Catechin grafted dandelion polysaccharide as well as preparation method and application thereof

Abstract

The invention discloses catechin grafted dandelion polysaccharide as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing dandelion polysaccharide and catechin in an acetic acid aqueous solution, fully dissolving, adding ascorbic acid in a nitrogen atmosphere, stirring until the ascorbic acid is dissolved, adding hydrogen peroxide, stirring for 24 hours, dialyzing for 72 hours, and freeze-drying to obtain the catechin grafted dandelion polysaccharide conjugate. Through the synergistic effect of catechin and dandelion polysaccharide, compared with the dandelion polysaccharide, the conjugate has the advantage that the inhibition capability on alpha-amylase is enhanced.

Description

Technical Field

[0001] This invention belongs to the field of drug modification technology, specifically relating to a method for modifying dandelion polysaccharides, and particularly to a catechin-grafted dandelion polysaccharide, its preparation method, and its application. Background Technology

[0002] Dandelion polysaccharides, as an important natural bioactive substance, have been shown in studies to have significant effects in antioxidation, antitumor activity, and antidiabetic activity. In recent years, the application of graft copolymerization in polysaccharide modification has received widespread attention. Grafting functional groups (especially phenolic compounds) onto polysaccharides not only combines the advantages of both polyphenols and polysaccharides but also exerts synergistic effects, such as producing enhanced physiological properties and altering functional properties, including solubility, rheology, and emulsification, thereby expanding their application potential in food, cosmetics, and pharmaceuticals.

[0003] The biological and pharmacological effects of catechins (CAs), including antioxidant, hypoglycemic, anti-inflammatory, antimutagenic, anticancer, and antimicrobial properties, have been studied. Research has shown that catechins have been successfully grafted onto chitosan, arabinoxylan, and glucan, with the increased bioactivity attributed to the function of the grafted catechins. However, to date, no reports have been made on conjugates of dandelion polysaccharides grafted with polyphenols and their antidiabetic activity. Summary of the Invention

[0004] The purpose of this invention is to provide a method for modifying dandelion polysaccharides, namely, a method for preparing dandelion polysaccharides grafted with catechins. The resulting dandelion polysaccharide-catechin conjugate allows each of its effects to be exerted, and further enhances its hypoglycemic activity through synergistic effects.

[0005] In a first aspect, this invention provides a method for preparing catechin-grafted dandelion polysaccharide conjugates, wherein dandelion polysaccharides are coupled to catechins via a free radical catalytic method, and the specific steps are as follows: (1) Dissolve catechins and dandelion polysaccharides in acetic acid solution to obtain solution A; (2) Under a nitrogen atmosphere, ascorbic acid was added to solution A and stirred at room temperature for 1-2 h to obtain solution B; (3) Add hydrogen peroxide to solution B and stir at room temperature for 12-24 h to obtain solution C; dialyze and freeze dry to obtain catechin-grafted dandelion polysaccharide conjugate.

[0006] The above-mentioned method for preparing a catechin-grafted dandelion polysaccharide conjugate, wherein the dandelion polysaccharide is a dandelion polysaccharide that has undergone protein removal treatment.

[0007] Furthermore, the dandelion polysaccharide is obtained by hot water extraction of the whole dandelion plant, followed by graded alcohol precipitation and deproteinization treatment using the Sevag method.

[0008] Specifically, after defatting the whole dandelion plant, hot water extraction was performed at 90℃ for 2 hours using a material-to-liquid ratio of 1 / 30. The extract was concentrated by rotary evaporation and then precipitated separately with anhydrous ethanol at final concentrations of 20%, 40%, 60%, and 80% to obtain crude dandelion polysaccharides. Previous studies have shown that dandelion polysaccharides precipitated with 60% ethanol exhibit superior hypoglycemic activity compared to those precipitated at other concentrations. The 60% ethanol-precipitated dandelion polysaccharide was then deproteinized using Sevag reagent (prepared with chloroform:n-butanol in a 4:1 ratio and polysaccharide in a 4:1 ratio). The mixture was vigorously shaken for 30 minutes, repeated until no protein layer was observed. The resulting product was then rotary evaporated, dialyzed, and freeze-dried to obtain deproteinized dandelion polysaccharides.

[0009] In the above-mentioned method for preparing a catechin-grafted dandelion polysaccharide conjugate, preferably, in step (1), the weight ratio of dandelion polysaccharide to catechin is 1-10:1; preferably 5-10:1; the volume percentage of acetic acid solution is 1%; and the concentration of dandelion polysaccharide in acetic acid solution is 1-5 mg / mL.

[0010] In the above-mentioned method for preparing a catechin-grafted dandelion polysaccharide conjugate, preferably, in step (2), the mass ratio of ascorbic acid to dandelion polysaccharide is 1-3:5.

[0011] In the above-mentioned method for preparing a catechin-grafted dandelion polysaccharide conjugate, preferably, in step (3), hydrogen peroxide is added as a free radical initiator, wherein the molar ratio of hydrogen peroxide to ascorbic acid is 29-30:1; preferably, the molar concentration of hydrogen peroxide is 1 M.

[0012] In the above-mentioned method for preparing a catechin-grafted dandelion polysaccharide conjugate, preferably, in step (3), the dialysis is performed in a dialysis bag with a specification of 3500 Da, during which distilled water is changed every 12 hours, and after the dialysis is completed, the catechin-grafted dandelion polysaccharide conjugate is obtained by freeze drying.

[0013] A second aspect of the present invention provides a catechin-grafted dandelion polysaccharide conjugate prepared by the above method.

[0014] The in vitro hypoglycemic activity of catechin-grafted dandelion polysaccharide conjugates was evaluated by inhibiting α-amylase activity. The results showed that the inhibitory activity of the conjugates on α-amylase was significantly higher than that of the physically mixed conjugates, suggesting that the hypoglycemic activity of dandelion polysaccharide was significantly enhanced after catechin grafting modification.

[0015] Comparison of conjugates with different grafting ratios revealed that the conjugate grafted with dandelion polysaccharide at a mass ratio of 5:1 to catechin showed the highest inhibition rate against α-amylase activity, followed by the conjugate grafted at a ratio of 10:1. This suggests that the synergistic effect of catechin grafting onto dandelion polysaccharide enhances the hypoglycemic effect, with the optimal hypoglycemic effect observed within the grafting range of 10:1 to 5:1.

[0016] In view of the above activity evaluation results, the third aspect of the present invention provides the use of the above-mentioned catechin-grafted dandelion polysaccharide conjugate in the preparation of products for controlling blood sugar and / or controlling weight.

[0017] Furthermore, the application of the catechin-grafted dandelion polysaccharide conjugate in the preparation of α-amylase inhibitors.

[0018] Compared with the prior art, the advantages and technical effects of the present invention are as follows: The raw material used in this invention, dandelion polysaccharide, is highly water-soluble and non-toxic. Dandelion is a plant with both medicinal and edible uses, found worldwide. Due to its excellent biological activity, catechins are grafted onto it, enhancing its hypoglycemic activity. This provides a feasible path from laboratory to industrialization for the high-value utilization of natural products, and also provides an important material basis for innovation in functional foods, pharmaceuticals, and new materials. Attached Figure Description

[0019] Figure 1 Ultraviolet full-wavelength scan images of catechin grafted onto dandelion polysaccharide before and after grafting.

[0020] Figure 2 Fourier transform infrared images of catechin grafted onto dandelion polysaccharide before and after grafting.

[0021] Figure 3 The standard curve for catechin polyphenol content.

[0022] Figure 4 The inhibition rate of α-amylase activity before and after catechin grafting onto dandelion polysaccharide. Detailed Implementation

[0023] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to enable researchers in the art to better understand it.

[0024] The dandelion polysaccharide described in the following embodiments of the present invention was prepared by the following method, with specific steps as follows: The whole dandelion plant was dried, pulverized, and sieved (50 mesh). The powder was weighed and added to anhydrous ethanol at a ratio of 100 g: 3000 mL. The mixture was stirred with a magnetic stirrer for 12 h, allowed to stand, the supernatant was removed, and the mixture was dried. The defatted dandelion whole plant powder was added to distilled water at a ratio of 1:30 and heated in a water bath at 90°C for 3 h. The supernatant was centrifuged and rotary evaporated to 1 / 4 of its original volume. Anhydrous ethanol was used to adjust the final volume concentrations to 20%, 40%, and 60%, respectively. The polysaccharides were precipitated separately for 12 h. After centrifugation, the precipitate was dried, dissolved in water, and freeze-dried to obtain crude dandelion polysaccharide. The α-amylase inhibitory activity test results of the crude dandelion polysaccharide obtained by 60% ethanol precipitation showed the best inhibitory activity. The crude dandelion polysaccharide obtained by 60% ethanol precipitation was deproteinized using Sevag reagent, which was prepared by chloroform: n-butanol = 4:1 and polysaccharide = 4:1. The mixture was shaken vigorously for 30 min and repeated until no protein layer was observed. The polysaccharide was then lyophilized by rotary evaporation and dialyzed using a 3500 Da dialysis bag to obtain deproteinized dandelion polysaccharide, denoted as DPH-2.

[0025] Example 1

[0026] The preparation method of dandelion polysaccharide grafted with catechins is as follows: Dandelion polysaccharide and catechin were added to 10 mL of 1% acetic acid solution at a 1:1 ratio (50 mg: 50 mg) and stirred at 25 °C until fully dissolved. Then, 30 mg of ascorbic acid was added and nitrogen gas was introduced. The mixture was stirred at 25 °C for 1 h. Finally, 5 mL of 1 M hydrogen peroxide was added to initiate the conjugation reaction, which was carried out at 25 °C for 24 h. After dialyzing with distilled water for 72 h, the mixture was freeze-dried to obtain the catechin-grafted dandelion polysaccharide conjugate, denoted as DPH-2:CA=1:1.

[0027] Example 2

[0028] The preparation method of dandelion polysaccharide grafted with catechins is as follows: Dandelion polysaccharide and catechin were added to 10 mL of 1% acetic acid solution at a ratio of 5:1 (50 mg: 10 mg). The mixture was stirred at 25 °C until fully dissolved. Then, 30 mg of ascorbic acid was added and nitrogen gas was introduced. The mixture was stirred at 25 °C for 1 h. Finally, 5 mL of 1 M hydrogen peroxide was added to initiate the conjugation reaction, which was carried out at 25 °C for 24 h. After dialyzing with distilled water for 72 h, the mixture was freeze-dried to obtain the catechin-grafted dandelion polysaccharide conjugate, denoted as DPH-2:CA=5:1.

[0029] like Figure 1The UV-Vis spectra of DPH-2, CA, and DPH-2:CA = 5:1 couplings were compared. CA and DPH-CA couplings exhibited a characteristic absorption band at 277 nm, indicating the formation of covalent bonds in the DPH-CA coupling.

[0030] like Figure 2 The FT-IR spectra of DPH-2 and the DPH-2:CA = 5:1 conjugate are shown. The FT-IR spectra of the DPH-2 and DPH-CA conjugates are at 3421.74 cm⁻¹. -1 3426 cm -1 The strong signal at 2921.86 cm -1 2935.17 cm -1 The weak absorption bands at 1746.2 cm⁻¹ can be attributed to the stretching vibrations of the OH and CH bonds, respectively, both characteristic absorption peaks of polysaccharides. Compared to the DPH-CA conjugate, DPH-2 shows a stronger absorption band at 1746.2 cm⁻¹. -1 The disappearance of the absorption peak at 1545.5 cm⁻¹ may be due to the consumption of the carboxyl groups of the acidic polysaccharide through esterification or free radical grafting. Compared with DPH-2, the DPH-CA conjugate showed a higher absorption peak at 1545.5 cm⁻¹. -1 The enhancement of the aromatic ring peak at the point indicates that the benzene ring structure of catechin is introduced into the polysaccharide system.

[0031] Example 3

[0032] The preparation method of dandelion polysaccharide grafted with catechins is as follows: Dandelion polysaccharide and catechin were added to 10 mL of 1% acetic acid solution at a ratio of 10:1 (50 mg: 5 mg). The mixture was stirred at 25 °C until fully dissolved. Then, 30 mg of ascorbic acid was added and nitrogen gas was introduced. The mixture was stirred at 25 °C for 1 h. Finally, 5 mL of 1 M hydrogen peroxide was added to initiate the conjugation reaction, which was carried out at 25 °C for 24 h. After dialyzing with distilled water for 72 h, the mixture was freeze-dried to obtain the catechin-grafted dandelion polysaccharide conjugate, denoted as DPH-2:CA=10:1.

[0033] Comparative Example 1

[0034] Dandelion polysaccharide and catechin were added to 10 mL of 1% acetic acid solution in a 1:1 ratio (50 mg: 50 mg). After being stirred and dissolved at 25°C, the mixture was freeze-dried to obtain a physically mixed substance, denoted as DPH-2+CA.

[0035] Example 4

[0036] The polyphenol grafting rate was determined using the Folin-Ciocalteu method.

[0037] Solution preparation: Accurately weigh 2.5 mg of catechin standard into a 50 mL volumetric flask and dilute to volume to prepare a 50 μg / mL standard solution. Using a pipette, transfer 0 μL, 20 μL, 40 μL, 60 μL, 80 μL, 100 μL, 150 μL, and 200 μL of the standard solution into centrifuge tubes, respectively. Make up to 500 μL with distilled water. Prepare a 20 μg / mL DPH-CA solution and add it to 500 μL of centrifuge tubes. Add 0.5 mL of 10% Folin-Ciocalteu solution to each tube, shake well, and react for 3 min. Add 0.4 mL of 7.5% sodium carbonate solution, and make up to 1 mL with distilled water. Shake well and let stand for 1 h. Measure the absorbance at 765 nm. Plot a catechin standard curve with catechin concentration on the x-axis and absorbance on the y-axis. Figure 3 The regression equation was calculated as follows: y =0.0097 x +0.0626, R 2 =0.9947, the polyphenol content of DPH-2:CA=1:1 is 1.61, the grafting rate is 19.07%, the polyphenol content of DPH-2:CA=5:1 is 1.728, the grafting rate is 13.57%, and the polyphenol content of DPH-2:CA=10:1 is 0.89, the grafting rate is 10.31%.

[0038] Example 5

[0039] Determination of α-amylase activity using DPH-2 and DPH-CA grafted at different ratios.

[0040] 25 μL of sample solutions of different concentrations (0.2–1 mg / mL) were mixed with 25 μL of α-amylase solution (50 U / mL, prepared with 0.1 mol / L, pH 6.8 PBS) and incubated at 37 °C for 10 min. Then, 25 μL of starch solution (20 mg / mL) was added, and the mixture was reacted in a boiling water bath for 10 min, immediately cooled to room temperature in an ice-water bath. 50 μL of dinitrosalicylic acid (DNS) reagent was added, and the mixture was heated at 95 °C for 5 min to develop a colorimetric reaction, immediately cooled to room temperature in an ice-water bath. The mixture was then added to 750 μL of deionized water and centrifuged at 1000 g for 5 min. The supernatant (200 μL) was transferred to a 96-well plate and measured at 540 nm using a microplate reader. The inhibitory activity of α-amylase was expressed as a percentage of inhibition and calculated using the following formula:

[0041] α-Amylase inhibition rate (%) =

[0042] In the formula, ODa represents a mixture of sample, starch, and amylase; ODb represents a mixture of 0.1 M PBS (in place of amylase), sample, and starch; and ODc represents a mixture of 0.1 M PBS (in place of sample), starch, and amylase.

[0043] Figure 4 The inhibitory effect on α-amylase activity increased with increasing concentrations of DPH-2 and DPH-CA conjugates. The DPH-CA conjugate showed inhibition of α-amylase activity close to that of DPH-2 at a concentration of 0.6 mg / mL, and with further increases in concentration, its inhibitory effect on α-amylase activity was higher than that of DPH-2 and higher than that of catechins. Among the conjugates obtained by grafting DPH-2 and CA at different ratios, the 5:1 grafted conjugate showed a higher inhibition rate of α-amylase activity than the 10:1 and 1:1 grafted conjugates, with the 1:1 grafted conjugate showing the lowest inhibition rate among these groups. In summary, the hypoglycemic activity of the grafted conjugates was stronger than that of catechins and dandelion polysaccharides, as well as the activity of a physical mixture of both. This indicates that catechin grafting with dandelion polysaccharides has a synergistic effect, resulting in a stronger hypoglycemic effect, with the optimal hypoglycemic effect observed within the 10:1-5:1 grafting range.

[0044] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a catechin-grafted Taraxacum polysaccharide conjugate, characterized by, The specific steps include: (1) dissolving catechin and dandelion polysaccharide in acetic acid solution to obtain solution A; (2) adding ascorbic acid to solution A under nitrogen atmosphere, stirring at room temperature for 1-2 h to obtain solution B; (3) adding hydrogen peroxide to solution B, stirring at room temperature for 12-24 h to obtain solution C; dialysis, freeze-drying to obtain catechin grafted dandelion polysaccharide conjugate.

2. The method for preparing the catechin-grafted Taraxacum polysaccharide conjugate according to claim 1, characterized in that, The dandelion polysaccharide is obtained after deproteinization treatment.

3. The method for preparing the catechin-grafted dandelion polysaccharide conjugate according to claim 2, characterized in that, The dandelion polysaccharide is obtained by hot water extraction method from dandelion whole plant, fractional alcohol precipitation, and deproteinization treatment by Sevag method.

4. The method for preparing the catechin-grafted dandelion polysaccharide conjugate according to claim 3, characterized in that, The fractional alcohol precipitation is: after concentration of the extract, adding anhydrous ethanol to make the volume fraction of ethanol 20%, 40%, 60% and 80% respectively, fractional precipitation, and using 60% alcohol precipitated dandelion polysaccharide for deproteinization treatment by Sevag method.

5. The method for preparing the catechin-grafted dandelion polysaccharide conjugate according to claim 1, characterized in that, In step (1), the mass ratio of dandelion polysaccharide to catechin is 1-10:

1.

6. The method for preparing the catechin-grafted dandelion polysaccharide conjugate according to claim 1, characterized in that, In step (2), the mass ratio of ascorbic acid to dandelion polysaccharide is 1-3:

5.

7. The method for preparing the catechin-grafted dandelion polysaccharide conjugate according to claim 1, characterized in that, In step (3), the molar ratio of hydrogen peroxide to ascorbic acid is 29-30:

1.

8. The catechin grafted dandelion polysaccharide conjugate prepared by the method according to any one of claims 1-7.

9. Use of the catechin grafted dandelion polysaccharide conjugate according to claim 8 in the preparation of products for controlling blood glucose and / or controlling body weight.

10. Use of the catechin grafted dandelion polysaccharide conjugate according to claim 8 in the preparation of α-amylase inhibitors.

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