the following steps may be further performed to obtain a highly pure carboxymethylated lotus root polysaccharide-folic acid complex: adjusting the mixed solution of carboxymethylated lotus root polysaccharide-folic acid complex to alkalinity by using sodium carbonate solution, centrifuging to remove supernatant, washing precipitate with distilled water to remove residual dimethyl sulfoxide, and freeze-drying to obtain the carboxymethylated lotus root polysaccharide-folic acid complex.
Step S30, grafting the carboxymethylated lotus root polysaccharide-folic acid compound to the trichostatin A to obtain the carboxymethylated lotus root polysaccharide-trichostatin A conjugate.
When step S30 is implemented, it can be implemented by: dissolving the carboxymethylated lotus root polysaccharide-folic acid compound obtained in the step S20 in dimethyl sulfoxide to obtain a solution to be reacted; and adding the dimethyl sulfoxide solution of the trichostatin A into the solution to be reacted, and reacting to obtain carboxymethylated lotus root polysaccharide-trichostatin A conjugate. Wherein, the reaction is carried out under the uniform stirring of a magnetic stirrer, and the reaction time is 8-16 h.
In addition, the carboxymethylated lotus root polysaccharide used in the steps is prepared by self, and the polyethyleneimine-polyethylene glycol copolymer can be purchased from the market or prepared by self. Specifically, the preparation method of the two is as follows:
the preparation method of carboxymethylated lotus root polysaccharide comprises the following steps: placing the un-lyophilized rhizoma Nelumbinis polysaccharide in a flask, adding sodium hydroxide solution to dissolve rhizoma Nelumbinis polysaccharide completely, and refluxing. Chloroacetic acid was then added to the reaction and continued under reflux by condensation. The solution obtained above was adjusted to neutral with hydrochloric acid, and then dialyzed with running water. Concentrating the reaction solution after dialysis treatment, uniformly mixing the concentrated solution with ethanol, storing at low temperature overnight, centrifuging to remove ethanol, and freeze-drying to obtain carboxymethylated lotus root polysaccharide powder.
Similarly, the lotus root polysaccharide is also obtained by self-preparation. When prepared by itself, the preparation method can be as follows:
weighing lotus root powder, placing the lotus root powder in a flask, adding ethanol, uniformly mixing, placing in a water bath kettle, and performing condensation reflux to remove impurities such as lipid in lotus root polysaccharide. Centrifuging the solution obtained by condensation reflux, removing ethanol, adding water, performing condensation reflux in water bath to extract polysaccharide, and obtaining lotus root polysaccharide leaching liquor. Centrifuging the obtained polysaccharide leaching solution by using a centrifuge, and then filtering to remove lotus root powder residues. And (3) concentrating the centrifuged polysaccharide leaching liquor by using a rotary evaporator at a certain rotating speed to obtain viscous lotus root polysaccharide leaching liquor. And (3) placing the concentrated polysaccharide leaching liquor and an equivalent volume of Savage solution into a separating funnel, uniformly mixing, standing for layering, and keeping a supernatant. Transferring the obtained supernatant into a conical flask, adding ethanol, mixing uniformly, storing at 4 ℃ overnight, centrifuging and removing ethanol to obtain the crude lotus root polysaccharide. In order to improve the purity of the obtained lotus root polysaccharide, the lotus root polysaccharide after alcohol precipitation is completely dissolved by taking a proper amount of ultrapure water, is placed in a water bath kettle for condensation and reflux, is mixed with equivalent volume of Savage solution again after concentration, is kept stand for layering, and the supernatant is reserved. And putting the supernatant into a conical flask, adding ethanol, precipitating with ethanol at 4 ℃ overnight, centrifuging, and removing the ethanol to obtain the lotus root polysaccharide. And carrying out alcohol precipitation on the obtained lotus root polysaccharide again to obtain the lotus root polysaccharide with higher purity.
The preparation method of the polyethyleneimine-polyethylene glycol copolymer comprises the following steps: dissolving polyethylene glycol 2000 in dry chloroform, adding succinic anhydride, and heating to react to obtain monomethyl ether polyethylene glycol succinate. And (3) reacting the monomethyl ether polyethylene glycol succinate with N-hydroxysuccinimide and N, N-dicyclohexylcarbodiimide to obtain an intermediate product polyethylene glycol succinimide ester. Dissolving polyethyleneimine (with relative molecular weight of 2000) in phosphate buffer solution, adding polyethylene glycol succinimide ester, performing running water dialysis treatment on the reaction solution by using a dialysis bag (with molecular weight cutoff of 3500) after reaction, centrifuging the retention solution, and freeze-drying to obtain a white powdery product, namely a polyethyleneimine-polyethylene glycol copolymer material.
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
Weighing 0.5g of carboxymethylated lotus root polysaccharide, placing the carboxymethylated lotus root polysaccharide in a 50mL flask, measuring 25mL of phosphate buffer solution (pH is 6.0) by using a 25mL pipette, stirring to uniformly disperse the carboxymethylated lotus root polysaccharide in the buffer solution, precisely weighing 80mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride by using an electronic balance, adding the 80mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction system to activate the carboxymethylated lotus root polysaccharide, adding 0.5g of polyethyleneimine-polyethylene glycol copolymer, uniformly mixing, keeping the reaction process away from light, placing the mixture in a magnetic stirrer at room temperature for uniform stirring reaction for 36 hours, transferring the reaction liquid to a dialysis bag (the molecular weight cutoff is 8000-14000), carrying out distilled water dialysis treatment on the reaction liquid for 36 hours, removing the supernatant by centrifugation, and carrying out freeze drying treatment to obtain the carboxymethylated lotus root polysaccharide compound.
Weighing 1.0g of folic acid, placing the folic acid into a 50mL flask, uniformly mixing 0.5mL of triethylamine and 20mL of dimethyl sulfoxide, adding the mixture into the flask, performing ultrasonic treatment to completely dissolve the folic acid, respectively weighing 0.52g N, N-dicyclohexylcarbodiimide and 0.26g of N-hydroxysuccinimide, dissolving the two in 10mL of dimethyl sulfoxide, adding the mixture into the flask while stirring to activate the folic acid, keeping out of the sun, placing the mixture in a magnetic stirrer, and performing uniform stirring reaction for 12 hours. The reaction by-product dicyclohexylurea was removed by suction filtration, 2000mL of ultrapure water was added to the obtained filtrate with stirring to precipitate a yellow precipitate, which was filtered off with suction after standing overnight, and then lyophilized.
Grinding the freeze-dried crude product of the folic acid active ester into powder, repeatedly washing with hot water for 2-3 times, soaking in appropriate amount of acetone and ultrasonically treating for 10min, removing unreacted folic acid and reaction by-product dicyclohexylurea, filtering, and freeze-drying to obtain light yellow powder.
Accurately weighing 0.2g carboxymethylated lotus root polysaccharide complex, placing in 50mL round bottom flask, adding 25mL anhydrous dimethyl sulfoxide to uniformly disperse polysaccharide, and slowly adding 2.5mL folic acid under the action of magnetic stirrerThe mixture was washed with light and dimethyl sulfoxide (29mg/mL) to react for 12 hours. After the reaction is finished, 0.1mol/L Na is used2Adjusting pH of CO3 solution to 9.0, centrifuging to remove supernatant, washing precipitate with distilled water to remove residual dimethyl sulfoxide, and freeze drying to obtain carboxymethylated lotus root polysaccharide-folic acid complex.
Accurately weighing a proper amount of carboxymethylated lotus root polysaccharide-folic acid obtained by freeze drying, placing the carboxymethylated lotus root polysaccharide-folic acid into a 50mL flask, adding 20-30 mL of dimethyl sulfoxide for dissolving to enable the dimethyl sulfoxide to be uniformly distributed, simultaneously using the dimethyl sulfoxide as a solvent, preparing 1mg/mL of solution from trichostatin A, diluting 500 mu L of trichostatin A solution to 5mL, adding the diluted trichostatin A solution into the flask, stirring for 12 hours at a constant speed under the action of a magnetic stirrer, and freeze drying to obtain the carboxymethylated lotus root polysaccharide-trichostatin A conjugate. Characterization analysis of grafted carboxymethylated lotus root polysaccharide
(1) Ultraviolet spectral analysis
The carboxymethylated lotus root polysaccharide used in example 1 and the prepared carboxymethylated lotus root polysaccharide complex were scanned by an ultraviolet spectrophotometer with a wavelength scan range of 200-600 nm, and the results are shown in fig. 2. As can be seen from the figure, the lotus root polysaccharide and carboxymethylated lotus root polysaccharide have weaker absorption peaks at 280nm, the absorbance is obviously increased after the polyethyleneimine-polyethylene glycol is grafted, and a certain degree of red shift occurs. The results of uv scanning of carboxymethylated lotus root polysaccharide and carboxymethylated lotus root polysaccharide complex were similar, so it was believed that the molecular structure of the polysaccharide was not destroyed after grafting polyethyleneimine-polyethylene glycol. However, the absorbance of the polysaccharide complex at 300-600 nm is obviously higher than that of carboxymethylated lotus root polysaccharide, which indicates that the particle size of the material is large, but the dispersibility is good, and the agglomeration phenomenon does not occur.
Scanning carboxymethylated lotus root polysaccharide-folic acid compound by using an ultraviolet spectrophotometer within the wavelength range of 200-600 nm, wherein the scanning result is shown in figure 3. In the wavelength range, a strong absorption peak of carboxymethylated lotus root polysaccharide-folic acid compound at 290nm can be observed, and a maximum absorption peak also exists at 350nm, which corresponds to a characteristic absorption peak of folic acid, so that the carboxymethylated lotus root polysaccharide compound is judged to be successfully grafted with folic acid, and the main structure in the carboxymethylated lotus root polysaccharide compound is not damaged according to the analysis of a map, which shows that the synthesis process has feasibility.
The carboxymethylated lotus root polysaccharide-folic acid complex and the carboxymethylated lotus root polysaccharide-trichostatin A conjugate are scanned within the wavelength of 200-600 nm, and the scanning result is shown in figure 4. The two compounds have absorption peaks at 290nm, and the absorption spectra have no obvious difference, which can indicate that the structures of the compounds are not obviously changed after the compounds are grafted with the trichostatin A. In addition, as can be seen from FIG. 3, this absorption peak is a characteristic absorption peak of folic acid.
(2) Infrared spectroscopic analysis
Using Fourier transform infrared spectrometer at 4000cm-1-500cm-1And performing infrared spectrum characterization on the carboxymethylated lotus root polysaccharide compound in the range, and comparing the infrared spectrum characterization with the carboxymethylated lotus root polysaccharide infrared spectrum for analysis. The infrared scan results are shown in fig. 5. 3400cm-1The absorption peak is formed by N-H and O-H stretching vibration, the characteristic peak is narrowed from a broad peak, and the weakening indicates that the carbonyl group and the amino group of the carboxymethylated lotus root polysaccharide are subjected to derivatization reaction. 1600cm-1The absorption peak near the position is the characteristic absorption peak of C ═ O of carboxymethylated lotus root polysaccharide, and the absorption peak of C ═ O is enhanced in the complex carboxymethylated lotus root polysaccharide complex. 2930cm-1Near the absorption peak is-CH2Is formed by stretching vibration of 1140cm-1The absorption peak near the peak is formed by C-O stretching vibration, and it can be seen from FIG. 5 that the carboxymethylated lotus root polysaccharide complex is 2930cm-1And 1140cm-1The absorption peak intensity of the compound is obviously stronger than that of carboxymethylated lotus root polysaccharide, so that the carboxymethylated lotus root polysaccharide and the polyethyleneimine-polyethylene glycol copolymer material are considered to have a grafting reaction.
Scanning carboxymethylated Nelumbo Nucifera polysaccharide-Folic acid complex and carboxymethylated Nelumbo Nucifera polysaccharide-Trigonamycin A conjugate with wavelength of 4000cm-1-500cm-1As can be seen from FIG. 6, after the grafting of folic acid, the intensity of the infrared absorption peak was reduced compared to that before the grafting, but the infrared spectrum and carboxymethylated lotus root polysaccharide complex were observedSimilarly, the characteristic peak of folic acid is not obvious probably because the carboxymethylated lotus root polysaccharide complex is a high molecular polymer and the molecular weight of folic acid is relatively small, and the infrared absorption peak intensity is enhanced after the grafting of the trichostatin A, but no new characteristic peak appears, which indicates that the functional groups of the complex are not damaged after the carboxymethylated lotus root polysaccharide complex is grafted with the folic acid and the trichostatin A in sequence.
(3) SEM detection
Comparison of results of scanning electron microscopy observations of carboxymethylated lotus root polysaccharide-folate complex and carboxymethylated lotus root polysaccharide-trichostatin a conjugate, as shown in fig. 7, a 1-A3 are magnified scan images of carboxymethylated lotus root polysaccharide-folate complex at 500 times (100 μm), 1000 times (50 μm) and 2500 times (20 μm), and B1-B3 are magnified scan images of carboxymethylated lotus root polysaccharide-trichostatin a conjugate at 500 times (100 μm), 1000 times (50 μm) and 2500 times (20 μm). According to the SEM detection result, folic acid and the trichostatin A are agglomerated with carboxymethylated lotus root polysaccharide in a particle form due to the action of hydrogen bonds and are uniformly distributed on the surface of the carboxymethylated lotus root polysaccharide particles.
Research on antitumor activity of (di) carboxymethylated lotus rhizome polysaccharide-trichostatin A conjugate
The ovarian cancer cell A2780 is used for constructing a tumor cell model, the antitumor activity of carboxymethylated lotus root polysaccharide-trichostatin A conjugate is used as an evaluation index, a blank control group and a carboxymethylated lotus root polysaccharide-folic acid compound control group are arranged for comparison, and the activity of the tumor cells after sample treatment is detected by a CCK-8 method. The test method and results are as follows:
(1) the experimental method comprises the following steps:
step 1 cell recovery
Taking out the freezing tube, immediately placing in a 37 deg.C water bath, water bathing for 1min, blowing the liquid in the freezing tube with a pipette gun, mixing well, transferring to a 1.5mL centrifuge tube, centrifuging at 1000rpm for 3min, discarding the supernatant, adding 1mL complete culture solution, resuspending the cells, transferring to a culture dish, adding 3mL complete culture solution, mixing the cells in the culture dish, transferring the cells to 37 deg.C, and CO2Culturing in a 5% incubator;
When the confluence degree of the cells reaches 90%, carrying out passage and expanded culture on the cells, sucking out the culture solution on the upper layer of the cells by using a pipette gun, rinsing the surfaces of the cells by using phosphate buffer solution, removing the phosphate buffer solution, carefully blowing down the cells along the bottom of a culture dish by using the pipette gun, adding the complete culture solution to 4mL, blowing and uniformly mixing, transferring the cells to 37 ℃, and carrying out CO (carbon monoxide) separation2Culturing in 5% incubator.
Step 3 cell cryopreservation
After the confluence degree of the cells reaches 80%, discarding the culture solution, rinsing the cells with phosphate buffer solution, discarding phosphate buffer solution, absorbing the culture solution on the upper layer of the cells by using a pipette gun, rinsing the surfaces of the cells with phosphate buffer solution, discarding phosphate buffer solution, carefully blowing down the cells along the bottom of a culture dish by using the pipette gun according to the basic culture solution: serum: dimethyl sulfoxide is 7: 2: 1 preparing a freezing medium, discarding supernatant after centrifugation, re-suspending cells in the freezing medium, adding 1mL of the freezing medium into each freezing tube, marking on the freezing tube, performing gradient freezing, respectively standing at 4 ℃ and-20 ℃ for 2h, storing at-80 ℃ overnight, and then transferring to liquid nitrogen for storage.
Step 4 cell Activity assay
Digesting the cells reaching confluency, adding 100 μ L of complete culture solution into each well, culturing in an incubator for 24h, taking out the 96-well plate from the incubator, discarding the culture solution, washing twice with the basic culture solution, using cell culture solution of the treated group containing carboxymethylated lotus root polysaccharide-folic acid complex and carboxymethylated lotus root polysaccharide-trichostatin A conjugate (with concentrations of 0, 100 μ g/mL, 200 μ g/mL, 300 μ g/mL, 400 μ g/mL, 500 μ g/mL, respectively), and (3) incubating in an incubator for 24h, taking out a 96-well plate from the incubator, discarding the culture solution, washing the cells twice with the basic culture solution, adding 10 mu L of CCK-8 solution and 100 mu L of the basic culture solution into each well, incubating in the incubator for 1-4 h, and measuring the absorbance at the wavelength of 450nm by using an enzyme-labeling instrument.
(2) Results
Ovarian cancer cells A2780 are used for evaluating the antitumor activity of the carboxymethylated lotus root polysaccharide-trichostatin A conjugate, a blank control group and a carboxymethylated lotus root polysaccharide-folic acid complex control group are used for comparison, and then the CCK-8 method is used for detecting the cell activity, wherein the experimental result is shown in figure 8. The sample concentration reaches more than 200 mug/mL, the carboxymethylated lotus rhizome polysaccharide-trichostatin A conjugate treatment group has the effect of inhibiting the survival of tumor cells, and the effect of inhibiting the survival of the tumor cells is optimal when the sample concentration is 400 mug/mL. In the concentration range of 100-500 mug/mL, the carboxymethylated lotus root polysaccharide-folic acid compound does not show the effect of inhibiting the activity of tumor cells, so that the carboxymethylated lotus root polysaccharide-folic acid compound and the trichostatin A can be judged to be successfully synthesized. The experimental data are analyzed, and the antitumor activity of the sample and the concentration of the sample have no obvious dependence in the concentration range of 100-500 mu g/mL.
Example 2
Weighing 0.4g of carboxymethylated lotus root polysaccharide, placing the carboxymethylated lotus root polysaccharide in a 50mL flask, measuring 25mL of phosphate buffer solution (pH is 6.0) by using a 25mL pipette, stirring to uniformly disperse the carboxymethylated lotus root polysaccharide in the buffer solution, precisely weighing 60mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride by using an electronic balance, adding the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction system to activate the carboxymethylated lotus root polysaccharide, adding 0.4g of polyethyleneimine-polyethylene glycol copolymer, uniformly mixing, keeping the reaction process away from light, placing the mixture in a magnetic stirrer at room temperature for uniform stirring reaction for 24 hours, transferring the reaction solution to a dialysis bag (the molecular weight cutoff is 8000-14000), carrying out distilled water dialysis treatment on the reaction solution for 24 hours, removing the supernatant by centrifugation, and carrying out freeze drying treatment to obtain the carboxymethylated lotus root polysaccharide compound.
Weighing 0.75g of folic acid, placing the folic acid into a 50mL flask, uniformly mixing 0.5mL of triethylamine and 20mL of dimethyl sulfoxide, adding the mixture into the flask, performing ultrasonic treatment to completely dissolve the folic acid, respectively weighing 0.375g N, dissolving N-dicyclohexylcarbodiimide and 0.2g of N-hydroxysuccinimide in 10mL of dimethyl sulfoxide, adding the mixture into the flask while stirring to activate the folic acid, keeping out of the sun, placing the mixture in a magnetic stirrer, and performing uniform stirring reaction for 8 hours. The reaction by-product dicyclohexylurea was removed by suction filtration, 2000mL of ultrapure water was added to the obtained filtrate with stirring to precipitate a yellow precipitate, which was filtered off with suction after standing overnight, and then lyophilized.
Grinding the freeze-dried crude product of the folic acid active ester into powder, repeatedly washing with hot water for 2-3 times, soaking in appropriate amount of acetone and ultrasonically treating for 10min, removing unreacted folic acid and reaction by-product dicyclohexylurea, filtering, and freeze-drying to obtain light yellow powder.
Accurately weighing 0.18g of carboxymethylated lotus root polysaccharide complex, placing in a 50mL round-bottom flask, adding 25mL of anhydrous dimethyl sulfoxide to uniformly disperse the polysaccharide, slowly adding 2mL of dimethyl sulfoxide solution (29mg/mL) of folic acid active ester under the action of a magnetic stirrer, and reacting for 8h in a dark place. After the reaction is finished, 0.1mol/L Na is used2Adjusting pH of CO3 solution to 9.0, centrifuging to remove supernatant, washing precipitate with distilled water to remove residual dimethyl sulfoxide, and freeze drying to obtain carboxymethylated lotus root polysaccharide-folic acid complex.
Accurately weighing a proper amount of carboxymethylated lotus root polysaccharide-folic acid obtained by freeze drying, placing the carboxymethylated lotus root polysaccharide-folic acid into a 50mL flask, adding 20-30 mL of dimethyl sulfoxide for dissolving to enable the dimethyl sulfoxide to be uniformly distributed, simultaneously using the dimethyl sulfoxide as a solvent, preparing 1mg/mL of solution from trichostatin A, diluting 500 mu L of trichostatin A solution to 5mL, adding the diluted trichostatin A solution into the flask, stirring for 8 hours at a constant speed under the action of a magnetic stirrer, and freeze drying to obtain the carboxymethylated lotus root polysaccharide-trichostatin A conjugate.
Example 3
Weighing 0.6g of carboxymethylated lotus root polysaccharide, placing the carboxymethylated lotus root polysaccharide in a 50mL flask, measuring 25mL of phosphate buffer solution (pH is 6.0) by using a 25mL pipette, stirring to uniformly disperse the carboxymethylated lotus root polysaccharide in the buffer solution, precisely weighing 100mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride by using an electronic balance, adding the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction system to activate the carboxymethylated lotus root polysaccharide, adding 0.6g of polyethyleneimine-polyethylene glycol copolymer, uniformly mixing, keeping the reaction process away from light, placing the mixture in a magnetic stirrer at room temperature for uniform stirring reaction for 48 hours, transferring the reaction solution to a dialysis bag (the molecular weight cutoff is 8000-14000), carrying out distilled water dialysis treatment on the reaction solution for 48 hours, removing the supernatant by centrifugation, and carrying out freeze drying treatment to obtain the carboxymethylated lotus root polysaccharide compound.
Weighing 1.25g of folic acid, placing the folic acid into a 50mL flask, uniformly mixing 0.5mL of triethylamine and 20mL of dimethyl sulfoxide, adding the mixture into the flask, performing ultrasonic treatment to completely dissolve the folic acid, respectively weighing 0.625g N, N-dicyclohexylcarbodiimide and 0.3g of N-hydroxysuccinimide, dissolving the two in 10mL of dimethyl sulfoxide, adding the mixture into the flask while stirring to activate the folic acid, keeping out of the sun, placing the mixture in a magnetic stirrer, and performing uniform stirring reaction for 16 hours. The reaction by-product dicyclohexylurea was removed by suction filtration, 2000mL of ultrapure water was added to the obtained filtrate with stirring to precipitate a yellow precipitate, which was filtered off with suction after standing overnight, and then lyophilized.
Grinding the freeze-dried crude product of the folic acid active ester into powder, repeatedly washing with hot water for 2-3 times, soaking in appropriate amount of acetone and ultrasonically treating for 10min, removing unreacted folic acid and reaction by-product dicyclohexylurea, filtering, and freeze-drying to obtain light yellow powder.
Accurately weighing 0.22g of carboxymethylated lotus root polysaccharide complex, placing in a 50mL round-bottom flask, adding 25mL of anhydrous dimethyl sulfoxide to uniformly disperse the polysaccharide, slowly adding 3mL of dimethyl sulfoxide solution (29mg/mL) of folic acid active ester under the action of a magnetic stirrer, and reacting for 16h in a dark place. After the reaction is finished, 0.1mol/L Na is used2Adjusting pH of CO3 solution to 9.0, centrifuging to remove supernatant, washing precipitate with distilled water to remove residual dimethyl sulfoxide, and freeze drying to obtain carboxymethylated lotus root polysaccharide-folic acid complex.
Accurately weighing a proper amount of carboxymethylated lotus root polysaccharide-folic acid obtained by freeze drying, placing the carboxymethylated lotus root polysaccharide-folic acid into a 50mL flask, adding 20-30 mL of dimethyl sulfoxide for dissolving to enable the dimethyl sulfoxide to be uniformly distributed, simultaneously using the dimethyl sulfoxide as a solvent, preparing 1mg/mL of solution from trichostatin A, diluting 500 mu L of trichostatin A solution to 5mL, adding the diluted trichostatin A solution into the flask, stirring for 16h at a constant speed under the action of a magnetic stirrer, and freeze drying to obtain the carboxymethylated lotus root polysaccharide-trichostatin A conjugate.
Example 4
Weighing 0.4g of carboxymethylated lotus root polysaccharide, placing the carboxymethylated lotus root polysaccharide in a 50mL flask, measuring 25mL of phosphate buffer solution (pH is 6.0) by using a 25mL pipette, stirring to uniformly disperse the carboxymethylated lotus root polysaccharide in the buffer solution, precisely weighing 80mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride by using an electronic balance, adding the 80mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction system to activate the carboxymethylated lotus root polysaccharide, adding 0.6g of polyethyleneimine-polyethylene glycol copolymer, uniformly mixing, keeping the reaction process away from light, placing the reaction system in a magnetic stirrer at room temperature for uniform stirring reaction for 36 hours, transferring the reaction liquid to a dialysis bag (with the molecular weight cutoff of 8000-14000), carrying out distilled water dialysis treatment on the reaction liquid for 36 hours, removing the supernatant by centrifugation, and carrying out freeze drying treatment to obtain the carboxymethylated lotus root polysaccharide compound.
Weighing 0.75g of folic acid, placing the folic acid into a 50mL flask, uniformly mixing 0.5mL of triethylamine and 20mL of dimethyl sulfoxide, adding the mixture into the flask, performing ultrasonic treatment to completely dissolve the folic acid, respectively weighing 0.5g N, dissolving N-dicyclohexylcarbodiimide and 0.3g of N-hydroxysuccinimide in 10mL of dimethyl sulfoxide, adding the mixture into the flask while stirring to activate the folic acid, keeping out of the sun, placing the mixture in a magnetic stirrer, and performing uniform stirring reaction for 12 hours. The reaction by-product dicyclohexylurea was removed by suction filtration, 2000mL of ultrapure water was added to the obtained filtrate with stirring to precipitate a yellow precipitate, which was filtered off with suction after standing overnight, and then lyophilized.
Grinding the freeze-dried crude product of the folic acid active ester into powder, repeatedly washing with hot water for 2-3 times, soaking in appropriate amount of acetone and ultrasonically treating for 10min, removing unreacted folic acid and reaction by-product dicyclohexylurea, filtering, and freeze-drying to obtain light yellow powder.
Accurately weighing 0.2g carboxymethylated lotus root polysaccharide complex, placing in a 50mL round-bottom flask, adding 25mL anhydrous dimethyl sulfoxide to uniformly disperse the polysaccharide, slowly adding 2.5mL dimethyl sulfoxide solution (29mg/mL) of folic acid active ester under the action of a magnetic stirrer, and reacting for 12h in a dark place. After the reaction is finished, 0.1mol/L Na is used2Adjusting pH of CO3 solution to 9.0, centrifuging to remove supernatant, washing precipitate with distilled water to remove residual dimethyl sulfoxide, and freeze drying to obtain carboxymethylated lotus root polysaccharide-folic acid complex.
Accurately weighing a proper amount of carboxymethylated lotus root polysaccharide-folic acid obtained by freeze drying, placing the carboxymethylated lotus root polysaccharide-folic acid into a 50mL flask, adding 20-30 mL of dimethyl sulfoxide for dissolving to enable the dimethyl sulfoxide to be uniformly distributed, simultaneously using the dimethyl sulfoxide as a solvent, preparing 1mg/mL of solution from trichostatin A, diluting 500 mu L of trichostatin A solution to 5mL, adding the diluted trichostatin A solution into the flask, stirring for 12 hours at a constant speed under the action of a magnetic stirrer, and freeze drying to obtain the carboxymethylated lotus root polysaccharide-trichostatin A conjugate.
Example 5
Weighing 0.6g of carboxymethylated lotus root polysaccharide, placing the carboxymethylated lotus root polysaccharide in a 50mL flask, measuring 25mL of phosphate buffer solution (pH is 6.0) by using a 25mL pipette, stirring to uniformly disperse the carboxymethylated lotus root polysaccharide in the buffer solution, precisely weighing 80mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride by using an electronic balance, adding the 80mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride into a reaction system to activate the carboxymethylated lotus root polysaccharide, adding 0.4g of polyethyleneimine-polyethylene glycol copolymer, uniformly mixing, keeping the reaction process away from light, placing the reaction system in a magnetic stirrer at room temperature, stirring at a constant speed for reaction for 36 hours, transferring the reaction liquid to a dialysis bag (the molecular weight cutoff is 8000-14000), carrying out distilled water dialysis treatment on the reaction liquid for 36 hours, removing the supernatant through centrifugation, and carrying out freeze drying treatment to obtain the carboxymethylated lotus root polysaccharide compound.
Weighing 1.25g of folic acid, placing the folic acid into a 50mL flask, uniformly mixing 0.5mL of triethylamine and 20mL of dimethyl sulfoxide, adding the mixture into the flask, performing ultrasonic treatment to completely dissolve the folic acid, respectively weighing 0.5g N, dissolving N-dicyclohexylcarbodiimide and 0.2g of N-hydroxysuccinimide in 10mL of dimethyl sulfoxide, adding the mixture into the flask while stirring to activate the folic acid, keeping out of the sun, placing the mixture in a magnetic stirrer, and performing uniform stirring reaction for 12 hours. The reaction by-product dicyclohexylurea was removed by suction filtration, 2000mL of ultrapure water was added to the obtained filtrate with stirring to precipitate a yellow precipitate, which was filtered off with suction after standing overnight, and then lyophilized.
Grinding the freeze-dried crude product of the folic acid active ester into powder, repeatedly washing with hot water for 2-3 times, soaking in appropriate amount of acetone and ultrasonically treating for 10min, removing unreacted folic acid and reaction by-product dicyclohexylurea, filtering, and freeze-drying to obtain light yellow powder.
0.2g carboxymethylation was accurately weighedPlacing the lotus root polysaccharide compound in a 50mL round-bottom flask, adding 25mL anhydrous dimethyl sulfoxide to uniformly disperse the polysaccharide, slowly adding 3mL dimethyl sulfoxide solution (29mg/mL) of folic acid active ester under the action of a magnetic stirrer, and reacting for 12h in a dark place. After the reaction is finished, 0.1mol/L Na is used2Adjusting pH of CO3 solution to 9.0, centrifuging to remove supernatant, washing precipitate with distilled water to remove residual dimethyl sulfoxide, and freeze drying to obtain carboxymethylated lotus root polysaccharide-folic acid complex.
Accurately weighing a proper amount of carboxymethylated lotus root polysaccharide-folic acid obtained by freeze drying, placing the carboxymethylated lotus root polysaccharide-folic acid into a 50mL flask, adding 20-30 mL of dimethyl sulfoxide for dissolving to enable the dimethyl sulfoxide to be uniformly distributed, simultaneously using the dimethyl sulfoxide as a solvent, preparing 1mg/mL of solution from trichostatin A, diluting 500 mu L of trichostatin A solution to 5mL, adding the diluted trichostatin A solution into the flask, stirring for 12 hours at a constant speed under the action of a magnetic stirrer, and freeze drying to obtain the carboxymethylated lotus root polysaccharide-trichostatin A conjugate.
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 (5)
1. A method for preparing carboxymethylated lotus root polysaccharide-trichostatin a conjugate, which comprises the following steps:
uniformly dispersing carboxymethylated lotus root polysaccharide in a phosphate buffer solution, then sequentially adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and a polyethyleneimine-polyethylene glycol copolymer, uniformly mixing, and reacting in the dark to obtain a reaction solution, wherein the weight ratio of the carboxymethylated lotus root polysaccharide, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the polyethyleneimine-polyethylene glycol copolymer is (40-60): (6-10): (40-60);
transferring the reaction solution to a dialysis bag, carrying out running water dialysis treatment, taking the retention solution for centrifugation, discarding the supernatant, and drying the precipitate to obtain carboxymethylated lotus root polysaccharide complex;
grafting folic acid on the carboxymethylated lotus root polysaccharide complex to obtain a carboxymethylated lotus root polysaccharide-folic acid complex;
grafting the carboxymethylated lotus root polysaccharide-folic acid compound to the trichostatin A to obtain carboxymethylated lotus root polysaccharide-trichostatin A conjugate;
grafting folic acid on the carboxymethylated lotus root polysaccharide complex to obtain the carboxymethylated lotus root polysaccharide-folic acid complex, which comprises the following steps:
dissolving folic acid in a mixed solution of triethylamine and dimethyl sulfoxide to obtain a first mixed solution;
dissolving N, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide in dimethyl sulfoxide to obtain a second mixed solution;
adding the second mixed solution into the first mixed solution, and reacting in a dark place to obtain folic acid active ester;
dissolving carboxymethylated lotus root polysaccharide complex and folic acid active ester in dimethyl sulfoxide, and reacting in the dark to obtain carboxymethylated lotus root polysaccharide-folic acid complex;
the weight ratio of folic acid, N-dicyclohexylcarbodiimide and N-hydroxysuccinimide is (30-50): 15-25): 8-12);
the weight ratio of the carboxymethylated lotus root polysaccharide complex to the folic acid active ester is (18-22) to (6-9).
2. The method of claim 1, wherein the carboxymethylated lotus rhizome polysaccharide-trichostatin A conjugate is prepared by uniformly dispersing carboxymethylated lotus rhizome polysaccharide in phosphate buffer, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and polyethyleneimine-polyethylene glycol copolymer, mixing, and reacting under dark conditions to obtain a reaction solution: the reaction time is 24-48 h; and/or the presence of a gas in the gas,
transferring the reaction solution to a dialysis bag, and performing running water dialysis treatment, wherein the method comprises the following steps: the time of running water dialysis treatment is 24-48 h.
3. The method of preparing carboxymethylated lotus rhizome polysaccharide-trichostatin a conjugate according to claim 1, wherein the step of adding the second mixed solution into the first mixed solution and reacting away from light to obtain active folic acid ester comprises: the reaction time is 8-16 h; and/or the presence of a gas in the gas,
dissolving carboxymethylated lotus root polysaccharide complex and folic acid active ester in dimethyl sulfoxide, and reacting in a dark place to obtain carboxymethylated lotus root polysaccharide-folic acid complex: the reaction time is 8-16 h.
4. The method of claim 1, wherein the step of grafting the carboxymethylated lotus root polysaccharide-folic acid complex to the trichostatin a to obtain the carboxymethylated lotus root polysaccharide-trichostatin a conjugate comprises:
dissolving carboxymethylated lotus root polysaccharide-folic acid compound in dimethyl sulfoxide to obtain a solution to be reacted;
and adding the dimethyl sulfoxide solution of the trichostatin A into the solution to be reacted, and reacting to obtain carboxymethylated lotus root polysaccharide-trichostatin A conjugate.
5. The method of claim 4, wherein the step of adding a solution of trichostatin A in DMSO to the solution to be reacted to obtain carboxymethylated lotus root polysaccharide-trichostatin A conjugate comprises: the reaction time is 8-16 h.
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