WO2022155792A1

WO2022155792A1 - Sodium alginate-based hydrogel loaded with lutein, and preparation method therefor and use thereof

Info

Publication number: WO2022155792A1
Application number: PCT/CN2021/072739
Authority: WO
Inventors: 宋江峰; 戴竹青; 徐鹏翔; 李大婧; 刘春泉; 冯蕾; 张钟元
Original assignee: 江苏省农业科学院
Priority date: 2021-01-19
Filing date: 2021-01-19
Publication date: 2022-07-28

Abstract

Provided are a sodium alginate-based hydrogel loaded with lutein, and a preparation method therefor and the use thereof. The sodium alginate-based hydrogel loaded with lutein comprises the following ingredients in parts by weight: 3.12-10.53 parts by weight of lutein, 46.8-47.7 parts by weight of chickpea protein isolate, 120-125 parts by weight of sodium alginate, 8.4-42 parts by weight of Ca2+-EGTA, and 7.12-35.6 parts by weight of D-gluconolactone. Further provided is a method for preparing the sodium alginate-based hydrogel loaded with lutein. The obtained sodium alginate-based hydrogel loaded with lutein has a lutein loading capacity of 760 μg/g and an encapsulation efficiency of 96% or more, and shows good drug loading performance and sustained release performance. Further provided is the use thereof in the preparation of a drug for preventing or treating diseases caused by inflammatory factor levels and intestinal microbial structures.

Description

[Title of invention formulated by ISA pursuant to Rule 37.2] Lutein-loaded sodium alginate-based hydrogel, and its preparation and use

technical field

The invention relates to the technical field of hydrogel preparation, in particular to a lutein-loaded sodium alginate-based hydrogel and a preparation method and application thereof.

Background technique

Lutein is a functional small molecule compound that is beneficial to human health, with potential effects such as anti-inflammatory, anti-cancer and improving cardiovascular disease. On the other hand, there have been many scientific studies proving the efficacy of lutein and zeaxanthin in preventing AMD, and studies have shown that a daily intake of 6 mg of lutein can reduce the prevalence of age-related macular degeneration by 57%. However, in the daily diet, the content of lutein in fruits, vegetables and some grains is relatively low, and the bioavailability rate is very low, it is difficult to ensure sufficient intake, resulting in the total amount of lutein entering the human body cannot be effective concentrations in the body. At present, the most common way of supplementation is through lutein dietary supplements. Dietary supplements with relatively high lutein content have been launched in the market, but lutein products that can effectively improve bioavailability are rarely seen. In recent years, the global market value of carotenoids has continued to grow, with a market value of US$1.4 billion in 2017 and is expected to reach US$2 billion in 2022, of which lutein accounts for 23% of the market share and lutein as a newly developed vitamin product market The prospects are very broad. Improving the absorption rate or bioavailability of lutein dietary supplements is a difficult and hot spot for current research in enterprises and academia. If lutein can maintain good stability, digestion and absorption in the body and controllable release characteristics, The dose of lutein ingested by the human body from the outside world is lower, which will be very cost-effective.

The dispersibility of lutein in the aqueous system is often increased by means of nanometerization, but external mechanical force or external factors such as strong acid and alkali will destroy the structure of the nanometer system, making it difficult to control the release behavior of lutein. Therefore, colloidal encapsulation systems such as liposomes, emulsions and gels were developed for the delivery of lutein. Among them, hydrogel carriers have advantages in improving the bioavailability of lipophilic active substances and controlling the release of active substances in the gastrointestinal tract. The network structure of sodium alginate hydrogel can be loaded with active substances. By adjusting the mechanical properties of the hydrogel and the concentration of cross-linking agent, the encapsulated substances in the hydrogel can be slowly released, and the hydrogel can respond to changes in pH. Controlled release of encapsulates. At present, there is no research or report on loading lutein in sodium alginate-based hydrogel. The present invention develops a sodium alginate-based hydrogel loaded with lutein with good stability and biocompatibility.

SUMMARY OF THE INVENTION

An object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages which will be explained later.

Another object of the present invention is to provide a lutein-loaded sodium alginate-based hydrogel, which fills the nano-encapsulated lutein from chickpea protein isolate into the sodium alginate hydrogel to obtain leaf The highest loading amount of flavin was 770.88μg/g, and the encapsulation efficiency reached 99.39%.

In order to achieve these objects and other advantages according to the present invention, a lutein-loaded sodium alginate-based hydrogel is provided, which comprises the following components in parts by weight: lutein 3.12-10.53 parts by weight, eagle 46.8-47.8 parts by weight of soybean protein isolate, 120-125 parts by weight of sodium alginate, 8.4-42 parts by weight of Ca2+-EGTA and 7.12-35.6 parts by weight of D-gluconolactone.

Preferably, the chickpea protein isolate and the lutein are prepared into a chickpea protein isolate lutein nanoparticle conjugate; the D-gluconolactone induces Ca ²⁺ -Ca ²⁺ in EGTA Release and in situ gelation of sodium alginate to form a cross-linked hydrogel, the chickpea protein isolate lutein nanoparticle conjugate is loaded in the hydrogel to prepare lutein-loaded sodium alginate-based water gel.

The invention uses sodium alginate, Ca ²⁺ -EGTA and D-gluconolactone as raw materials, and induces the release of Ca ²⁺ in Ca ²⁺ -EGTA through D-glucono lactone, and the formation of in situ gelation of sodium alginate. Cross-linking hydrogels, further loading chickpea protein isolate lutein nanoparticle conjugates in hydrogels to obtain lutein-loaded sodium alginate-based hydrogels to solve the ubiquitous stability of lutein Low, fast release of embedded material and other issues.

The present invention also provides a preparation method of the lutein-loaded sodium alginate-based hydrogel, which comprises the following steps:

Step (1) Weigh a certain amount of chickpea protein isolate sample in 10mM phosphate buffer solution, fully stir and then overnight at 4°C to fully hydrate the chickpea protein isolate to obtain a mass volume percentage of 1.0 % chickpea protein isolate solution;

Step (2) The chickpea protein isolate solution described in step (1) is stirred at room temperature for 2h in a magnetic stirrer with a stirring speed of 500rpm, and then homogenized for 1-3 times with a high-pressure microfluidizer with a pressure of 12000-15000psi , the homogenized chickpea protein isolate solution was refrigerated and centrifuged at 10,000 rpm for 10 min, and the supernatant was taken to obtain a chickpea protein isolate nano-solution;

Step (3) Add 5-15 mL of lutein ethanol solution dropwise to 100 mL of the chickpea protein isolate nano-solution described in step (2), and magnetically stir at 500 rpm for 20 min to make it fully mixed, and then reduce the temperature at 40 °C. pressure concentration to remove absolute ethanol, and then homogenize for 1-3 times with a pressure of 10000-15000psi high pressure microfluidizer to obtain a chickpea protein isolate lutein nanoparticle conjugate solution;

Step (4) Mix the chickpea protein isolate lutein nanoparticle conjugate solution prepared in step (3) with 25 mg/mL sodium alginate solution, stir magnetically evenly, and sequentially add 100 mM Ca ²⁺ -EGTA solution, The D-gluconolactone solution was placed at room temperature for 24 hours to completely gel the sodium alginate, and then the surface of the hydrogel was rinsed with deionized water to remove the unencapsulated chickpea protein isolate lutein nanoparticles. The particles are combined to obtain a lutein-loaded sodium alginate-based hydrogel.

Preferably, the mass volume concentration of lutein in the chickpea protein isolate lutein nanoparticle conjugate solution in the step (3) is 0.8-2.4 mg/mL.

Preferably, in the step (4), the molar ratio of Ca ²⁺ to D-gluconolactone is 1:2, and the molar concentration of Ca ²⁺ is 5.5-15 mM.

Preferably, the loading amount of lutein in the hydrogel can reach 760 μg/g, and the encapsulation efficiency is greater than 96%.

The present invention also provides the use of the lutein-loaded sodium alginate-based hydrogel in preparing a medicine for preventing or treating diseases caused by the level of inflammatory factors and the structure of intestinal microbes. The lutein-loaded sodium alginate-based hydrogel is beneficial for lutein to relieve intestinal inflammation by regulating the level of inflammatory factors and the structure of intestinal microbes.

Beneficial effects:

1. The lutein-loaded sodium alginate-based hydrogel of the present invention uses chickpea protein isolate nano-encapsulation to fill the lutein into the sodium alginate hydrogel, thereby realizing the loaded lutein The highest loading amount of lutein in the sodium alginate-based hydrogel was as high as 770.88 μg/g, and the encapsulation efficiency reached 99.39%. Structural characterization of the hydrogel by DSC and FTIR showed that the lutein nanoparticles were mainly physically filled in the hydrogel network structure, and the cross-linking of the lutein nanoparticles and excess Ca ²⁺ reduced the thermodynamic stability of the hydrogel sex.

2. The lutein-loaded sodium alginate-based hydrogel of the present invention realizes that the release rate of lutein in the gastric juice environment is close to 0, and the lutein is mainly released in the intestinal juice environment. In the pH≥6.8 environment, the increase of Ca ²⁺ concentration reduced the burst release of lutein in the hydrogel, while during the simulated digestion process, the digestive enzyme caused a rapid decrease in the surface structure of the hydrogel, and the higher the concentration of Ca ²⁺ cross-linked water The high content of lutein retained on the surface of the gel resulted in a significant burst release effect of lutein. Ca ²⁺ with a concentration of more than 7.5 mM affected the formation of lutein mixed micelles and reduced the bioavailability of lutein. The bioavailability of lutein was up to 30% after LAH-7.5 hydrogel digestion.

3. The lutein-loaded alginate-based hydrogel of the present invention maintains the integrity of the intestinal barrier by reducing fecal heme content and colon tissue damage, increasing the expression of intestinal tight junction protein, and inhibiting the NF-κB pathway. , decreased the expression and secretion of inflammatory factors such as TNF-α, iNOS, NLRP3 and IL-1β, and alleviated the mouse experiments induced by sodium dextran sulfate by down-regulating Desulfovibrionaceae and up-regulating the relative abundance of Erysipelotrichaceae and Rikenellaceae. Colitis. In addition, the adhesion and controlled release properties of the hydrogel carrier prolonged the residence time of lutein in the colon and greatly improved the alleviation of colitis symptoms in mice.

Other advantages, objects, and features of the present invention will appear in part from the description that follows, and in part will be appreciated by those skilled in the art from the study and practice of the invention.

Description of drawings

Fig. 1 shows the loading amount of lutein in the lutein-loaded sodium alginate-based hydrogel described in various embodiments of the present invention;

Fig. 2 shows the encapsulation efficiency of lutein in the lutein-loaded sodium alginate-based hydrogel described in various embodiments of the present invention;

Fig. 3 shows the infrared spectrogram of the lutein-loaded sodium alginate-based hydrogel of the present invention;

Fig. 4 shows the infrared spectrum of blank sodium alginate-based hydrogel;

Fig. 5 shows the bioavailability of lutein after in vitro simulated digestion of the lutein-loaded sodium alginate-based hydrogel in various embodiments of the present invention;

Figure 6 shows a comparison diagram of the content of inflammatory factor interferon-γ (IFN-γ) in the serum of mice with different treatments in the present invention;

Figure 7 shows the comparison diagram of the content of inflammatory factor tumor necrosis factor-α (TNF-α) in the serum of mice with different treatments in the present invention;

Figure 8 shows a comparison diagram of the inflammatory factor monocyte chemoattractant protein-1 (MCP-1) content in the serum of mice with different treatments in the present invention;

Figure 9 shows the comparison diagram of the content of inflammatory factor interleukin-6 (IL-6) in the serum of mice with different treatments in the present invention;

Figure 10 shows a comparison diagram of the content of inflammatory factor interleukin-1β (IL-1β) in the serum of mice with different treatments in the present invention;

Figure 11 shows a graph showing the comparison of the expression levels of the gene TNF-α in the colon tissue of mice by different treatments in the present invention;

Figure 12 shows a graph showing the comparison of the expression levels of the gene IL-1β in mouse colon tissue by different treatments in the present invention;

Figure 13 shows a graph showing the comparison of the expression levels of the gene IL-6 in the colon tissue of mice with different treatments in the present invention;

Figure 14 shows a graph showing the comparison of the expression levels of the gene IFN-γ in the colon tissue of mice by different treatments in the present invention;

Figure 15 shows a graph showing the comparison of the expression levels of genes iNOS (inducible nitric oxide synthase) in mouse colon tissue with different treatments in the present invention;

Figure 16 shows a graph comparing the expression levels of the gene COX-2 (cyclooxygenase-2) in mouse colon tissue by different treatments in the present invention;

Figure 17 shows a graph showing the comparison of the expression levels of the gene MCP-1 in mouse colon tissue by different treatments in the present invention;

Figure 18 shows a graph showing the comparison of the expression levels of the gene TLR-4 (TOLL-like receptor-4) in mouse colon tissue by different treatments in the present invention;

Figure 19 shows a graph comparing the expression levels of the gene NLRP3 (NOD-like receptor 3) in mouse colon tissue by different treatments in the present invention;

Fig. 20 shows the expression level comparison diagram of gene ZO-1 in mouse colon tissue by different treatments in the present invention;

Figure 21 shows a graph showing the comparison of the expression levels of the gene Occludin in mouse colon tissue by different treatments in the present invention;

Figure 22 shows a graph of the expression level comparison of gene Claudin-1 in mouse colon tissue by different treatments in the present invention;

Figure 23 shows a graph comparing the expression levels of the gene GPR41 (G protein-coupled receptor 41) in mouse colon tissue by different treatments in the present invention;

Figure 24 shows a graph comparing the expression levels of the gene GPR43 (G protein-coupled receptor 43) in mouse colon tissue by different treatments in the present invention;

Figure 25 shows the distribution characteristics of the intestinal flora of mice based on the phylum level of different treatments of the present invention;

Figure 26 shows the family-level distribution characteristics of the intestinal flora of mice with different treatments of the present invention.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it with reference to the description.

It should be understood that terms such as "having", "comprising" and "including" as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

The present invention will be further elaborated below in conjunction with the embodiments.

Example 1

The preparation method of the lutein-loaded sodium alginate-based hydrogel comprises: weighing 1.2 g of a chickpea protein isolate sample into 120 mL of a 10 mM phosphate buffer solution, fully stirring, and keeping it overnight at 4°C, The chickpea protein isolate was fully hydrated to obtain a chickpea protein isolate solution with a mass volume percentage of 1%. The chickpea protein isolate solution was stirred at room temperature for 2 h in a magnetic stirrer with a stirring speed of 500 rpm, and then homogenized twice with a high-pressure microfluidizer with a pressure of 12,000 psi. The homogenized chickpea protein isolate solution was refrigerated and centrifuged at 10,000 rpm. After 10 min, the supernatant was taken to obtain the chickpea protein isolate nano-solution. To 100 mL of chickpea protein isolate nanosolution, 15 mL of lutein ethanol solution with a mass volume concentration of 16 mg/mL was added dropwise, and stirred magnetically at 500 rpm for 20 min to make it fully mixed, and then concentrated under reduced pressure at 40 °C Remove absolute ethanol, and then homogenize for 3 times with a pressure of 12000 psi high pressure microfluidizer to obtain a chickpea protein isolate lutein nanoparticle conjugate solution.

Take 3.9 mL of the prepared chickpea protein isolate lutein nanoparticle conjugate solution and mix it with 0.5 mL of sodium alginate solution with a concentration of 25 mg/mL, and then add 0.275 mL of 100 mM Ca ²⁺ -EGTA solution, 0.1 mL D-gluconolactone solution, so that the molar ratio of Ca ²⁺ to D-glucono lactone is 1:2, and the molar concentration of Ca ²⁺ is 5.5mM, add water to the volume of 5mL, and leave it at room temperature for 24h to make the seaweed After the sodium gelation was complete, the surface of the hydrogel was rinsed with deionized water to remove the unencapsulated chickpea protein isolate lutein nanoparticle conjugate to obtain lutein-loaded sodium alginate-based water gel. The loading amount of lutein in the hydrogel can reach 760.45 μg/g, and the encapsulation efficiency can reach 97.47%.

Example 2

The preparation method of the lutein-loaded sodium alginate-based hydrogel comprises: weighing 1.2 g of a chickpea protein isolate sample into 120 mL of a 10 mM phosphate buffer solution, fully stirring, and keeping it overnight at 4°C, The chickpea protein isolate was fully hydrated to obtain a chickpea protein isolate solution with a mass volume percentage of 1%. The chickpea protein isolate solution was stirred at room temperature for 2 h in a magnetic stirrer with a stirring speed of 500 rpm, and then homogenized once with a high-pressure microfluidizer with a pressure of 15,000 psi, and the homogenized chickpea protein isolate solution was refrigerated and centrifuged at 10,000 rpm. After 10 min, the supernatant was taken to obtain the chickpea protein isolate nano-solution. To 100 mL of chickpea protein isolate nano-solution, 10 mL of lutein ethanol solution with a mass volume concentration of 20 mg/mL was added dropwise, and magnetically stirred at 500 rpm for 20 min to make it fully mixed, and then concentrated under reduced pressure at 40 °C Remove absolute ethanol, and then homogenize twice with a pressure of 15000 psi high pressure microfluidizer to obtain a chickpea protein isolate lutein nanoparticle conjugate solution.

Take 3.9 mL of the prepared chickpea protein isolate lutein nanoparticle conjugate solution and mix it with 0.5 mL of sodium alginate solution with a concentration of 25 mg/mL, and then add 0.375 mL of 100 mM Ca ²⁺ -EGTA solution, 0.1 mL D-gluconolactone solution, so that the molar ratio of Ca ²⁺ to D-glucono lactone is 1:2, the molar concentration of Ca ²⁺ is 7.5mM, add water to the volume of 5mL, and leave it at room temperature for 24h to make the seaweed After the sodium gelation was complete, the surface of the hydrogel was rinsed with deionized water to remove the unencapsulated chickpea protein isolate lutein nanoparticle conjugate to obtain lutein-loaded sodium alginate-based water gel. The loading amount of lutein in the hydrogel can reach 770.88 μg/g, and the encapsulation efficiency can reach 99.39%.

Example 3

The preparation method of the lutein-loaded sodium alginate-based hydrogel comprises: weighing 1.2 g of a chickpea protein isolate sample into 120 mL of a 10 mM phosphate buffer solution, fully stirring, and keeping it overnight at 4°C, The chickpea protein isolate was fully hydrated to obtain a chickpea protein isolate solution with a mass volume percentage of 1%. The chickpea protein isolate solution was stirred at room temperature for 2 h in a magnetic stirrer with a stirring speed of 500 rpm, and then homogenized twice with a high-pressure microfluidizer with a pressure of 12,000 psi. The homogenized chickpea protein isolate solution was refrigerated and centrifuged at 10,000 rpm. After 10 min, the supernatant was taken to obtain the chickpea protein isolate nano-solution. To 100 mL of chickpea protein isolate nano-solution, 10 mL of lutein ethanol solution with a mass volume concentration of 20 mg/mL was added dropwise, and magnetically stirred at 500 rpm for 20 min to make it fully mixed, and then concentrated under reduced pressure at 40 °C Remove absolute ethanol, and then homogenize once again with a high pressure microfluidizer at a pressure of 10,000 psi to obtain a chickpea protein isolate lutein nanoparticle conjugate solution.

Take 3.9 mL of the prepared chickpea protein isolate lutein nanoparticle conjugate solution and mix it with 0.5 mL of sodium alginate solution with a mass volume concentration of 25 mg/mL, and then add 0.5 mL of 100 mM Ca ²⁺ -EGTA solution, 0.1 mL D-gluconolactone solution, so that the molar ratio of Ca ²⁺ to D-glucono lactone is 1:2, the molar concentration of Ca ²⁺ is 10mM, add water to the volume of 5mL, and leave it at room temperature for 24h to make the alginic acid After the sodium gelation was complete, the surface of the hydrogel was rinsed with deionized water to remove the unencapsulated chickpea protein isolate lutein nanoparticle conjugate to obtain a lutein-loaded sodium alginate-based hydrogel. glue. The loading amount of lutein in the hydrogel can reach 765.36 μg/g, and the encapsulation efficiency can reach 96.64%.

Example 4

The preparation method of the lutein-loaded sodium alginate-based hydrogel comprises: weighing 1.2 g of a chickpea protein isolate sample into 120 mL of a 10 mM phosphate buffer solution, fully stirring, and keeping it overnight at 4°C, The chickpea protein isolate was fully hydrated to obtain a chickpea protein isolate solution with a mass volume percentage of 1%. The chickpea protein isolate solution was stirred at room temperature for 2 h in a magnetic stirrer with a stirring speed of 500 rpm, and then homogenized 3 times with a high-pressure microfluidizer with a pressure of 15,000 psi. The homogenized chickpea protein isolate solution was refrigerated and centrifuged at 10,000 rpm. After 10 min, the supernatant was taken to obtain the chickpea protein isolate nano-solution. 10 mL of lutein ethanol solution with a mass volume concentration of 16 mg/mL was added dropwise to 100 mL of chickpea protein isolate nano-solution, and stirred magnetically at 500 rpm for 20 min to make it fully mixed, and then concentrated under reduced pressure at 40 °C Remove absolute ethanol, and then homogenize twice with a pressure of 12000 psi high-pressure microfluidizer to obtain a chickpea protein isolate lutein nanoparticle conjugate solution.

Take 3.9 mL of the prepared chickpea protein isolate lutein nanoparticle conjugate solution and mix it with 0.5 mL of sodium alginate solution with a mass/volume concentration of 25 mg/mL, and then add 0.75 mL of 100 mM Ca ²⁺ -EGTA solution, 0.1 mL D-gluconolactone solution, so that the molar ratio of Ca ²⁺ to D-glucono lactone is 1:2, the molar concentration of Ca ²⁺ is 15mM, add water to a volume of 5mL, and leave it at room temperature for 24h to make the alginic acid After the sodium gelation was complete, the surface of the hydrogel was rinsed with deionized water to remove the unencapsulated chickpea protein isolate lutein nanoparticle conjugate to obtain a lutein-loaded sodium alginate-based hydrogel. glue. The loading amount of lutein in the hydrogel can reach 767.84 μg/g, and the encapsulation efficiency can reach 97.10%.

Example 5

The preparation method of the lutein-loaded sodium alginate-based hydrogel comprises: other steps are the same as above, wherein, taking 3.9 mL and 0.5 mL of the prepared chickpea protein isolate lutein nanoparticle conjugate solution with a mass volume concentration of 0.5 mL Mix 25mg/mL sodium alginate solution evenly, add 0.2mL 100mM Ca ²⁺ -EGTA solution and 0.1mL D-gluconolactone solution in sequence, so that the molar ratio of Ca ²⁺ to D-glucono lactone is 1 : 2, the molar concentration of Ca ²⁺ is 4.0mM, add water to a volume of 5mL, and leave it at room temperature for 24h to make the sodium alginate gel completely, and then rinse the hydrogel surface with deionized water to remove the unencapsulated water. The chickpea protein isolate lutein nanoparticle conjugates to obtain lutein-loaded sodium alginate-based hydrogels.

Example 6

The preparation method of the lutein-loaded sodium alginate-based hydrogel comprises: other steps are the same as above, wherein, taking 3.9 mL and 0.5 mL of the prepared chickpea protein isolate lutein nanoparticle conjugate solution with a mass volume concentration of 0.5 mL Mix 25mg/mL sodium alginate solution evenly, add 1.0mL 100mM Ca ²⁺ -EGTA solution and 0.1mL D-gluconolactone solution in sequence, so that the molar ratio of Ca ²⁺ to D-glucono lactone is 1 : 2, the molar concentration of Ca ²⁺ is 20mM, add water to a volume of 5mL, and leave it at room temperature for 24h to make the sodium alginate gel completely, and then rinse the surface of the hydrogel with deionized water to remove the unencapsulated Chickpea isolate protein lutein nanoparticle conjugates to obtain lutein-loaded sodium alginate-based hydrogels.

Comparative ratio

The blank sodium alginate-based hydrogels cross-linked with different concentrations of Ca2+ were prepared, wherein the molar concentrations of Ca2 ⁺ were 4.0 mM, 5.5 mM, 7.5 mM, 10 mM, 15 mM and 20 mM in sequence.

Based on the above examples, the lutein-loaded sodium alginate-based hydrogel of the present invention is analyzed below.

1. By extracting and measuring the loading amount and encapsulation rate of lutein in the hydrogel system, the results are shown in Figure 1 and Figure 2. In Figure 1 and Figure 2, AH4.0, AH5.5, AH7.5, AH10, AH15, AH20 represent blank sodium alginate-based hydrogels cross-linked with different concentrations of Ca2+ in the comparative example, LAH4.0, LAH5. 5. LAH7.5, LAH10, LAH15, and LAH20 represent the lutein-loaded sodium alginate-based hydrogels cross-linked with different concentrations of Ca2+ corresponding to each example. It can be seen from Figure 1 that the loading of lutein in the hydrogel can reach up to 770.88 μg/g. With the increase of Ca ²⁺ concentration, the loading of lutein shows an increasing trend, but different concentrations of Ca 2+ cross The difference in loading between the linked hydrogels did not reach a significant level (p﹥0.05). It can be seen from Figure 2 that the encapsulation rate of lutein also maintains a high level, up to 99.39%. Therefore, the lutein nanoparticles can be effectively filled into the hydrogel within the research range.

2. Hydrogel FTIR Analysis

The possible interactions between the blank control hydrogel LP and the sodium alginate hydrogel were analyzed by FTIR assay, and the results are shown in Figures 3 and 4. In the infrared spectrum of the blank control, the characteristic peak of lutein at 1660-1600cm-1 representing the C=C stretching vibration was detected at 1651cm-1, and the paper showed that the absorption band near 1532cm-1 was the stretching of C-C in the benzene ring. formed by extensional vibration, but disappeared in the blank control LP, indicating that the lutein molecule was encapsulated by chickpea protein isolate. The peak at 1038 cm-1 is assigned to -CH=CH- (trans-conjugated olefin), the absorption peak of free -OH at 3336 cm-1, and the absorption peak of -OH vibration at 2958 cm-1. The absorption peaks at 2917cm-1 and 2849cm-1 represent the asymmetric and symmetric stretching vibrations of -CH2 and -CH3, respectively. The absorption peak of the scissor vibration of -CH2 at 1435cm-1 shows that the absorption peak at 1363cm-1 It is formed by the cleavage of the dimethyl group. The absorption peak at 1038 cm-1 represents -CH- in the plane, and the absorption peak at 962 cm-1 is the deformation vibration in the -CH=CH- plane. The above characteristic peaks are consistent with the chemical structure of lutein. Therefore, the -NH2 stretching vibration of chickpea protein isolate has a strong absorption peak at 3270cm-1, the change of this peak in lutein nanoparticles, and the broadening of the absorption peak of lutein free-OH, indicating that leaf The hydroxyl and free amino groups of flavin may have reacted. Amide I and II are the two most prominent vibrational bands in the protein backbone. Amide I is caused by the stretching vibration of C-O and C-N, while amide II is formed by the bending vibration of N-H and the stretching vibration of C-N. In the infrared spectrum of lutein nanoparticles, the intensities of the absorption peaks of amide I (1750-1600cm-1) and amide II (1550-1510cm-1) were significantly reduced, indicating the interaction between lutein and chickpea protein isolate. The effect is mainly electrostatic interaction.

As shown in Figure 3, taking AH-7.5 as an example, the absorption peak at 1025cm-1 represents the stretching vibration of C-O, and this characteristic peak is the absorption peak shared by polysaccharides, which is closely related to galacturonic acid and paleo in AH Llucuronic acid related. The broad absorption peak at 3246 cm-1 represents the asymmetric stretching vibration of -OH in the polysaccharide. The absorption peaks at 1593 cm-1 and 1404 cm-1 represent the symmetric and asymmetric stretching vibrations of AH's characteristic COO-, respectively. The absorption peaks at 1081 cm-1 and 815 cm-1 represent the symmetric stretching vibration of C-O-C, and the absorption peak at 1025 cm-1 represents the inverse symmetric stretching vibration of C-O-C. The absorption peak at 2927 cm-1 represents the stretching vibration of C-H in the methylene group. The absorption peak at 1304 cm-1 represents the stretching vibration of C=O in the carboxyl group. As the concentration of Ca2+ increased from 4.0mM to 20mM, the absorption peak at 1304cm-1 shifted to the high wavenumber direction, and shifted from 1296cm-1 to 1318cm-1, indicating that with the increase of Ca2+ concentration, the concentration of Ca2+ and sodium alginate in the The Na+ substitution effect is strengthened.

As shown in Figure 4, taking LAH-7.5 as an example, the absorption peaks of C-O, -OH, COO-, C-O-C, C-H and C=O are consistent with the AH infrared spectrum. Compared with AH-7.5, the absorption band representing the C=O stretching vibration is shifted towards higher wavenumber. A new absorption peak appeared at 2988cm-1, which was not shown in AH-4.0 and AH-5.5, and appeared in AH-7.5, AH-10, AH-15 and AH-20. Compared with LP, the peak shape of LAH at 3240 cm-1 is broader, and most of the characteristic peaks associated with LP are not shown in the LAH infrared spectrum. The above results indicated that the sodium alginate hydrogel successfully achieved the encapsulation of lutein nanoparticles.

3. The bioavailability of lutein was experimentally analyzed, and the lutein-loaded sodium alginate hydrogel of the present invention had an effect on the content of inflammatory factors in mouse serum, the expression level of related genes in colon tissue, and the effect of intestinal flora. Compositional Impact

3.1 Select 60 SPF grade 6-week-old male C57BL/6Cnc mice to freely eat and drink water to adapt to the rearing environment for a week, and then randomly divide the mice into 6 groups according to body weight, which are marked as the control group with normal feed and drinking water (BLK group), respectively. 1.5% dextran sodium sulfate solution treatment group (DSS group), lutein gavage group (LUT group), lutein nanoparticle gavage group (LP group), blank hydrogel gavage group (AH group) and lutein-loaded hydrogel gavage group (LAH group). Lutein preparations interfered with the growth of mice throughout the whole process. Each mouse was given intragastric administration once a day. The intragastric dose was determined by 20 mg/kg bw of lutein. The intragastric dose of the AH group was referred to that of the LAH group. The DSS intervention was started on the 8th day of the formal experiment. Except for the BLK group, 1.5% DSS was added to the drinking bottle of the mice in the other groups. During the induction of colitis, the feces and intestinal bleeding of the mice were observed to determine the stop time of DSS. In order to reduce the experimental error, the same gavage operation was performed on the BLK group and the DSS group with distilled water instead of the gavage every day. Mice were treated for a total of 17 days depending on the feeding situation. During the feeding period of the mice, the body weight, food intake, water intake and solid stool quality of the mice were recorded every day, and the content of inflammatory factors in the serum of the mice, the expression levels of related genes in the colon tissue, and the composition of the intestinal flora were analyzed.

3.2 The bioavailability of lutein in different treatments after simulated digestion in vitro was determined, and the results are shown in Figure 5. In the simulated small intestine and colon stages in vitro, the lutein in the lutein-loaded hydrogel gavage group (LAH group) was continuously released with the stretching and degradation of the hydrogel network structure. The released lutein must be dissolved in mixed micelles before it can be absorbed by intestinal cells, and the bioavailable rate of lutein usually refers to the content dissolved in mixed micelles of intestinal digesta. As shown in Fig. 5, the bioavailability of lutein in nanoparticles was 21%, while the bioavailability of lutein in LAH-7.5 was up to 30%, and the hydrogel carrier improved the bioavailability of lutein Rate. However, with the increase of Ca2+ concentration, the bioavailability of lutein gradually decreased, so the hydrogel with excessive cross-linking degree during the digestion process had a negative impact on the bioavailability of lutein.

3.3 The effects of different treatments on inflammatory factors in mouse serum were determined, and the results are shown in Figures 6-10. As can be seen from the figure, DSS treatment increased serum interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin- 6 (IL-6) and interleukin-1β (IL-1β) content. Compared with DSS group, LUT significantly decreased IFN-γ and IL-6 contents, LP significantly decreased IFN-γ, TNF-α and IL-6 contents, AH significantly decreased IFN-γ, JEMCP-1 and IL-6 -6 content (p<0.05). LAH significantly reduced the content of all the above-mentioned inflammatory factors (p<0.05), and there was no significant difference between the levels of inflammatory factors in the BLK group (p>0.05). Compared with LUT, LAH significantly inhibited JEMCP-1 and TNF-α content (p<0.05). Compared with LP, LAH significantly inhibited IFN-γ, IL-6 and MCP-1 content (p<0.05). Compared with AH, LAH significantly decreased the levels of IFN-γ, IL-1β and TNF-α (p<0.05). This indicates that there may be a synergistic effect between sodium alginate hydrogel and lutein nanoparticles to reduce the level of inflammatory factors in serum.

3.4 Determination of the expression levels of colon tissue-related genes in mice by different treatments, and the mRNA expression levels of colon tissue-related genes in mice by qPCR, including TNF-α, IL-1β, IL-6, IFN-γ, iNOS (inducible Nitric oxide synthase), COX-2 (cyclooxygenase-2), MCP-1, TLR-4 (TOLL-like receptor-4), NLRP3 (NOD-like receptor 3), ZO-1, Occludin, Claudin-1, GPR41 (G protein coupled receptor 41) and GPR43 (G protein coupled receptor 43), the results are shown in Figures 11-24. As shown in Figure 11-Figure 19, compared with the BLK group, the expression levels of inflammatory factors TNF-α, IL-1β, IFN-γ, iNOS and COX-2 in the group treated with DSS on days 8-14 of the experiment were all significant increased (p<0.05). The DSS group significantly increased the levels of all inflammatory factors (p<0.05), including TNF-α, IL-1β, IL-6, IFN-γ, iNOS, COX-2, MCP-1, TLR-4 and NLRP3. Compared with DSS group, LUT group significantly inhibited the mRNA expression levels of IL-6, IFN-γ, iNOS, TLR-4 and NLRP3; LP group significantly inhibited TNF-α, IL-6, IFN-γ, COX-2, MCP-1, TLR-4 and NLRP3 mRNA expression levels; AH group only significantly inhibited IL-6, IFN-γ and NLRP3 mRNA expression levels; LAH group significantly inhibited TNF-α, IL-1β, IL-6, IFN-γ , iNOS, TLR-4 and NLRP3 mRNA expression levels, indicating that the presence of lutein in the preparation plays a role in inhibiting the expression of inflammation-related genes. Compared with the LUT group, the LP group significantly decreased the mRNA expression levels of IL-1β, IL-6, IFN-γ, MCP-1 and NLRP3; 6. The mRNA expression levels of IFN-γ, iNOS, MCP-1 and TLR-4. Therefore, lutein nanoparticles help lutein to play a role in inhibiting the expression level of inflammatory factors. The filling of lutein nanoparticles in sodium alginate hydrogel significantly improved the effect of lutein on inhibiting the expression level of inflammatory factors, but there was no significant difference.

As shown in Figures 20-24, compared with BLK, the DSS group significantly suppressed the mRNA expression levels of intestinal tight junction proteins ZO-1, Occludin and Claudin-1. Compared with the DSS group, the LAH group significantly increased the mRNA expression levels of ZO-1 and Claudin-1 and the expression of short-chain fatty acid receptor genes GRP41 and GRP43 genes. In conclusion, lutein-loaded sodium alginate hydrogels modulate mouse colonic inflammatory response by modulating the mRNA expression of the gut tight junction protein gene.

3.5 According to the species annotation results, sort the species at the phylum level of each sample in descending order of relative abundance, and draw the first 10 species at the phylum level to obtain the distribution characteristics of microorganisms in mouse feces. As shown in Figure 25, the fecal flora of each mouse is mainly composed of Actinobacteria, Bacteroidetes, Candidatus_Saccharibacteria, Cyanobacteria/Chloroplast, Deferribacteres, Firmicutes, Proteobacteria, Tenericutes, Verrucomicrobia and Unassigned are composed of 10 parts. The main dominant flora were Bacteroidetes and Firmicutes, and a small amount of Proteobacteria, which accounted for more than 90% of the fecal flora. Compared with the BLK group, the DSS group significantly increased the relative abundance of Verrucomicrobia in the fecal microbiota. Compared with the DSS group, the relative abundance of Verrucomicrobia in the AH group was further increased, accounting for 7.45%, which was greater than that in the other groups. The relative abundance of Proteobacteria in the LAH group, LUT group and AH group was significantly smaller than that in the DSS group. Compared with the DSS group, there was no significant difference in the remaining dominant flora (p>0.05).

Since there were only significant differences in individual phyla at the phylum level, the distribution of the fecal flora of mice in each group was analyzed at the family level, and the results are shown in Figure 26. As can be seen from Figure 26, the 13 family-level species with the highest relative abundance are Porphyromonadaceae, Lachnospiraceae, Ruminococcaceae, and Bacteroidaceae. ), Prevotellaceae, Rikenellaceae, Erysipelotrichaceae, Verrucomicrobiaceae, Deferribacteraceae, Desulfovibrionaceae , Sutterellaceae, Lactobacillaceae and Helicobacteraceae. Compared with the BLK group, the DSS group significantly decreased the relative abundances of Rikenellaceae, Eysipelotrichaceae, Clostridiales_Incertae_SedisXIII, and significantly increased the relative abundances of Ruminococcaceae, Bacteroidaceae, Verrucomicrobiaceae, Deferribacteraceae and Helicobacteraceae, and all other groups significantly increased the relative abundance of Bacteroidaceae. Compared with the DSS group, the LAH group significantly decreased the relative abundances of Lachnospiraceae, Ruminococcaceae and Desulfovibrionaceae, and significantly increased the relative abundances of Rikenellaceae and Eysipelotrichaceae; the LP group significantly decreased the relative abundance of Deferribacteraceae and significantly increased the relative abundances of Prevotellaceae, Rikenellaceae and Erysipelotrichaceae. The LUT group significantly decreased the relative abundance of Desulfovibrionaceae and Coriobacteriaceae and increased the relative abundance of Rikenellaceae; the AH group significantly decreased the relative abundance of Rikenellaceae, Verrucomicrobiaceae and Bifidobacteriaceae, and significantly increased the relative abundance of Ruminococcaceae, Deferribacteraceae, Desulfovibrionacea and Bdellovibrionaceae.

Therefore, the lutein-loaded sodium alginate-based hydrogel of the present invention is beneficial for lutein to relieve intestinal inflammation by regulating the level of inflammatory factors and the structure of intestinal microbes. The lutein-loaded sodium alginate-based hydrogel can be used in the preparation of a medicament for preventing or treating diseases caused by the level of inflammatory factors and the structure of intestinal microbes.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the application listed in the description and the embodiment, and it can be applied to various fields suitable for the present invention. For those skilled in the art, it can be easily Therefore, the invention is not limited to the specific details and illustrations shown and described herein without departing from the general concept defined by the appended claims and the scope of equivalents.

Claims

The lutein-loaded sodium alginate-based hydrogel is characterized by comprising the following components in parts by weight: 3.12-10.53 parts by weight of lutein, 46.8-47.7 parts by weight of chickpea protein isolate, sodium alginate 120-125 parts by weight, Ca2+-EGTA 8.4-42 parts by weight and D-gluconolactone 7.12-35.6 parts by weight.
The lutein-loaded sodium alginate-based hydrogel according to claim 1, wherein the chickpea protein isolate and the lutein are prepared into chickpea protein isolate lutein nanoparticles and combined thing;

The D-gluconolactone induces the release of Ca 2+ in Ca 2+ -EGTA, and the in-situ gelation of sodium alginate forms a cross-linked hydrogel, and the chickpea protein isolate lutein nanoparticle conjugate is loaded In the hydrogel, a lutein-loaded sodium alginate-based hydrogel was prepared.
The preparation method of the lutein-loaded sodium alginate-based hydrogel according to any one of claims 1-2, characterized in that, comprising the following steps:

Step (1) Weigh a sample of chickpea protein isolate in proportion and add it to a 10 mM phosphate buffer solution. After fully stirring, keep it at 4°C overnight to fully hydrate the chickpea protein isolate, and the obtained mass volume percentage is: 1% chickpea protein isolate solution;

Step (2) The chickpea protein isolate solution prepared in step (1) was placed in a magnetic stirrer with a stirring speed of 500 rpm and stirred at room temperature for 2h, and then homogenized with a high-pressure microfluidizer with a pressure of 12000-15000psi for 1- 3 times, the homogenized chickpea protein isolate solution was refrigerated and centrifuged at 10,000 rpm for 10 min, and the supernatant was taken to obtain chickpea protein isolate nano-solution;

Step (3) Add lutein ethanol solution dropwise to 100 mL of the chickpea protein isolate nano-solution described in step (2), and magnetically stir at 500 rpm for 20 min to make it fully mixed, and then concentrate under reduced pressure at 40 ° C to remove no residue. water ethanol, and then homogenize for 1-3 times with a high-pressure microfluidizer with a pressure of 10000-15000 psi to obtain a chickpea protein isolate lutein nanoparticle conjugate solution;

Step (4) Mix the chickpea protein isolate lutein nanoparticle conjugate solution prepared in step (3) with a sodium alginate solution with a mass volume concentration of 25 mg/mL, stir magnetically evenly, and add 100 mM Ca 2+ in sequence - EGTA solution and D-gluconolactone solution were placed at room temperature for 24 hours to make the sodium alginate gel completely, and then rinse the surface of the hydrogel with deionized water to obtain a lutein-loaded sodium alginate-based hydrogel glue.
The method for preparing a lutein-loaded sodium alginate-based hydrogel according to claim 3, wherein in the step (3), the chickpea protein isolate lutein nanoparticle conjugate solution The mass volume concentration of mid-lutein is 0.8-2.4 mg/mL.
The preparation method of lutein-loaded sodium alginate-based hydrogel according to claim 3, in the step (4), the molar concentration of Ca 2+ in the Ca 2+ -EGTA solution is 5.5-15mM ; Make the molar ratio of Ca 2+ and D-gluconolactone in the solution to be 1:2.
According to the preparation method of the lutein-loaded sodium alginate-based hydrogel according to claim 3, the loading amount of lutein in the hydrogel can reach 760 μg/g, and the encapsulation rate is greater than 96%.
Use of the lutein-loaded sodium alginate-based hydrogel according to any one of claims 1-2 in the preparation of a medicament for preventing or treating diseases caused by the level of inflammatory factors and the structure of intestinal microbes.
The use of the lutein-loaded sodium alginate-based hydrogel according to claim 7, wherein the disease caused by the level of inflammatory factors and the structure of intestinal microbes includes intestinal inflammation.