Mulberry twig polysaccharide zinc complex, preparation method thereof and application of mulberry twig polysaccharide zinc complex in preparation of product with antioxidant and / or anti-inflammatory effect

Abstract

The invention relates to the technical field of biological medicines, and particularly discloses a preparation method of a ramulus mori polysaccharide zinc complex, which comprises the following steps: dissolving ramulus mori polysaccharide in water to obtain a ramulus mori polysaccharide aqueous solution; under a stirring condition, dropwise adding a zinc salt solution into the ramulus mori polysaccharide aqueous solution to obtain a mixed solution; an alkali solution is added into the mixed solution, the pH is adjusted to 6-7, stirring continues to be conducted for 2-4 h at the temperature of 60-80 DEG C, and a reaction solution is obtained; cooling the reaction solution, concentrating to obtain a concentrated solution, and dialyzing the concentrated solution; and adding ethanol into the dialysate for alcohol precipitation, and drying the solid after alcohol precipitation to obtain the mulberry twig polysaccharide zinc complex. Researches show that the mulberry twig polysaccharide zinc complex prepared by the method has antioxidant activity and anti-inflammatory activity; particularly, the anti-inflammatory activity is obviously higher than that of ramulus mori polysaccharide, so that obvious technical progress is achieved; the important application value is realized.

Description

Technical Field

[0001] The present invention relates to the field of biological medicine technology, and particularly relates to a mulberry twig polysaccharide zinc complex, a preparation method thereof, and an application thereof in the preparation of a product having antioxidant and / or anti-inflammatory effects. Background Art

[0002] Mulberry twig is one of the main by-products of mulberry trees and is rich in polysaccharide components. However, except for mulberries and mulberry leaves, mulberry twigs have not been effectively utilized, which is likely to cause great waste of resources. As a traditional Chinese medicine, mulberry twig contains rich chemical components including flavonoids, polysaccharides, alkaloids, and amino acids, and has pharmacological activities such as hypoglycemic, hypolipidemic, anti-inflammatory, immune, and antiviral effects. Among them, mulberry twig polysaccharide is composed of monosaccharides such as mannose, rhamnose, glucuronic acid, arabinose, glucose, galactose, and galacturonic acid. Modern pharmacological studies have shown that mulberry twig polysaccharide has pharmacological effects such as relieving diabetes, protecting the liver, and enhancing immunity.

[0003] In addition, polysaccharide-metal complexes have significant biological activities, including antioxidant, antifungal, antibacterial and other functions, showing broad application prospects in the pharmaceutical, cosmetic, and food industries. Various complexes formed by glycyrrhizin polysaccharide extracted from the traditional Chinese medicine licorice and metal cations not only retain the respective advantages of glycyrrhizin polysaccharide and metal ions, but also have an obvious superposition enhancement effect (CN101328222A). The polysaccharide-zinc ion complex extracted from Prunus serrulata var. lannesiana has a significant effect on inhibiting the growth of Escherichia coli when applied to baked products (CN118146398A).

[0004] In recent years, mulberry twig polysaccharide has also been well studied. Among them, a separated mulberry twig polysaccharide RMPW-1 has obvious inhibitory effects on the growth of human cervical cancer cells Hela and human gastric cancer cells SGC-7901, and can be used for the research and development of anti-tumor drugs (CN105294878A). Another mulberry twig polysaccharide SZP has certain antioxidant effects and strong inhibitory activity against α-glucosidase, and has a certain delaying effect on the blood glucose value and oral glucose tolerance of diabetic mice, and can be used for the preparation of hypoglycemic drugs (CN118930674A).

[0005] Polysaccharide-metal complexes have significant antioxidant and antibacterial biological activities, and the research and application of mulberry twig polysaccharide-metal complexes have not been reported yet. Summary of the Invention

[0006] In order to overcome at least one of the technical problems existing in the prior art, the present invention provides a mulberry twig polysaccharide zinc complex, a preparation method thereof, and an application thereof in the preparation of a product having antioxidant and / or anti-inflammatory effects.

[0007] The technical solution of the present invention is as follows: The present invention provides a method for preparing a mulberry twig polysaccharide zinc complex, which comprises the following steps: Take mulberry twig polysaccharide and dissolve it in water to obtain an aqueous solution of mulberry twig polysaccharide; Under stirring conditions, dropwise add a zinc salt solution to the aqueous solution of mulberry twig polysaccharide, and obtain a mixed solution after the addition is completed; Add an alkali solution to the mixed solution, adjust the pH to 6 - 7, and continue stirring at 60 - 80 °C for 2 - 4 h to obtain a reaction solution; Cool the reaction solution and then concentrate it to obtain a concentrated solution. Dialyze the concentrated solution; Add ethanol to the dialyzed solution for alcohol precipitation, and dry the solid after alcohol precipitation to obtain the mulberry twig polysaccharide zinc complex.

[0008] Preferably, the dosage ratio of mulberry twig polysaccharide to water is 0.8 - 1.2 g: 15 - 25 mL.

[0009] More preferably, the dosage ratio of mulberry twig polysaccharide to water is 1 g: 20 mL.

[0010] Preferably, the concentration of the zinc salt solution is 30 - 60 mg / mL.

[0011] Most preferably, the concentration of the zinc salt solution is 50 mg / mL.

[0012] Preferably, the zinc salt solution is a zinc sulfate solution or a zinc chloride solution.

[0013] Preferably, the volume of the concentrated solution is 1 / 4 - 1 / 6 of the volume of the reaction solution.

[0014] Preferably, the ethanol is anhydrous ethanol; the volume dosage of anhydrous ethanol is 1 - 3 times the volume of the dialyzed solution.

[0015] Most preferably, the volume dosage of anhydrous ethanol is 2 times the volume of the dialyzed solution.

[0016] The present invention also provides a mulberry twig polysaccharide zinc complex prepared by the above - mentioned preparation method.

[0017] The present invention also provides an application of the above - mentioned mulberry twig polysaccharide zinc complex in the preparation of a product with antioxidant effect.

[0018] The present invention also provides an application of the above - mentioned mulberry twig polysaccharide zinc complex in the preparation of a product with anti - inflammatory effect.

[0019] Preferably, the product is a drug or a nutritional supplement.

[0020] Beneficial effects: The present invention provides a novel method for preparing mulberry twig polysaccharide zinc complex; research shows that the mulberry twig polysaccharide iron complex prepared by the method of the present invention has antioxidant activity and anti-inflammatory activity; in particular, its anti-inflammatory activity is significantly higher than that of mulberry twig polysaccharide, achieving significant technological progress; it has important application value. Description of the Drawings

[0021] Figure 1 It is the infrared spectrum diagram of mulberry twig polysaccharide.

[0022] Figure 2 It is the infrared spectrum diagram of mulberry twig polysaccharide zinc complex.

[0023] Figure 3 It is the experimental result diagram of measuring antioxidant activity by 1,10-phenanthroline method. Among them, Figure 3 a. in it is the blank group, Figure 3 b. in it is the control group, Figure 3 c. in it is the experimental group (1mg / mL mulberry twig polysaccharide zinc complex), Figure 3 d. in it is the experimental color comparison of mulberry twig polysaccharide zinc complex with different addition amounts.

[0024] Figure 4 It is the experimental result diagram of the free radical scavenging effect of mulberry twig polysaccharide and mulberry twig polysaccharide zinc complex.

[0025] Figure 5 It is the experimental result diagram of the anti-inflammatory and cytotoxicity of mulberry twig polysaccharide and mulberry twig polysaccharide zinc complex; among them, Figure 5 a in it is the anti-inflammatory experiment, Figure 5 b in it is the cytotoxicity experiment. Detailed Embodiments

[0026] The following further explains the present invention in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form.

[0027] The mulberry twig polysaccharide in the present invention and the following embodiments is prepared by the following method: (1) Dissolve 50 g of mulberry twig extract powder with about 50% polysaccharide content in 150 mL of pure water, add 24 mL of chloroform and 6 mL of n-butanol, stir vigorously for 15 minutes and then let it stand for liquid separation, and take the supernatant; repeat 3 - 4 times until there is no white precipitate; collect the supernatant and concentrate it to 50 mL; (2) Take 50 mL of the above concentrated solution, dropwise add 150 mL of anhydrous ethanol while stirring, then place it in an ice-water bath to cool and precipitate, collect the precipitate and remove the filtrate; dissolve the precipitate again in 50 mL of pure water, repeat the operation 3 - 4 times to obtain the precipitate, collect it and wash the filter cake with anhydrous ethanol and acetone, and dry it to obtain 20 g of pure mulberry twig polysaccharide.

[0028] Physicochemical properties of the obtained product: Mulberry twig polysaccharide is a brownish-gray powdery solid, odorless and tasteless, easily soluble in water, its aqueous solution is a brown transparent solution, with a neutral pH, and insoluble in organic solvents such as absolute methanol, ethanol, and ether.

[0029] Structure of the product: From Figure 1 The infrared spectrogram of the mulberry twig polysaccharide shown, its characteristic peak data are: 3376.52 (-OH), 2925.22 (-CH2-), 1636.52 (-C=O), 1417.39, 1362.61 (-O-H, -C-H), 1151.30 (-C-O-C), 1075.65, 1023.48 (C-O-H). Among them, 1151.30 cm -1 , 1075.65 cm -1 are the characteristic absorption peaks of pyranose, and 926.96 cm -1 , 848.09 cm -1 are the characteristic absorption peaks of β-pyranose and α-pyranoside bonds respectively.

[0030] The following further explains the present invention in conjunction with specific embodiments, but the embodiments do not limit the present invention in any form.

[0031] The mulberry twig polysaccharide in the present invention and the following embodiments is prepared by the following method: (1) Dissolve 50 g of mulberry twig extract powder with a polysaccharide content of about 50% in 150 mL of pure water, add 24 mL of chloroform and 6 mL of n-butanol, stir vigorously for 15 minutes and then let it stand for liquid separation, and take the supernatant; repeat 3 - 4 times until there is no white precipitate; collect the supernatant and concentrate it to 50 mL; (2) Take 50 mL of the above concentrated solution, dropwise add 150 mL of absolute ethanol while stirring, then place it in an ice-water bath to cool and precipitate, collect the precipitate and remove the filtrate; dissolve the precipitate again in 50 mL of pure water, repeat the operation 3 - 4 times to obtain the precipitate, collect it and wash the filter cake with absolute ethanol and acetone, and dry it to obtain 20 g of pure mulberry twig polysaccharide.

[0032] Physicochemical properties of the obtained product: Mulberry twig polysaccharide is a brownish-gray powdery solid, odorless and tasteless, easily soluble in water, its aqueous solution is a brown transparent solution, with a neutral pH, and insoluble in organic solvents such as absolute methanol, ethanol, and ether.

[0033] Structure of the product: From Figure 1From the infrared spectrum of the mulberry twig polysaccharide shown, the characteristic peak data are as follows: 3376.52 (-OH), 2925.22 (-CH2-), 1636.52 (-C=O), 1417.39, 1362.61 (-O-H, -C-H), 1151.30 (-C-O-C), 1075.65, 1023.48 (C-O-H). Among them, 1151.30 cm -1 , 1075.65 cm -1 are characteristic absorption peaks of pyranose, and 926.96 cm -1 , 848.09 cm -1 are characteristic absorption peaks of β-pyranose and α-pyranoside bonds respectively.

[0034] Example 1 Preparation of Mulberry Twig Polysaccharide-Zinc Complex Weigh 1.0 g of mulberry twig polysaccharide and dissolve it in 20 mL of pure water. While stirring, add zinc sulfate solution (200 mg of zinc sulfate dissolved in 4 mL of pure water). Adjust the pH of the mixed solution to 6 with 0.5 M sodium hydroxide solution. Continue stirring at 70 °C for 3 h. After cooling, concentrate the reaction solution to about 5 mL and dialyze it with a dialysis bag. After dialysis and concentration, add 2 volumes of anhydrous ethanol while stirring, cool and let stand, filter to collect the solid, wash it with ethanol, and dry it to obtain 700 mg of a brownish powder solid, which is the mulberry twig polysaccharide-zinc complex; from Figure 2 the infrared spectrum of the mulberry twig polysaccharide-zinc complex shown, the characteristic peak data are as follows: 3384.35 (-OH), 2920.00 (-CH2-), 1641.74 (-C=O), 1414.78 (-O-H, -C-H), 1148.70 (-C-O-C), 1075.65, 1020.87 (C-O-H). The Zn element content is determined to be 3.2% by ICP-OES.

[0035] After the mulberry twig polysaccharide forms a complex with zinc ions, some of its main absorption peaks have obvious displacements, confirming that the zinc ions have a coordination effect with the complex polysaccharide. In the infrared spectrum of the mulberry twig polysaccharide, the broad associated peak at 3376.52 cm -1 is a strong stretching vibration peak absorption peak of hydroxyl groups. After modification and coordination, the peak shape changes and the absorption peak weakens, probably because the hydroxyl groups of the polysaccharide participate in the complexation reaction. The absorption peak at 2920 cm -1 appears due to the stretching vibration of asymmetric C-H of plant sugars. The strong absorption peak at 1641 cm -1 belongs to the stretching vibration of C=O. The absorption peak at about 1414 cm -1 is the vibration absorption peak of C-H. The absorption peaks at 1148 cm -1 and 1075 cm -1The absorption peaks at this position belong to the stretching vibrations of C-O-C and C-O-H. From Figure 2 it can be seen that compared with mulberry twig polysaccharide, the absorption peaks of its zinc complex at these positions are all weakened, indicating that the presence of Zn 2+ may form coordination bonds with these groups, thereby resulting in the weakening of its absorption peaks. The changes in these peak shapes and the appearance of new absorption peaks all indicate that zinc forms a complex with mulberry twig polysaccharide.

[0036] Example 2 Preparation of Mulberry Twig Polysaccharide Zinc Complex Weigh 1.0 g of mulberry twig polysaccharide and dissolve it in 20 mL of pure water. While stirring, add dropwise a zinc chloride solution (200 mg of zinc chloride dissolved in 4 mL of pure water). The mixed solution is adjusted to pH 7 with 0.5 M sodium hydroxide solution, and stirring is continued at 70 °C for 3 h. After cooling, the reaction solution is concentrated to about 5 mL and dialyzed with a dialysis bag for 24 h. After dialysis and concentration, add 2 volumes of anhydrous ethanol while stirring, cool and let stand, filter to collect the solid, wash it with ethanol, and dry it to obtain 400 mg of a brownish powder solid, which is the mulberry twig polysaccharide zinc complex.

[0037] Example 3 Antioxidant Activity Test of Mulberry Twig Polysaccharide Zinc Complex 1,10-Phenanthroline method: Prepare 1 mg / mL aqueous ferric chloride solution, 1 mg / mL aqueous ferrous chloride solution, 1 mg / mL ethanol solution of 1,10-phenanthroline (Cas: 66-71-7) and 1, 2, 4 mg / mL aqueous solution of mulberry twig polysaccharide zinc complex respectively. The experiments are as follows: ① Blank group: 0.2 mL of Fe 3+ aqueous solution + 0.2 mL of pure water + 0.1 mL of 1,10-phenanthroline solution; ② Control group: 0.2 mL of Fe 2+ solution + 0.2 mL of pure water + 0.1 mL of 1,10-phenanthroline solution; ③ Experimental group: Add 0.2 mL of the above three concentrations of mulberry twig polysaccharide zinc complex solution to 0.2 mL of Fe 3+ aqueous solution respectively. After shaking well and standing for 10 minutes, add 0.1 mL of 1,10-phenanthroline ethanol solution, and observe the color change of the solution.

[0038] As Figure 3 shown, compared with the blank group, the control group quickly turns red, and the experimental group gradually turns from light yellow to red and deeper with the increase of the concentration of the polysaccharide zinc complex. Samples are taken from the blank group, control group and experimental group, and identified by liquid chromatography. The results show that the characteristic absorption peak ( 2+ produced by the complex of Fe and 1,10-phenanthroline at a wavelength of 510 nm is detected in both the control group and the experimental group ( Figure 3 b in Figure 3 c). This result confirms that the mulberry twig polysaccharide zinc complex can reduce Fe3+ Reduced to Fe 2+ , indicating that it has significant antioxidant activity.

[0039] Example 4 Free radical scavenging experiments of mulberry twig polysaccharide and mulberry twig polysaccharide zinc complex DPPH free radical scavenging experiment method (spectrophotometry, wavelength 517 nm): Prepare a 0.2 mg / mL DPPH (Cas: 1898 - 66 - 4) free radical solution with absolute ethanol. Dilute mulberry twig polysaccharide with pure water into 5 gradient concentrations of 0.5, 1, 2, 4, and 6 mg / mL. Respectively prepare 180 μL of the test solution + 60 μL of the free radical solution, shake well and incubate at room temperature for 30 min, and measure the OD value at 517 nm. Calculate the free radical scavenging rate according to the following formula: DPPH free radical scavenging rate (%) = [1 - (As - A0) / A1] × 100 Where, As: absorbance value of mulberry twig polysaccharide + DPPH ethanol solution; A0: absorbance value of mulberry twig polysaccharide + absolute ethanol; A1: absorbance value of absolute ethanol + DPPH ethanol solution. Using the same experimental operation, prepare a solution of mulberry twig polysaccharide zinc complex (zinc content 3.2%) and vitamin C (positive control), and test and calculate the free radical scavenging rate of the mulberry twig polysaccharide zinc complex.

[0040] As Figure 4 shown, the results indicate that both mulberry twig polysaccharide and its zinc complex can directly scavenge free radicals and exhibit antioxidant activity. With the increase in concentration, their ability to scavenge DPPH free radicals gradually increases. Among them, the mulberry twig polysaccharide zinc complex shows more excellent antioxidant activity and has a stronger free radical scavenging ability compared to mulberry twig polysaccharide.

[0041] Example 5 Anti - inflammatory and cytotoxicity experiments of mulberry twig polysaccharide and mulberry twig polysaccharide zinc complex 1. Cell viability detection (CCK - 8 experiment) Seed RAW 264.7 cells into a 96 - well culture plate at a density of 1×10 5 cells per well and incubate at 37°C and 5% CO2 for 24 hours. Subsequently, add media containing different concentrations of mulberry twig polysaccharide (0.5, 1.0, 1.5, 2.0, 2.5, and 3 mg / mL) or mulberry twig polysaccharide zinc complex (0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mg / mL) to each well and continue to incubate for 24 hours. After incubation, add 10 μL of CKK - 8 solution to each well, incubate at 37°C in the dark for 1 hour, and measure the absorbance at 450 nm wavelength to evaluate cell viability.

[0042] 2. NO production inhibition experiment (Griess reagent detection) To detect the inhibitory activity of NO production, RAW 264.7 cells (1×10 5 cells / mL) were seeded in 96-well plates and incubated with lipopolysaccharide (LPS, final concentration 1 μg / mL) and different concentrations of mulberry twig polysaccharide (0.5–3.0 mg / mL) or mulberry twig polysaccharide-zinc complex (0.5–3.0 mg / mL) for 24 hours, with aspirin (ASP) as the positive control. 50 μL of the culture supernatant of each group was collected, mixed with 100 μL of Griess reagent, and the absorbance at 540 nm was measured after standing for 10 minutes to calculate the amount of NO production respectively.

[0043] As Figure 5 shown, the results of the CCK-8 cell viability experiment showed that mulberry twig polysaccharide and its zinc complex had no obvious toxicity to RAW 264.7 cells in the concentration range of 0.5–1.5 mg / mL and had good biosafety; the results of the Griess reagent method detection showed that both mulberry twig polysaccharide and mulberry twig polysaccharide-zinc complex could significantly inhibit LPS-induced NO production, and the inhibitory effect of mulberry twig polysaccharide-zinc complex was stronger, indicating its more excellent anti-inflammatory activity.

[0044] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification without departing from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A preparation method of mulberry twig polysaccharide zinc complex, characterized in that, It comprises the following steps: Take mulberry twig polysaccharide and dissolve it in water to obtain an aqueous solution of mulberry twig polysaccharide; Under stirring conditions, dropwise add a zinc salt solution to the aqueous solution of mulberry twig polysaccharide, and obtain a mixed solution after the addition is completed; Add an alkali solution to the mixed solution, adjust the pH to 6 - 7, and continue stirring at 60 - 80 °C for 2 - 4 h to obtain a reaction solution; Cool the reaction solution and then concentrate it to obtain a concentrated solution. Dialyze the concentrated solution; Add ethanol to the dialyzed solution for alcohol precipitation, and dry the solid after alcohol precipitation to obtain the mulberry twig polysaccharide zinc complex.

2. The preparation method of the mulberry twig polysaccharide zinc complex according to claim 1, characterized in that, The dosage ratio of mulberry twig polysaccharide to water is 0.8 - 1.2 g: 15 - 25 mL.

3. The preparation method of the mulberry twig polysaccharide zinc complex according to claim 2, characterized in that, The dosage ratio of mulberry twig polysaccharide to water is 1 g: 20 mL.

4. The preparation method of the mulberry twig polysaccharide zinc complex according to claim 1, characterized in that, The concentration of the zinc salt solution is 30 - 60 mg / mL.

5. The preparation method of the mulberry twig polysaccharide zinc complex according to claim 1, characterized in that, The zinc salt solution is a zinc sulfate solution or a zinc chloride solution.

6. The preparation method of the mulberry twig polysaccharide zinc complex according to claim 1, wherein, The volume of the concentrated solution is 1 / 4 - 1 / 6 of the volume of the reaction solution.

7. The preparation method of the mulberry twig polysaccharide zinc complex according to claim 1, characterized in that, The ethanol is anhydrous ethanol; the volume dosage of anhydrous ethanol is 1 - 3 times the volume of the dialyzed solution.

8. The mulberry twig polysaccharide zinc complex prepared by the preparation method according to any one of claims 1 - 7.

9. Use of the mulberry twig polysaccharide zinc complex according to claim 8 in the preparation of a product having an antioxidant effect.

10. Use of the mulberry twig polysaccharide zinc complex according to claim 8 in the preparation of a product having an anti - inflammatory effect.

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