A kind of Artemisia seaweed polyphenol nanoparticles and preparation method thereof

Abstract

The present application discloses a kind of sargassum polyphenol nanoparticles and a preparation method thereof, and relates to the technical field of pharmaceutical preparation preparation. The prescription of the sargassum polyphenol nanoparticles comprises the following components by mass percentage: 1% to 20% of sargassum polyphenols, 10% to 35% of gelatin, 5% to 20% of chitosan, 30% to 70% of water, 0.025% to 0.2% of glacial acetic acid, and an appropriate amount of 10% sodium hydroxide aqueous solution. The sargassum polyphenol nanoparticles of the present invention can significantly improve the stability of sargassum polyphenols, and provide a dosage form selection for the application of sargassum polyphenols.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to sargassum seaweed polyphenol nanoparticles and a preparation method thereof. Background Art

[0002] Sargassum pallidum belongs to the Sargassum family of the Phaeophyta and is widely distributed in coastal areas such as Shandong and Liaoning in China. It is an edible and medicinal plant. Sargassum pallidum polyphenols are the representative active ingredients of Sargassum pallidum and have multiple pharmacological activities.

[0003] Polyphenol active compounds are very sensitive to oxygen, light and heat during use and storage, and have the characteristics of low boiling point, high volatility and poor stability. For the further application of polyphenols, these defects should be overcome. Gelatin nanoparticles (GNPs) were first reported in the 1980s. This drug delivery system has many advantages, including simple preparation process, enhanced drug stability, odor masking, controlled release rate, improved drug lipid solubility and water solubility, and effectively enhanced drug bioavailability.

[0004] At present, most marine natural products face great challenges in dosage form research due to their large molecular weight, poor stability, poor absorption, etc., and there are very few research reports. Therefore, researching and developing dosage forms and drug delivery systems for seaweed polyphenols has significant scientific and economic value. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a new artemisia seaweed polyphenol nanoparticle with good stability in view of the deficiencies of the prior art.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the aforementioned Artemisia seaweed polyphenol nanoparticles.

[0007] The technical problem to be solved by the present invention is achieved by the following technical solution. The present invention is a sea wormwood polyphenol nanoparticle, which is characterized by:

[0008] The nanoparticles include the following components in mass percentage: 1%-20% of sea wormwood polyphenol, 10%-35% of gelatin, 5%-20% of chitosan, 30%-70% of water, 0.025%-0.2% of glacial acetic acid and an appropriate amount of 10% sodium hydroxide aqueous solution.

[0009] A further preferred technical solution of the above-mentioned Artemisia seaweed polyphenol nanoparticles is that the purity of polyphenols in the Artemisia seaweed polyphenols is 50% to 100%.

[0010] The present invention also discloses a method for preparing artemisia seaweed polyphenol nanoparticles, the steps of which are as follows:

[0011] (1) Dissolve the prescribed amount of chitosan in 0.5% acetic acid solution at room temperature, stir overnight, and filter with a 0.45 μm filter membrane;

[0012] (2) dissolving the prescribed amount of Artemisia seaweed polyphenols in water at room temperature and filtering with a 0.45 μm filter membrane;

[0013] (3) mixing the sargassum polyphenol solution and the chitosan solution and setting aside;

[0014] (4) Dissolve the prescribed amount of gelatin in water at 40°C and filter through a 0.45 μm filter membrane;

[0015] (5) The solution obtained in step (3) is slowly injected into the gelatin solution, placed in a constant temperature magnetic stirrer, stirred at room temperature for 10 min at a speed of 300 to 800 rpm, the remaining water is added, and then the pH value is adjusted to 5.4, stirred for another 30 min, and then heated in constant temperature water at 60° C. for 10 to 30 min to obtain seaweed polyphenol nanoparticles.

[0016] Preferably, the concentration of the chitosan solution in step (1) is 0.25-3 mg / mL.

[0017] Preferably, the concentration of the Sargassum seaweed polyphenol solution in step (2) is 3 to 7 mg / mL.

[0018] Preferably, the concentration of the gelatin solution in step (4) is 1 to 5 mg / mL.

[0019] Preferably, the storage temperature of the Artemisia seaweed polyphenol nanoparticles prepared in step (5) is 4°C.

[0020] Beneficial Effects

[0021] (1) The present invention provides a formulation composition and preparation method of Artemisia seaweed polyphenol nanoparticles.

[0022] (2) The sargassum polyphenol nanoparticles prepared by this method can significantly improve the stability of sargassum polyphenol. DETAILED DESCRIPTION

[0023] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] Example 1

[0025] The preparation method of Artemisia seaweed polyphenols is as follows:

[0026] (1) freeze-dry fresh sea wormwood for 40 hours, cut it into pieces, put it into a grinder and pass it through a 60-mesh sieve to obtain sea wormwood powder; (2) accurately weigh a certain amount of sea wormwood powder, add 50% ethanol aqueous solution with a solid-liquid ratio of 1:35 (w / v), mix and soak thoroughly;

[0027] (3) extracting polyphenols using a numerically controlled ultrasonic cleaner, with an ultrasonic power of 500 W, an ultrasonic frequency of 40 kHz, an extraction time of 40 min, an extraction temperature of 65° C., filtering, and extracting the residue again under the same conditions, and filtering;

[0028] (4) The filtrates obtained from the two extractions were combined and concentrated by vacuum rotary evaporation at 40° C. to obtain a polyphenol extract of Artemisia seaweed.

[0029] (5) Using gallic acid as the standard, the polyphenol content was determined using the Folin phenol reagent colorimetric method according to GB / T8313-2008: 7.5 mg GAE / g dry seaweed.

[0030] (6) The crude extract of sea wormwood polyphenols was initially purified using macroporous resin LS-305A. The sample was loaded at a flow rate of 1.0 BV / h, allowed to stand for adsorption for 1 h, and impurities were washed away with 1 column volume of deionized water. The extract was eluted with 2.5 column volumes of 50% ethanol, and the eluate was collected and concentrated by vacuum rotary evaporation at 40°C. The pure powder of sea wormwood polyphenols was obtained by freeze-drying.

[0031] (7) The total polyphenols of Artemisia marinum purified by macroporous resin were accurately weighed, ultrasonically dissolved with a small amount of water, centrifuged, and the supernatant was loaded onto a Sephadex LH-20 chromatography column and eluted with water, 50% ethanol, and anhydrous ethanol, respectively. The eluates of each component were rotary evaporated and freeze-dried to obtain the refined powder of Artemisia marinum polyphenols. The purity of the Artemisia marinum polyphenols in the 50% ethanol component was the highest, at 70.5%.

[0032] Embodiments 2 to 4

[0033] The sargassum seaweed polyphenols extracted in Example 1 are used to prepare sargassum seaweed polyphenol nanoparticles.

[0034] Name of raw materials Example 2 Dosage Example 3 Dosage Example 4 Dosage Sargassum polyphenols 12g 6g 14g gelatin 24g 12g 28g Chitosan 12g 6g 14g water 51.88g 75.94g 43.86g acetic acid 0.12g 0.06g 0.14g 10% sodium hydroxide aqueous solution Moderate Moderate Moderate

[0035] The dosage of raw materials and auxiliary materials of Artemisia seaweed polyphenol nanoparticles is as shown in the above table, and the preparation process is as follows:

[0036] (1) Dissolve the prescribed amount of chitosan in 0.5% acetic acid solution at room temperature, stir overnight, and filter with a 0.45 μm filter membrane to prepare a concentration of 0.5 mg / ml;

[0037] (2) dissolving the prescribed amount of seaweed polyphenols in water at room temperature, filtering with a 0.45 μm filter membrane, and preparing a concentration of 4 mg / ml;

[0038] (3) mixing the sargassum polyphenol solution and the chitosan solution and setting aside;

[0039] (4) Dissolve the prescribed amount of gelatin in water at 40°C and filter with a 0.45 μm filter membrane to a concentration of 3 mg / ml;

[0040] (5) The solution obtained in step (3) was slowly injected into the gelatin solution, placed in a constant temperature magnetic stirrer, stirred at room temperature for 10 min at a speed of 500 rpm, the remaining amount of water was added, and then the pH value was adjusted to 5.4, stirred for another 30 min, and then heated in constant temperature water at 60° C. for 20 min to obtain seaweed polyphenol nanoparticles.

[0041] Example 5

[0042] The sargassum seaweed polyphenols extracted in Example 1 are used to prepare sargassum seaweed polyphenol nanoparticles.

[0043] Name of raw materials Dosage Sargassum polyphenols 12g gelatin 24g Chitosan 12g water 51.88g acetic acid 0.12g 10% sodium hydroxide aqueous solution Moderate

[0044] The dosage of raw materials and auxiliary materials of Artemisia seaweed polyphenol nanoparticles is as shown in the above table, and the preparation process is as follows:

[0045] (1) Dissolve the prescribed amount of chitosan in 0.5% acetic acid solution at room temperature, stir overnight, and filter with a 0.45 μm filter membrane to prepare a concentration of 0.5 mg / ml;

[0046] (2) dissolving the prescribed amount of seaweed polyphenols in water at room temperature, filtering with a 0.45 μm filter membrane, and preparing a concentration of 6 mg / ml;

[0047] (3) mixing the sargassum polyphenol solution and the chitosan solution and setting aside;

[0048] (4) Dissolve the prescribed amount of gelatin in water at 40°C and filter with a 0.45 μm filter membrane to a concentration of 3 mg / ml;

[0049] (5) The solution obtained in step (3) was slowly injected into the gelatin solution, placed in a constant temperature magnetic stirrer, stirred at room temperature for 10 min at a speed of 500 rpm, the remaining amount of water was added, and then the pH value was adjusted to 5.4, stirred for another 30 min, and then heated in constant temperature water at 60° C. for 20 min to obtain seaweed polyphenol nanoparticles.

[0050] Test example

[0051] Test content 1: Encapsulation efficiency

[0052] Take the prepared Artemisia seaweed polyphenol nanoparticle sample, centrifuge the solution at a speed of 12000r / min for 30min, take the supernatant after the centrifugation, and use an ultraviolet spectrophotometer to measure the absorbance value of the supernatant after centrifugation to calculate the content of Artemisia seaweed polyphenol in it. The encapsulation efficiency is calculated according to the following formula:

[0053] Encapsulation rate = (total amount of sargassum polyphenols input - amount of sargassum polyphenols in the supernatant after centrifugation) / total amount of sargassum polyphenols input × 100%

[0054] Table 1 Encapsulation efficiency of Examples 2-5

[0055] Group Example 2 Example 3 Example 4 Example 5 Encapsulation efficiency 80.64％ 63.63％ 55.10％ 73.29％

[0056] The prescriptions of Examples 2-4 are different, but the preparation methods are the same. The prescriptions of Example 2 and Example 5 are the same, except that the concentrations of the sargassum polyphenol solution are different during the preparation process. It can be seen from the test results of Examples 2 to 4 above that changes in the amounts of the main raw and auxiliary materials, sargassum polyphenol, gelatin, and chitosan, affect the encapsulation rate of the product, and the encapsulation rate of Example 2 is the highest, at 80.64%. It can be seen from the test results of Examples 2 and 5 that the concentration of the sargassum polyphenol solution during the preparation process affects the encapsulation rate of the product, and the encapsulation rate of the sample prepared in Example 2 with a concentration of 4 mg / ml is higher.

[0057] Test content 2: Particle size, polydispersity index (PDI) and Zeta potential

[0058] The particle size, PDI and Zeta potential of Artemisia seaweed polyphenol nanoparticles were analyzed by a particle size analyzer. The test results are shown in Table 2.

[0059] Table 2 Particle size, PDI and zeta potential of Examples 2-5

[0060] Group Example 2 Example 3 Example 4 Example 5 Particle size (nm) 30.63 70.68 91.20 35.17 PDI 0.183 0.243 0.252 0.195 Zeta potential (mV) 45.9 35.9 32.9 42.8

[0061] From the test results of Examples 2 to 4, it can be seen that changes in the amount of the main raw and auxiliary materials, sea wormwood polyphenols, gelatin, and chitosan in the prescription affect the particle size, PDI, and zeta potential values ​​of the product, among which Example 2 has the smallest particle size, the smallest PDI value, and the largest zeta potential value, indicating that the sample of Example 2 is the most stable. From the test results of Examples 2 and 5, it can be seen that the solution concentration of sea wormwood polyphenols during the preparation process affects the particle size, PDI, and zeta potential values ​​of the product, and the sample prepared in Example 2 with a concentration of 4 mg / ml exhibits higher stability.

[0062] Test content 3: Antioxidant stability study

[0063] From the above physical and chemical evaluation, it can be seen that the sample prepared in Example 2 has the highest encapsulation rate and the best stability, and the sample prepared in Example 2 is selected for the antioxidant stability study.

[0064] The ABTS method is currently the most commonly used method for measuring the antioxidant properties of compounds. It can not only indirectly test hydrophilic substances, but also lipophilic substances. After being oxidized, ABTS can form a blue-green ABTS cation with a maximum absorption wavelength of 405nm. The antioxidant component can react chemically with the ABTS cation, causing the reaction system to become lighter in color, and the absorbance decreases at the maximum absorption wavelength. The change in absorbance is positively correlated with the removal of free radicals. The sea wormwood polyphenols and sea wormwood polyphenol nanoparticles were prepared into solutions of the same concentration, and the oxidative stability of the products was tested according to the operating requirements of the kit. The test results are shown in Table 3.

[0065] Table 3 ABTS inhibitory activity of sargassum polyphenols and sargassum polyphenol nanoparticles at 4°C and 37°C (%)

[0066]

[0067] From the above test results, we can know that: (1) The ABTS inhibitory activity of wormwood polyphenols gradually decreases with the extension of time, and the inhibitory activity is very low after 12 hours, while the ABTS inhibitory activity of wormwood polyphenol nanoparticles gradually increases with the extension of time, basically reaching the optimal inhibitory activity at 8 hours, and can be stably maintained up to 36 hours; (2) Compared with 37°C, the ABTS inhibition rate of wormwood polyphenols at 4°C decreases more slowly, and the ABTS inhibition rate of wormwood polyphenol nanoparticles at 4°C is slightly higher.

[0068] It can be seen that nanoparticle technology can improve the antioxidant stability of Artemisia seaweed polyphenols, and storage at 4°C is more conducive to maintaining its activity.

[0069] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A kind of Artemisia seaweed polyphenol nanoparticles, characterized by: The nanoparticles include the following components in mass percentage: 1% to 20% of sea wormwood polyphenol, 10% to 35% of gelatin, 5% to 20% of chitosan, 30% to 70% of water, 0.025% to 0.2% of glacial acetic acid, and an appropriate amount of 10% sodium hydroxide aqueous solution; the preparation method of the sea wormwood polyphenol nanoparticles is as follows: (1) Dissolve the prescribed amount of chitosan in 0.5% acetic acid solution at room temperature, stir overnight, and filter with a 0.45 μm filter membrane; (2) dissolving the prescribed amount of Artemisia seaweed polyphenols in water at room temperature and filtering with a 0.45 μm filter membrane; (3) mixing the sargassum polyphenol solution and the chitosan solution and setting aside; (4) Dissolve the prescribed amount of gelatin in water at 40°C and filter through a 0.45 μm filter membrane; (5) The solution obtained in step (3) is slowly injected into the gelatin solution, placed in a constant temperature magnetic stirrer, stirred at room temperature at a speed of 300 to 800 rpm for 10 min, the remaining water is added, and then the pH value is adjusted to 5.4, stirred for another 30 min, and then heated in constant temperature water at 60° C. for 10 to 30 min to obtain seaweed polyphenol nanoparticles.

2. The sargassum seaweed polyphenol nanoparticles according to claim 1, characterized in that: The polyphenol purity in the sea wormwood polyphenol is 50% to 100%.

3. The method for preparing the Artemisia seaweed polyphenol nanoparticles according to any one of claims 1 to 2, characterized in that: The steps are as follows: (1) Dissolve the prescribed amount of chitosan in 0.5% acetic acid solution at room temperature, stir overnight, and filter with a 0.45 μm filter membrane; (2) dissolving the prescribed amount of Artemisia seaweed polyphenols in water at room temperature and filtering with a 0.45 μm filter membrane; (3) mixing the sargassum polyphenol solution and the chitosan solution and setting aside; (4) Dissolve the prescribed amount of gelatin in water at 40°C and filter through a 0.45 μm filter membrane; (5) The solution obtained in step (3) is slowly injected into the gelatin solution, placed in a constant temperature magnetic stirrer, stirred at room temperature for 10 minutes at a speed of 300 to 800 rpm, the remaining water is added, and then the pH value is adjusted to 5.4, stirred for another 30 minutes, and then heated in constant temperature water at 60° C. for 10 to 30 minutes to obtain seaweed polyphenol nanoparticles.

4. The method for preparing artemisia seaweed polyphenol nanoparticles according to claim 3, wherein the concentration of the chitosan solution in step (1) is 0.25-3 mg / ml.

5. The method for preparing sargassum polyphenol nanoparticles according to claim 3, wherein the concentration of the sargassum polyphenol solution in step (2) is 3-7 mg / ml.

6. The method for preparing artemisia seaweed polyphenol nanoparticles according to claim 3, wherein the concentration of the gelatin solution in step (4) is 1 to 5 mg / ml.

7. The method for preparing sargassum seaweed polyphenol nanoparticles according to claim 3, wherein the sargassum seaweed polyphenol nanoparticles prepared in step (5) are stored at a temperature of 4°C.

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