Aminated lentinan as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of polysaccharide preparation, and particularly relates to aminated lentinan as well as a preparation method and application thereof. The method comprises the following steps: dissolving lentinan in an organic solvent, then adding succinic anhydride, and carrying out a first reaction to obtain LNT-COOH after the reaction is finished; the preparation method comprises the following steps: dissolving lentinan in an organic solvent, sequentially adding HATU, HOBT and DIEA, stirring, reacting, adding H2N-NHBOC after the reaction is finished, carrying out an amination reaction, adding trifluoroacetic acid after the amination reaction is finished, and stirring to obtain aminated lentinan. The method disclosed by the invention has the characteristics of simplicity and convenience in operation and high reaction efficiency, and the obtained aminated lentinan has better solubility, biocompatibility and remarkable biological activity, and is suitable for development and application of medicines, health-care products and other biological materials.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of polysaccharide preparation, and particularly relates to aminated lentinan and a preparation method and application thereof. BACKGROUND

[0002] Lentinan (LNT) is a natural polysaccharide extracted from Lentinus edodes, which has significant biological activity and is widely used in the fields of medicine, health care products and food. Studies have shown that lentinan has immunomodulatory, antitumor, antiviral, antioxidant and other biological effects, and is therefore considered an important natural medicine and health care product raw material. Lentinan not only can enhance the immune function of the body, but also can promote the activity of white blood cells and inhibit the growth of tumor cells, and has a wide application prospect.

[0003] However, the direct application of lentinan faces some problems, mainly in its poor water solubility, poor stability and low bioavailability. Lentinan is usually difficult to be effectively absorbed by the human body, and its biological activity in the body is limited. These defects limit its wide application in clinical treatment and daily health care. Therefore, how to effectively improve the biological activity of lentinan, improve its drug delivery effect and biocompatibility has become a problem to be solved by researchers. SUMMARY

[0004] The purpose of the present application is to at least solve one of the problems of the prior art, and specifically to provide an aminated lentinan and a preparation method and application thereof. The following technical solutions are specifically adopted: In a first aspect, the present application provides a preparation method of an aminated lentinan, comprising the following steps: Dissolve lentinan in an organic solvent, then add succinic anhydride for the first reaction, perform the first dialysis after the reaction is completed, concentrate, precipitate, redissolve, then perform the second dialysis, and freeze-dry to obtain LNT-COOH; Dissolve the LNT-COOH in an organic solvent, then sequentially add HATU, HOBT and DIEA for stirring, react, add H2N-NHBOC for amination reaction after the reaction is completed, stir with trifluoroacetic acid, perform the third dialysis, concentrate, and freeze-dry to obtain the aminated lentinan.

[0005] The present application first converts lentinan (LNT) into carboxylated lentinan (LNT-COOH), and then introduces amino groups into the structure of LNT-COOH through a chemical amination reaction to obtain aminated lentinan LNT-CONH-NH2. The reaction is carried out under mild conditions, and the reagents such as HATU, HOBT, DIEA and amino protecting reagent H2N-NHBOC used can effectively promote the amination reaction, while ensuring that the triple helix structure of lentinan is not damaged, so that the excellent biological activity thereof is maintained. Finally, LNT-CONH-NH2 is purified through steps such as dialysis, concentration and freeze-drying. The method of the present application has the characteristics of simple operation and efficient reaction, and the aminated lentinan obtained has good solubility, biocompatibility and significant biological activity, and is suitable for the development and application of medicines, health products and other biological materials.

[0006] As a further preferred embodiment, the mass ratio of the lentinan to succinic anhydride is 3-5:1.

[0007] As a further preferred embodiment, the pH in the first reaction is 8.5-9.

[0008] As a further preferred embodiment, the temperature in the first reaction is 30℃-40℃, and the time of the first reaction is 12 h-24 h.

[0009] As a further preferred embodiment, the organic solvent is DMSO.

[0010] As a further preferred embodiment, the amount ratio of HATU, HOBT, DIEA and H2N-NHBOC is 0.1 mmol:0.1 mmol:0.2 mmol:0.6 mmol.

[0011] As a further preferred embodiment, the concentration of trifluoroacetic acid is 0.4%-0.6%.

[0012] As a further preferred embodiment, the temperature of freeze-drying is -70℃~-80℃, and the time is 48-60 h.

[0013] In a second aspect, the present application provides aminated lentinan prepared by the above preparation method.

[0014] In a third aspect, the present application provides the use of the above aminated lentinan in the preparation of an antitumor drug.

[0015] The present application has the following advantages: The method has the characteristics of simple operation and efficient reaction, greatly preserves the triple helix structure of lentinan compared with the prior art, and the obtained aminated lentinan has good solubility, biocompatibility and significant biological activity, so that it can expand the grafting and compounding with other materials through the introduced amino group, and further expand the development and application in medicine, health care products and other biological materials. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings used in the embodiments will be briefly introduced as follows. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0017] Figure 1 The sugar acid content of LNT, LNT-COOH and LNT-CONH-NH2 in Example 1 is shown.

[0018] Figure 2 The Zeta potential of LNT, LNT-COOH and LNT-CONH-NH2 in aqueous solution is shown.

[0019] Figure 3 The color reaction photos of LNT and LNT-CONH-NH2 with hydrindantin are shown.

[0020] Figure 4 The Congo red test results of LNT and LNT-CONH-NH2 are shown.

[0021] Figure 5 The absorbance of 15 mg / mL LNT and LNT-CONH-NH2 solution at 600 nm is shown.

[0022] Figure 6 The effects of LNT and LNT-CONH-NH2 of different concentrations on the survival rate of HeLa cells are shown. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some embodiments of the present application, not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0024] Example 1 A preparation method of aminated lentinan, which specifically comprises the following steps: (1) Dissolution of Lentinan (LNT) and addition of succinic anhydride Dissolve 450 mg of LNT in 30 mL of DMSO and stir until completely dissolved to ensure that the LNT is completely dissolved; then, dissolve 135 mg of succinic anhydride in 1 mL of DMSO and slowly add it to the LNT solution under stirring; maintain the temperature of the solution at 35°C and the pH at 8.5 throughout the reaction process, which is adjusted by dropwise addition of NaOH solution, and react for 12 h to ensure that the succinic anhydride and LNT react sufficiently.

[0025] (2) pH adjustment and dialysis after reaction Adjust the pH of the solution after step (1) to 6.5 by acid to terminate the reaction completely, and then dialyze the solution (dialysis bag with a molecular weight cutoff of 8000-14000 Da) for 72 h to remove the byproducts and small molecular impurities generated during the reaction.

[0026] (3) Concentration and precipitation Concentrate the solution after step (2) dialysis; then, precipitate using 70% ethanol, separate the precipitate by centrifugation at a speed of 3000 rpm for 10 min, collect the precipitate, and wash the precipitate with anhydrous ethanol three times, each time using 50 mL of anhydrous ethanol, to remove excess solvent and impurities through this step.

[0027] (4) Dialysis and freeze-drying Resuspend the washed precipitate from step (3) in water and dialyze it using a dialysis bag with a molecular weight cutoff of 8000-14000 Da for 72 h to remove small molecular impurities in the solution; after dialysis, freeze-dry the solution to obtain LNT-COOH.

[0028] (5) Dissolution of LNT-COOH and addition of reaction reagents Dissolve LNT-COOH (500 mg) obtained in step (4) in 50 mL of DMSO, ensure complete dissolution, and then add 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 38.3 mg, 0.1 mmol), 1-hydroxybenzotriazole (HOBT, 14.3 mg, 0.1 mmol), and N,N-diisopropylethylamine (DIEA, 26.0 mmol) in sequence, stir the mixed solution for 3 minutes to ensure that the reagents are uniformly dissolved and fully reacted; next, add 100 mg (0.6 mmol) of tert-butoxycarbonyl hydrazine (H2N-NHBOC) and continue stirring for 2 h for the amination reaction.

[0029] (6) Removal of protecting group and final purification After the reaction of step (5) is completed, 50% trifluoroacetic acid (0.5 mL) is added and stirred for 1 h to remove the Boc protecting group; then, a dialysis bag with a molecular weight cut-off of 8000-14000 Da is used for dialysis for 72 h to remove excess reagents and by-products, after dialysis is completed, the solution is concentrated and freeze-dried, and finally amino-functionalized lentinan (LNT-CONH-NH2) is obtained.

[0030] Example 2 The successful preparation of LNT-CONH-NH2 is verified by the change in uronic acid content. The specific steps are as follows: Step one: weigh 5 mg of glucuronic acid standard into a 50 mL volumetric flask, and transfer 0, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the standard solution into test tubes, and add ultrapure water to 1 mL.

[0031] Step two: weigh 25 mg of carbazole in 25 mL of anhydrous ethanol to obtain a 0.1% carbazole-ethanol solution.

[0032] Step three: prepare 1 mg / mL solutions of LNT, LNT-COOH, and LNT-CONH-NH2, respectively.

[0033] Step four: add 6 mL of concentrated sulfuric acid to the standard and sample test tubes in an ice bath, then react in a 80°C water bath for 20 min, then add 0.2 mL of 0.1% carbazole-ethanol solution, mix and stand for 2 h, and measure the absorbance at 530 nm.

[0034] The results are shown in Figure 1 The uronic acid content of LNT-COOH is significantly higher than that of LNT, which confirms the success of the succinylation reaction. Compared with LNT-COOH, the uronic acid content of the amino-functionalized product LNT-CONH-NH2 is significantly reduced, further verifying the success of the -NH2 grafting.

[0035] Example 3 The successful preparation of LNT-CONH-NH2 is verified by the change in Zeta potential content. The specific steps are as follows: Step one: prepare 5 mg / mL solutions of LNT, LNT-COOH, and LNT-CONH-NH2, respectively.

[0036] Step two: Zeta potential of LNT, LNT-COOH, LNT-CONH-NH2 and LNT-Au were measured by using a particle size potential analyzer (Zetasizer Pro, Malvern Panaco LTD).

[0037] The results are shown in Figure 4. Compared with LNT, LNT-COOH showed lower Zeta potential due to the introduction of negatively charged -COOH. After amination, the Zeta potential increased, indicating the grafting of positively charged -NH2. Figure 2

[0038] Example 4 The successful preparation of LNT-CONH-NH2 was verified by hydration of the ninhydrin color reaction. The specific steps are as follows: Step one: LNT and LNT-CONH-NH2 solutions of 5 mg / mL were prepared respectively.

[0039] Step two: 5 mg / mL of ninhydrin solution was prepared (dissolved in anhydrous ethanol, containing 0.05% ascorbic acid).

[0040] Step three: 1 mL of LNT and LNT-CONH-NH2 solution was added to 2 mL of ninhydrin solution respectively, and heated at 100°C for 10 min to detect the presence of amino groups.

[0041] The results are shown in Figure 5. Compared with LNT, LNT-CONH-NH2 showed a clear blue-violet color after reaction with ninhydrin, indicating the successful preparation of aminated lentinan. Figure 3

[0042] Example 5 The retention of the triple helix structure of LNT-CONH-NH2 was verified by the Congo red experiment, and the specific steps are as follows: Step one: LNT and LNT-CONH-NH2 sample solutions of 1 mg / mL were prepared respectively, with water as a control.

[0043] Step two: 89 μmol / L of Congo red solution was prepared.

[0044] Step three: 4 mol / L of sodium hydroxide solution was prepared.

[0045] Step four: 0.5 mL of sample solution and 0.5 mL of Congo red solution were mixed, and a certain amount of sodium hydroxide solution was added to make the sodium hydroxide concentration of the system 0, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.5 mol / L respectively, and gently shaken to mix.

[0046] ​​Step five: After 15 min of reaction in dark condition, the maximum absorption wavelength of the reaction solution was determined by UV-Vis spectrophotometer.

[0047] The results are shown in Figure 4 Figure 4, similar to LNT, LNT-CONH-NH2 can form complex with Congo red, thus the absorption wavelength red-shifts compared with Congo red, and within a certain range of sodium hydroxide concentration, the maximum absorption wavelength sharply decreases, indicating that LNT-CONH-NH2 retains the triple helix structure of LNT.

[0048] Example 6 The improvement of solubility of LNT-CONH-NH2 was verified by turbidity method. The specific steps are as follows: Step one: LNT and LNT-CONH-NH2 solutions of 15 mg / mL were respectively configured.

[0049] Step two: The absorbance of LNT and LNT-CONH-NH2 solutions at 600 nm was determined by UV-Vis spectrophotometer.

[0050] The results are shown in Figure 5 Figure 5, the absorbance of LNT-CONH-NH2 at 600 nm is significantly lower than that of LNT, indicating that the solubility of LNT-CONH-NH2 is higher than that of LNT, and the water solubility is better.

[0051] Example 7 The improvement of bioavailability of LNT-CONH-NH2 was verified by detecting the cell viability of HeLa cells after incubation with LNT and LNT-CONH-NH2 respectively by CCK-8 kit. The specific steps are as follows: Step one: HeLa cells were placed in 9-well plates at a density of 1 x 10 4 cells / well, and cultured in an incubator for 12 h.

[0052] Step two: LNT and LNT-CONH-NH2 solutions of 0, 50, 100, 400, and 800 μg / mL were respectively configured in DMEM medium containing 10% FBS, and sterilized by filtration for standby.

[0053] Step three: The medium in each well of step one was aspirated, and 200 μL of LNT and LNT-CONH-NH2 solutions of different concentrations configured in step two were respectively added, and cultured in an incubator for 24 h.

[0054] Step four: The concentration of CCK-8 was diluted to 10% with DMEM medium.

[0055] Step five: aspirate the liquid in each well after step three culture, add 100 μL CCK-8 reaction solution prepared in step four to each well, react in the incubator for 30 min, then use the microplate reader to measure the absorbance at 450 nm of each well.

[0056] The results are shown in Figure 6 LNT-CONH-NH2 has more obvious inhibitory effect on HeLa than LNT, due to the better solubility of LNT-CONH-NH2, thus greatly improving its bioavailability.

[0057] The embodiments of the present application are described above in conjunction with the accompanying drawings, and the principles and implementation manners of the present application are described herein by applying specific examples, the above example descriptions are only used to help understand the core ideas of the present application, but the present application is not limited to the above specific implementation manners, the above specific implementation manners are only illustrative, but not restrictive, and those skilled in the art can make many forms under the inspiration of the present application without departing from the scope of the present application and the protection scope of the claims.

Claims

1. A method for preparing aminolated lentinan, characterized in that, Includes the following steps: Lentinan was dissolved in an organic solvent, and then succinic anhydride was added for the first reaction. After the reaction was completed, the mixture was dialyzed for the first time, concentrated, precipitated, reconstituted, dialyzed for the second time, and freeze-dried to obtain LNT-COOH. The LNT-COOH was dissolved in an organic solvent, and then HATU, HOBT and DIEA were added sequentially and stirred to react. After the reaction was completed, H2N-NHBOC was added to carry out an amination reaction. After the amination reaction was completed, trifluoroacetic acid was added and stirred. The mixture was dialyzed for the third time, concentrated, and freeze-dried to obtain the amination lentinan.

2. The preparation method according to claim 1, characterized in that, The mass ratio of lentinan to succinic anhydride is 3-5:

1.

3. The preparation method according to claim 2, characterized in that, The pH during the first reaction was 8.5-9.

4. The preparation method according to claim 3, characterized in that, The temperature during the first reaction is 30℃-40℃, and the reaction time is 12 h-24 h.

5. The preparation method according to claim 4, characterized in that, The organic solvent is DMSO.

6. The preparation method according to claim 1, characterized in that, The ratio of HATU, HOBT, DIEA and H2N-NHBOC is 0.1 mmol: 0.1 mmol: 0.2 mmol: 0.6 mmol.

7. The preparation method according to claim 1, characterized in that, The volume concentration of the trifluoroacetic acid is 0.4%-0.6%.

8. The preparation method according to claim 1, characterized in that, The freeze-drying temperature is -70℃ to -80℃, and the time is 48-60 h.

9. An aminolated lentinan, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The use of the aminolated lentinan according to claim 9 in the preparation of antitumor drugs.

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