Elagic acid-pomegranate peel acidic polysaccharide compound as well as preparation method and application thereof

Abstract

The invention relates to an ellagic acid-pomegranate peel acidic polysaccharide compound as well as a preparation method and application thereof. The preparation method specifically comprises the following steps: mixing ellagic acid with a pomegranate rind acidic polysaccharide solution, and reacting for 0.5-2.0 hours under the weakly alkaline condition at the temperature of 15-35 DEG C to obtain an ellagic acid-pomegranate rind acidic polysaccharide compound solution; wherein the mass ratio of pomegranate peel polysaccharide to ellagic acid is (1: 1)-(10: 1). The ellagic acid-pomegranate rind acidic polysaccharide compound prepared by the method disclosed by the invention takes naturally extracted pomegranate rind acidic polysaccharide as a carrier, so that the stability of ellagic acid is improved, and technical guidance is provided for developing ellagic acid related nutrition fortified food and health care products.

Description

Technical Field

[0001] The invention relates to an ellagic acid complex, in particular to an ellagic acid-pomegranate peel acidic polysaccharide complex and a preparation method and application thereof. Background Art

[0002] Ellagic acid (EA) exists in plants in nature mainly in the form of condensed ellagitannins or glycosides. 14 H6O8. Ellagic acid is widely found in various plants, especially in fruits such as grapes, apples, strawberries, and pomegranates, and is distributed in all parts of pomegranates. It is known for its powerful antioxidant properties, which can neutralize free radicals and reduce oxidative stress, thereby protecting cells from damage. In addition, ellagic acid also shows anti-inflammatory, antibacterial and anti-cancer biological activities. Studies have shown that it can inhibit the growth of certain cancer cells and slow the development of tumors by regulating cell signaling pathways. These properties of ellagic acid make it a promising and preferred substance for the research and development of new drugs and health products. However, ellagic acid has poor solubility and low absorption efficiency in the gastrointestinal tract, and often requires intestinal flora to convert it into urolithic acid to exert its biological activity.

[0003] Pomegranate peel polysaccharide is a natural polysaccharide extracted from pomegranate peel. In recent years, it has attracted widespread attention due to its diverse biological activities. Pomegranate peel polysaccharide has a significant antioxidant effect, can remove free radicals in the body, and slow down cell aging. In addition, it also shows anti-inflammatory and immunomodulatory properties, which helps to reduce inflammatory responses and enhance the body's immune defense capabilities. In anti-cancer research, pomegranate peel polysaccharides have also been shown to inhibit the proliferation of certain cancer cells and fight against the development of tumors by affecting the cell cycle and apoptosis pathways. Xinjiang pomegranates are of excellent quality and high yield, and pomegranate planting has become a pillar industry in some parts of Xinjiang. In addition to fresh consumption, some pomegranates will be processed into products for sale, but the processing process will produce a large amount of pomegranate peel by-products. By extracting natural polysaccharides from pomegranate peels and obtaining purified pomegranate peel acidic polysaccharides, it is possible to achieve comprehensive utilization of pomegranate resources and improve the economic and industrial benefits of Xinjiang.

[0004] At present, there is no report at home or abroad on the technology of improving the solubility of ellagic acid and enhancing the bioavailability of ellagic acid by preparing ellagic acid-pomegranate peel polysaccharide complex. Summary of the invention

[0005] The object of the present invention is to provide an ellagic acid-pomegranate peel acid polysaccharide complex and a preparation method thereof, and specifically to provide a method for improving the solubility and utilization rate of ellagic acid and an ellagic acid-pomegranate peel acid polysaccharide complex prepared by the preparation method thereof. The preparation method provided by the present invention uses pomegranate peel polysaccharide as a carrier, improves the bioavailability of ellagic acid and exerts health activity, and belongs to the protection technology of functionally active small molecules.

[0006] The present invention provides a method for improving the solubility and utilization rate of ellagic acid, that is, a method for preparing an ellagic acid-pomegranate peel acidic polysaccharide complex, which specifically comprises: mixing an ellagic acid solution with a pomegranate peel acidic polysaccharide solution, reacting for 0.5 to 2.0 hours under neutral or alkaline conditions at a temperature of 15 to 35° C., to obtain an ellagic acid-pomegranate peel acidic polysaccharide complex solution;

[0007] Among them, the mass ratio of pomegranate peel acidic polysaccharide to ellagic acid is 1:1 to 10:1.

[0008] The mass ratio specifically refers to the mass ratio of pomegranate peel acidic polysaccharide to the ellagic acid solution in the ellagic acid solution and the pomegranate peel acidic polysaccharide solution during the reaction process.

[0009] The ellagic acid solution is ethanol (80%), and the solvent of the pomegranate peel acid polysaccharide solution is water.

[0010] Preferably, the obtained ellagic acid-pomegranate peel acidic polysaccharide complex solution can be further freeze-dried to prepare ellagic acid-pomegranate peel acidic polysaccharide complex powder for storage.

[0011] The present invention provides a method for improving the solubility of ellagic acid and enhancing its bioaccessibility. The pomegranate peel acidic polysaccharide interacts with ellagic acid to improve the properties of ellagic acid. By preparing an ellagic acid-pomegranate peel acidic polysaccharide complex, the two synergize to enable ellagic acid to exert health effects, providing ideas for developing ellagic acid-related health foods and nutritionally fortified foods.

[0012] Wherein, the pomegranate peel acidic polysaccharide is preferably prepared by the following method:

[0013] The crude polysaccharide solution of pomegranate peel is purified by DEAE cellulose-52 method to obtain pomegranate peel acidic polysaccharide, and the pomegranate peel acidic polysaccharide is collected;

[0014] Preferably, a 1-10 mg / mL pomegranate peel crude polysaccharide solution is purified by DEAE cellulose-52 method to obtain pomegranate peel acidic polysaccharide, and then freeze-dried to obtain pomegranate peel acidic polysaccharide.

[0015] The crude pomegranate peel polysaccharide is prepared by the following method: freezing the pomegranate peel at -20°C, freeze-drying and then crushing to obtain pomegranate peel powder; dissolving the pomegranate peel powder in ultrapure water, extracting by a composite enzyme extraction method, and then purifying to obtain a crude pomegranate peel polysaccharide solution.

[0016] Preferably, the following method is specifically adopted:

[0017] 1. Take the pomegranate peel without mold, freeze-dry it and crush it with a grinder;

[0018] 2. Dissolve the pomegranate peel powder in ultrapure water, extract it with pectinase and cellulase, and then perform purification operations such as defatting, deproteinizing, decolorizing and dialysis.

[0019] Preferably, the concentration of the pomegranate peel crude polysaccharide solution is 1 to 10 mg / mL.

[0020] The present invention adopts DEAE cellulose-52 method to classify and obtain neutral polysaccharide and acidic polysaccharide respectively.

[0021] The present invention further proposes that the concentration of the ellagic acid solution is 0 to 0.5 mg / mL; preferably 0.1 to 0.5 mg / mL; most preferably 0.1 mg / mL.

[0022] The present invention further proposes that the concentration of the pomegranate peel acidic polysaccharide solution is 0-1 mg / mL; preferably 0.1-0.8 mg / mL; most preferably 0.6 mg / mL.

[0023] The present invention further proposes that the reaction temperature is room temperature, such as 20-30°C, and particularly preferably 25°C.

[0024] The present invention further proposes that the pH value of the reaction is 7.0 to 8.0, preferably 7.0 to 7.5. The present invention preferably conducts the reaction under neutral or weakly alkaline conditions.

[0025] In the preparation method provided by the present invention, in addition to optimizing the reaction concentration and reaction conditions, it is found that when the mass ratio of the added pomegranate peel acidic polysaccharide to ellagic acid is 6, the effect of the obtained ellagic acid-pomegranate peel acidic polysaccharide complex is particularly remarkable. In the fluorescence titration experiment, as the amount of pomegranate peel acidic polysaccharide added in the system increases progressively, the fluorescence intensity of the ellagic acid emission peak is gradually quenched. The result shows that pomegranate peel acidic polysaccharide interacts with ellagic acid to form a non-fluorescent complex, so that its fluorescence is reduced. It is found through fitting that the binding site between pomegranate peel acidic polysaccharide and ellagic acid is 5.34 ± 0.38.

[0026] Since the molecular weight of pomegranate peel acidic polysaccharide is much greater than that of ellagic acid, and the concentration of pomegranate peel acidic polysaccharide is much less than that of ellagic acid at the same mass, and since ellagic acid has poor water solubility, the mass of ellagic acid in the complex is less than that of acidic pomegranate peel polysaccharide. In the present invention, the mass ratio of pomegranate peel acidic polysaccharide to ellagic acid is preferably 1:1 to 10:1.

[0027] Preferably, the mass ratio of pomegranate peel acidic polysaccharide to ellagic acid is 1:1 to 6:1, especially 6:1.

[0028] The present invention provides an optimization scheme, and the preparation method comprises the following steps:

[0029] 1) preparing a 0-0.5 mg / mL ellagic acid solution;

[0030] 2) preparing a pomegranate peel acidic polysaccharide solution with a concentration of 0 to 1 mg / mL;

[0031] 3) slowly dripping the ellagic acid solution into the pomegranate peel acidic polysaccharide solution, and slowly stirring the reaction for 0.5 to 2.0 hours at room temperature under neutral or weakly alkaline conditions to obtain an ellagic acid-pomegranate peel acidic polysaccharide complex solution;

[0032] Among them, the mass ratio of pomegranate peel acidic polysaccharide to ellagic acid is 1:1 to 6:1.

[0033] In the actual process, the stirring time can be adjusted according to the dissolution situation to ensure complete dissolution.

[0034] The ellagic acid-pomegranate peel acidic polysaccharide complex solution prepared by the above preparation method, according to the fluorescence spectrum results, was fitted to find that the binding site between pomegranate peel acidic polysaccharide and ellagic acid was 5.34±0.38. The ellagic acid-pomegranate peel acidic polysaccharide complex solution can be freeze-dried to obtain ellagic acid-pomegranate peel acidic polysaccharide complex powder.

[0035] The preparation method of the present invention further comprises filtering;

[0036] The filtration is specifically as follows: using a 0.45 μm disposable needle filter membrane (organic system) for purification and sterilization.

[0037] The purpose of filtration is not only purification, but also sterilization.

[0038] The ellagic acid-pomegranate peel acidic polysaccharide complex prepared by the method of the present invention is clear and transparent, has good antioxidant capacity, and the solubility and bioaccessibility of ellagic acid are significantly improved. Pomegranate peel acidic polysaccharide as a carrier can protect ellagic acid and promote ellagic acid to exert its own health effects. It provides theoretical and technical support and guidance for the development and application of ellagic acid nutritional fortification. In addition, this technology has high application prospects and social benefits for the development of relevant functional foods and health products using functional ingredients in pomegranate by-products and increasing the added value of the pomegranate industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the infrared spectrum of crude polysaccharide from pomegranate peel;

[0040] Figure 2 This is the UV spectrum of crude polysaccharide from pomegranate peel;

[0041] Figure 3 This is the high performance gel permeation chromatogram of pomegranate peel acidic polysaccharide;

[0042] Figure 4 This is the fluorescence titration diagram of pomegranate peel acidic polysaccharide against ellagic acid;

[0043] Figure 5 This is the fluorescence fitting diagram of pomegranate peel acidic polysaccharide to ellagic acid. DETAILED DESCRIPTION

[0044] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0045] To further illustrate the present invention, the ellagic acid-pomegranate peel acid polysaccharide complex, preparation method and application provided by the present invention are described in detail below in conjunction with examples.

[0046] The reagents used in the following examples are all commercially available.

[0047] The pomegranate peel acidic polysaccharide used in the following examples was prepared in the following manner:

[0048] 1) taking pomegranate peel without mildew, freeze-drying it and then crushing it with a crusher;

[0049] 2) Take 10g and dissolve it in 40mL ultrapure water, add pectinase and cellulase to extract, inactivate the enzyme in boiling water, decolorize with H2O2, precipitate with alcohol and then dialysis for purification; finally, obtain a pomegranate peel crude polysaccharide solution, and freeze-dry to obtain pomegranate peel crude polysaccharide.

[0050] 3) Purifying the crude polysaccharide solution of pomegranate peel by DEAE cellulose-52 method, adding the pomegranate peel polysaccharide solution into a chromatography column, eluting with 0-0.5 mol / L NaCl solution, determining the polysaccharide content in the collection tube, collecting the eluate with a higher polysaccharide content, concentrating by rotary evaporation, and dialyzing to obtain pomegranate peel acidic polysaccharide;

[0051] 4) freeze-drying the obtained polysaccharide solution to collect pomegranate peel acidic polysaccharide.

[0052] Example 1

[0053] This embodiment provides a method for preparing an ellagic acid-pomegranate peel acidic polysaccharide complex, which is as follows:

[0054] 1) preparing a 0.1 mg / mL ellagic acid solution; weighing 0.1 g of ellagic acid and dissolving it in ethanol (80%);

[0055] 2) preparing a pomegranate peel acidic polysaccharide solution with a concentration of 0.6 mg / mL;

[0056] 3) slowly adding the ellagic acid aqueous solution to the pomegranate peel acidic polysaccharide aqueous solution; slowly stirring the reaction, the reaction time is 0.5h, the reaction temperature is 25°C, the reaction pH value is 7.0, and the ellagic acid-pomegranate peel acidic sugar complex solution is obtained;

[0057] Among them, the mass ratio of pomegranate peel acidic polysaccharide to ellagic acid is 6:1.

[0058] Example 2

[0059] This example provides a method for preparing an ellagic acid-pomegranate peel acidic polysaccharide complex, which is different from Example 1 only in that the mass ratio of pomegranate peel acidic polysaccharide to ellagic acid is 4:1.

[0060] Example 3

[0061] This example provides a method for preparing an ellagic acid-pomegranate peel acidic polysaccharide complex, which is different from Example 1 only in that the mass ratio of pomegranate peel acidic polysaccharide to ellagic acid is 2:1.

[0062] Example 4

[0063] This example provides a method for preparing an ellagic acid-pomegranate peel acidic polysaccharide complex, which is different from Example 1 only in that the mass ratio of pomegranate peel acidic polysaccharide to ellagic acid is 1:1.

[0064] Comparative Example 1

[0065] In this comparative example, 0.1 g of ellagic acid was dissolved in ethanol (80%) to obtain an ellagic acid solution.

[0066] Experimental results:

[0067] 1. Characterization of pomegranate peel polysaccharides

[0068] 1.1FITR

[0069] Weigh 1 mg of pomegranate peel polysaccharide, mix and grind with a certain volume of dried KBr, and use a tablet press to make uniform slices. -1 Infrared scanning within range.

[0070] 1.2 UV scanning

[0071] Dissolve 10 mg of pomegranate peel polysaccharide in 10 mL of deionized water, measure the UV spectrum at 200 nm to 400 nm, and use deionized water as a blank.

[0072] 1.3 Molecular weight of pomegranate peel acidic polysaccharide

[0073] The sample was dissolved in ultrapure water, filtered through a 0.22 μm filter membrane, and then measured on a gel chromatography instrument (model ELEOS System). The measurement conditions were: Shodex OH-pakSB-806 column, Water515 pump, laser detector (LS) (Wyatt DAWNHELEOS-Ⅱ), differential detector (DRI) (Wyatt Optilab-rEX), injection volume 500 μL, mobile phase ultrapure water + 0.2 mol / L potassium dihydrogen phosphate + 0.02% sodium azide, flow rate 0.5 mL / min.

[0074] from Figure 1 Infrared spectra show that at 3421cm -1 A strong broad absorption peak at 2932 cm-1 appeared due to the stretching vibration of hydroxyl groups. -1 The weak absorption peak at about 1617cm is due to the stretching vibration of the methyl group. -1 The appearance of the asymmetric stretching vibration peak at 1423 cm -1 The weak characteristic absorption peak at 1078cm is attributed to the combined result of CH and OH bending vibration. -1 and 1024cm -1 The strong absorption is due to the stretching vibration of the C-OH side group and the vibration of the glycosidic bond, which is consistent with the infrared spectrum of pomegranate peel polysaccharide in the literature.

[0075] from Figure 2 It can be seen from the UV spectrum that the 1 mg / mL crude polysaccharide solution has no characteristic absorption peaks at 260 nm and 280 nm, indicating that the content of protein and nucleic acid substances is very low.

[0076] according to Figure 3 The molecular weight of crude polysaccharide from pomegranate peel was measured to be 282486Da by high performance gel permeation chromatography (HPGPC).

[0077] 2. Fluorescence titration experiment

[0078] Use DMSO to prepare ellagic acid into a 0.5 mg / mL solution, and use water to prepare pomegranate peel acid polysaccharide into a 10 mg / mL solution. Place 1 mL of ellagic acid solution in a quartz cuvette, add 10 μL of pomegranate peel acid polysaccharide solution for titration, the final concentration of ellagic acid is 0.5 mg / mL, and the final concentration of pomegranate peel acid polysaccharide is 0-0.6 mg / mL. Measure the fluorescence emission spectrum of the sample scanned between 419 and 800 nm at an excitation wavelength of 400 nm.

[0079] In the fluorescence titration experiment, along with the increasing amount of pomegranate peel acidic polysaccharide addition in the system, the fluorescence intensity of ellagic acid emission peak is gradually quenched, and the maximum emission wavelength is red-shifted. This result shows that pomegranate peel acidic polysaccharide and ellagic acid interact to form a non-fluorescent complex, so that its fluorescence is reduced. The maximum emission wavelength red-shift of fluorescence (i.e., moving to a longer wavelength) generally means that the electronic energy level of the fluorescent molecule has changed, which may be caused by intermolecular interactions. In this case, the interaction of polysaccharide and ellagic acid may change the electronic structure of ellagic acid, making its fluorescence emission wavelength longer. It is found through fitting that the binding constant between pomegranate peel acidic polysaccharide and ellagic acid is 5.34 ± 0.38 for the binding site.

[0080] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing an ellagic acid-pomegranate peel acidic polysaccharide complex, characterized in that: The ellagic acid solution and the pomegranate peel acidic polysaccharide solution are mixed, and reacted for 0.5 to 2.0 hours under neutral or weakly alkaline conditions at a temperature of 15 to 35° C. to obtain an ellagic acid-pomegranate peel acidic polysaccharide complex solution; Among them, the mass ratio of ellagic acid to pomegranate peel acidic polysaccharide is 1:1 to 1:

10.

2. The preparation method according to claim 1, characterized in that: The pomegranate peel acidic polysaccharide is prepared by the following method: a crude pomegranate peel polysaccharide solution is purified by DEAE cellulose-52 method to obtain the pomegranate peel acidic polysaccharide, and the pomegranate peel acidic polysaccharide is collected; Preferably, the following method is specifically adopted: A pomegranate peel crude polysaccharide solution with a concentration of 1-10 mg / mL was prepared, and the pomegranate peel acidic polysaccharide was purified by DEAE cellulose-52 method, and then freeze-dried to obtain the pomegranate peel acidic polysaccharide.

3. The preparation method according to claim 1 or 2, characterized in that: The concentration of the ellagic acid solution is 0 to 0.5 mg / mL, preferably 0.1 to 0.5 mg / mL.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The concentration of the pomegranate acid polysaccharide solution is 0-1.0 mg / mL, preferably 0.1-0.8 mg / mL.

5. The preparation method according to any one of claims 1 to 4, characterized in that: The reaction temperature is 20-30°C.

6. The preparation method according to claim 1 or 5, characterized in that: The pH value of the reaction is 7.0-8.

0.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The mass ratio of pomegranate peel acidic polysaccharide to ellagic acid is 10:1 to 1:1, preferably 6:1 to 1:

1.

8. The preparation method according to claim 1 or 2, characterized in that: The steps include: 1) preparing a 0.1-0.5 mg / mL ellagic acid solution; 2) preparing a pomegranate peel acidic polysaccharide solution with a concentration of 0.1 to 0.8 mg / mL; 3) slowly dripping the ellagic acid solution into the pomegranate peel acidic polysaccharide solution, and slowly stirring the reaction for 0.5 to 2.0 hours at room temperature under neutral or weakly alkaline conditions to obtain an ellagic acid-pomegranate acidic polysaccharide complex; The mass ratio of pomegranate peel acidic polysaccharide to ellagic acid is 1:1 to 6:1; Preferably, filtering is further included after the reaction.

9. The ellagic acid-pomegranate peel acidic polysaccharide complex solution prepared by the preparation method according to any one of claims 1 to 8 or the ellagic acid-pomegranate peel acidic polysaccharide complex solution prepared by the preparation method according to any one of claims 1 to 8 is freeze-dried to prepare the ellagic acid-pomegranate peel acidic polysaccharide complex.

10. Use of the ellagic acid-pomegranate peel acidic polysaccharide complex solution or the ellagic acid-pomegranate peel acidic polysaccharide complex according to claim 9 in nutritionally fortified foods, health products or medicines.

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