Method for modifying lutein liposome with composite polyphenol

Abstract

The invention discloses a method for modifying lutein lipidosome by composite polyphenol, which comprises the following specific steps: (1) dissolving lutein and a lipid material to obtain a lutein lipid solution, and carrying out rotary evaporation to form a film; (2) dissolving the composite polyphenol in a thermal phosphate solution, and hydrating the xanthophyll film; (3) carrying out rotary evaporation after hydration to form a film, and adding a cholate solution for redissolving; and (4) after ultrasonic treatment, passing through a polycarbonate film and extruding to obtain the lutein liposome. The lutein liposome modified by the composite polyphenol has the characteristics of high stability and high bioavailability, and the accumulation of lutein in liver and plasma can be remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of functional food processing, and in particular relates to a method for modifying lutein liposomes with composite polyphenols. Background Art

[0002] Lutein is a natural pigment widely found in fruits, vegetables and other foods. It is one of the main components of macular pigment in the eyes. It has the function of absorbing blue light and enhancing retinal tissue. It has significant effects in protecting vision and preventing cataracts, and has attracted widespread attention. However, the carbon chain in the molecular structure of lutein is long and contains multiple hydrophobic groups, which makes it poorly soluble in water and has low bioavailability in the human body, limiting its application in food, medicine and other fields. Lutein is mixed with lipids in the body and wrapped in lipid droplets. After being transferred from lipid droplets, it is converted into water-soluble mixed liposomes together with bile salts, oils and phospholipids in the small intestine. The mixed liposomes have an affinity with the small intestinal epithelial cells and enter the cytoplasm through the brush border membrane of the small intestinal epithelial cells. The absorbed lutein is released from the liposome phase by the catalysis of various enzymes in the cell. After further ester hydrolysis to form monomers, it is wrapped in chylomicrons and released into the lymphatic system, and finally transported into the blood circulation and converted or stored in the liver. At present, the method for improving the bioavailability of lutein is mainly to improve its solubility, such as China Patent (CN111903846A) a stable, highly bioavailable lutein microcapsule and its preparation method and China Patent (CN111714466A) a preparation method of a lutein preparation with high bioavailability and stability. Although the above patents involve methods for improving the bioavailability of lutein, they are only carried out from a single perspective of improving solubility, which has certain limitations, because lutein is a complex absorption process in the human body and is affected by many factors. Lutein is mixed with lipids in the body and wrapped in fat droplets. It is released from the fat droplets in the small intestine through the action of lipase and exists in the gastrointestinal environment in the form of liposomes. It is then absorbed by the intestine through a pathway mediated by lipid transport-related proteins. Studies have found that connecting substances containing polyhydroxy groups to phospholipid molecules to expose some hydrophilic groups on the surface of liposomes can increase the stability of liposomes in the blood. EGCG, xanthohumol, anthocyanins, curcumin or quercetin are plant polyphenols rich in food. They are polyhydroxy active substances with multiple biological activities such as antioxidant, anti-inflammatory, anti-mutation and anti-cardiovascular disease.

[0003] This patent uses complex polyphenols such as EGCG, xanthohumol, anthocyanin, curcumin or quercetin to modify lutein, which can promote the hydrolysis of lutein liposomes and its stability in the gastrointestinal environment, promote the intestinal absorption of lutein by regulating the cellular absorption pathway mediated by receptor proteins, and thus significantly improve the in vivo bioavailability of lutein. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing lutein liposomes modified with composite polyphenols, wherein the lutein liposomes prepared by the method have good gastrointestinal stability, good intestinal absorption and high bioavailability. To achieve the above-mentioned object of the invention, the technical solution adopted by the present invention is:

[0005] (1) dissolving lutein and lipid phase in a chloroform-methanol solution with a volume ratio of 2:1 to obtain a lutein lipid solution;

[0006] (2) removing the organic solvent by rotary evaporation, so that the complex forms a uniform film at the bottom of the round-bottom flask, and increasing the vacuum degree to 0.1 MPa and continuing to pump dry for 30 min;

[0007] (3) dissolving the composite polyphenol in a hot phosphate solution to obtain a composite polyphenol solution, and dripping the composite polyphenol solution into the film obtained in step (2) at a hydration temperature of 35 to 65° C. for 30 to 150 min, and removing the organic solvent by rotary evaporation to form a uniform film of the composite at the bottom of the round-bottom flask;

[0008] (4) adding bile salt solution to dissolve the film obtained in step (3), ultrasonically treating the film in an ice-water bath with stirring, the ultrasonic power is 800 W, the pulse is 5 s / 5 s, the treatment time is 10 to 30 min, and stirring is continued for 4 to 6 h at a temperature of 35 to 40° C. and a stirring speed of 800 to 1200 rpm / min;

[0009] (5) centrifuging the dispersion obtained in step (4) at 12000 rpm / min for 15 min to remove unencapsulated lutein;

[0010] (6) The dispersion obtained in step (5) is extruded through a 200 nm polycarbonate membrane for 10 to 20 times, and excess polyphenols are removed through a G200 polysaccharide gel column to obtain polyphenol-lutein liposomes.

[0011] Preferably, the lipid phase in step (1) is composed of cholesterol, monoacylglycerol and oleic acid, the mass ratio of cholesterol, monoacylglycerol and oleic acid is 2:2:1, and the lutein concentration in the lutein lipid solution is 0.1-2.0%.

[0012] Preferably, the composite polyphenol in step (3) is one or more of EGCG, xanthohumol, anthocyanin, curcumin or quercetin, the molar ratio of lutein to the composite polyphenol is 1:2 to 1:10, the dripping speed is 10 to 30 drops per minute, and the stirring speed is 100 to 700 rpm / min.

[0013] Preferably, in step (4), the molar ratio of bile salt to lipid phase is 2:1 to 5:1.

[0014] The beneficial effects of the present invention are mainly reflected in:

[0015] (1) The present invention utilizes composite polyphenols to modify lutein liposomes, thereby avoiding the aggregation of liposomes and reducing the particle size of liposomes to 0.1 to 0.6 μM. After long-term storage, the average particle size increase ratio is less than 5%, and the penetration of oxygen, water molecules, etc. is effectively inhibited during storage. The lutein retention rate in the liposomes is ≥80%, which has a good protective effect on the lutein liposomes.

[0016] (2) The composite polyphenol and bile salt modified liposomes of the present invention improve the stability of lutein liposomes in the gastrointestinal tract. The prepared liposomes can resist the destruction of gastric enzymes, acid and bile in the gastrointestinal tract, inhibit the rapid release of lutein, and effectively improve the bioavailability of lutein.

[0017] (3) The present invention utilizes composite polyphenols to modify lutein liposomes, which facilitates the transfer of lutein to the upper layer of digestive fluid, thereby increasing the bioaccessibility of lutein liposomes by ≥1.5 times.

[0018] (4) The composite polyphenol and bile salt modified liposomes of the present invention significantly increase the expression of lutein cell transporter proteins and improve the absorption of lutein by intestinal cells; increase the abundance of intestinal flora related to lutein absorption, and increase the accumulation of lutein and its metabolites in mouse liver by ≥2 times and in mouse plasma by ≥20%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Effects of polyphenol modification on lutein bioaccessibility

[0020] Figure 2 Effects of polyphenol-modified lutein liposomes on lutein accumulation in mouse tissues DETAILED DESCRIPTION

[0021] The following examples are further detailed descriptions of the present invention, but are not intended to limit the present invention in any way.

[0022] Example 1

[0023] Lutein was dissolved in a chloroform-methanol solution with a volume ratio of 2:1, and 3mmol / L cholesterol, 3mmol / L oleic acid, 1.5mmol / L glyceride, etc. were added to obtain a lutein lipid solution. The organic solvent was blown dry by a nitrogen purge instrument to form a uniform film at the bottom of the test tube. EGCG and anthocyanins were dissolved in a hot sodium bicarbonate solution to obtain a polyphenol solution containing EGCG and anthocyanins. The polyphenol solution was added dropwise to the film, and after stirring at room temperature, the nitrogen purge instrument was blown dry to form a uniform film at the bottom of the test tube. The final molar ratio of lutein to composite polyphenol was 1:5. 15mmol / L sodium taurocholate solution was added to the composite film for hydration, the hydration temperature was 60℃, and the time was 120min. Ultrasound was performed under ice-water bath stirring conditions, the ultrasonic power was 800W, the pulse was 5s / 5s, the treatment time was 20min, and the temperature was 30℃ for 3h, and the stirring speed was 1000rpm / min. The dispersion was centrifuged at 10000 rpm / min for 10 min to remove unencapsulated lutein. The obtained dispersion was extruded through a 200 nm polycarbonate membrane 10 times, and excess polyphenols were removed through a G200 dextran gel column to obtain polyphenol-modified lutein liposomes.

[0024] Control group: Lutein was dissolved in a chloroform-methanol solution with a volume ratio of 2:1, and 3mmol / L cholesterol, 3mmol / L oleic acid, 1.5mmol / L glyceride, etc. were added to obtain a lutein lipid solution. The organic solvent was blown dry by a nitrogen purge instrument to form a uniform film at the bottom of the test tube. 15mmol / L sodium bicarbonate solution was added to the composite film for hydration, the hydration temperature was 60℃, and the time was 120min; ultrasonication was performed under stirring conditions in an ice-water bath, the ultrasonic power was 800W, the pulse was 5s / 5s, and the treatment time was 20min. After stirring for 3h at 30℃, the stirring speed was 1000rpm / min; the dispersion was centrifuged at a speed of 10000rpm / min for 10min to remove the unencapsulated lutein. The obtained dispersion was extruded 10 times through a 200nm polycarbonate membrane to obtain lutein liposomes, which were used as the control group.

[0025] The experimental group and the control group were placed in an in vitro digestion instrument and automatically injected with 10 mL of gastric juice (140 mmol / L NaCl, 5 mmol / L KCl, 10 mmol / L CaCl2·2H2O, 3.5 mmol / L KH2PO4), 2 mL of pepsin (adjusted to pH 2 with 1 mol / L HCl solution to a final concentration of 3.2 mg / mL), and the digestion temperature was set at 37°C, the speed was 150 r / min, and the time was 1 h. After the gastric digestion stage, the pH was adjusted to 7 with 1 mol / L NaHCO3, and 20 mL of intestinal juice (5 mg / mL pancreatic enzyme, 31.13 mg / mL bile salt, dissolved in 0.1 mol / L NaHCO3) was injected for digestion for 2 h.

[0026] The lutein content in the digestive juice was determined, and the results showed that polyphenol-modified lutein could significantly improve the bioaccessibility of lutein ( Figure 1 ), the bioaccessibility of lutein in the control group was only 3.0%, and the bioaccessibility of lutein after modification with EGCG and anthocyanins increased by 2.8 and 1.8 times, respectively. The liposome solutions obtained from the experimental group and the control group were measured for liposome particle size and ζ-potential by a nanoparticle size analyzer (Table 1). The experimental results showed that the experimental group could significantly reduce the particle size of lutein liposomes and increase the absolute value of ζ-potential, which was beneficial to maintain the intestinal stability of the liposomes. C57BL / 6 mice were divided into three groups: control group, EGCG and anthocyanin group, with 10 mice in each group. The experimental group was continuously gavaged with polyphenol-modified lutein liposomes once a day, 20 mg / kg each time, and the control group was given lutein liposomes in the same way. After 7 days, mouse tissues were taken and the lutein in the mouse tissues was extracted. The results of the study showed that compared with the control group, the experimental group significantly increased the accumulation of lutein in mouse tissues ( Figure 2 ), EGCG and anthocyanins increased the liver accumulation of lutein by 3.0 times and 2.4 times, respectively, and EGCG and anthocyanins increased the plasma accumulation of lutein by 50% and 20%, respectively.

[0027] Table 1 Liposome performance of polyphenol-modified lutein in gastrointestinal environment

[0028]

[0029]

[0030] Example 2

[0031] Lutein was dissolved in a chloroform-methanol solution with a volume ratio of 2:1, and 3mmol / L cholesterol, 3mmol / L oleic acid, 1.5mmol / L glyceride, etc. were added to obtain a lutein lipid solution. The organic solvent was blown dry by a nitrogen purge instrument to form a uniform film at the bottom of the test tube. EGCG, anthocyanins, and xanthohumol were dissolved in a hot sodium bicarbonate solution to obtain a polyphenol solution containing EGCG, anthocyanins, and xanthohumol. The polyphenol solution was added dropwise to the film, stirred at room temperature, and then blown dry by a nitrogen purge instrument to form a uniform film at the bottom of the test tube. The final molar ratio of lutein to composite polyphenols was 1:5. Add 15mmol / L sodium taurocholate solution to the composite film for hydration at 60℃ for 120min; ultrasonicate under ice-water bath stirring conditions, ultrasonic power 800W, pulse 5s / 5s, treatment time 20min, continue stirring at 30℃ for 3h, stirring speed 1000rpm / min; centrifuge the dispersion at 10000rpm / min for 10min to remove unencapsulated lutein. The obtained dispersion was extruded 10 times through a 200nm polycarbonate membrane, and excess polyphenols were removed through a G200 dextran gel column to obtain polyphenol-modified lutein liposomes.

[0032] Control group: Lutein was dissolved in a chloroform-methanol solution with a volume ratio of 2:1, and 1.5mmol / L cholesterol, 1.5mmol / L oleic acid, 0.75mmol / L glyceride, etc. were added to obtain a lutein lipid solution. The organic solvent was blown dry by a nitrogen purge instrument to form a uniform film at the bottom of the test tube. 7.5mmol / L sodium phosphate solution was added to the composite film for hydration, the hydration temperature was 60℃, and the time was 120min; ultrasonication was performed under stirring conditions in an ice-water bath, the ultrasonic power was 800W, the pulse was 5s / 5s, and the treatment time was 20min. After stirring for 3h at 30℃, the stirring speed was 1000rpm / min; the dispersion was centrifuged at a speed of 10000rpm / min for 10min to remove the unencapsulated lutein. The obtained dispersion was extruded 10 times through a 200nm polycarbonate membrane to obtain lutein liposomes, which were used as the control group.

[0033] The lipid hydrolysis rate and bioaccessibility of lutein liposomes were measured in an in vitro digestion system. After polyphenol modification, the lipid hydrolysis rate increased by 11-45%, and the bioaccessibility of lutein increased by 2.3-8.5 times. Caco-2 cell monolayer membranes were constructed to measure the cell absorption pathway and capacity of lutein liposomes. The results showed that after polyphenol modification, the expression of lutein cell transport receptors SR-BI and CD36 increased by 4-8 times, and cell absorption increased by 25%.

[0034] Example 3

[0035] Lutein was dissolved in a chloroform-methanol solution with a volume ratio of 2:1, and 3mmol / L cholesterol, 3mmol / L oleic acid, 1.5mmol / L glyceride, etc. were added to obtain a lutein lipid solution. The organic solvent was blown dry by a nitrogen purge instrument to form a uniform film at the bottom of the test tube. EGCG, xanthohumol, anthocyanin, curcumin and quercetin were dissolved in a hot sodium bicarbonate solution to obtain a composite polyphenol solution. The composite polyphenol solution was added dropwise to the film, stirred at room temperature, and then blown dry by a nitrogen purge instrument to form a uniform film at the bottom of the test tube. The final molar ratio of lutein to composite polyphenol was 1:5. Add 15mmol / L sodium taurocholate solution to the composite film for hydration at 60℃ for 120min; ultrasonicate under ice-water bath stirring conditions, ultrasonic power 800W, pulse 5s / 5s, treatment time 20min, continue stirring at 30℃ for 3h, stirring speed 1000rpm / min; centrifuge the dispersion at 10000rpm / min for 10min to remove unencapsulated lutein. The obtained dispersion was extruded 10 times through a 200nm polycarbonate membrane, and excess polyphenols were removed through a G200 dextran gel column to obtain composite polyphenol-modified lutein liposomes.

[0036] Control group: Lutein was dissolved in a chloroform-methanol solution with a volume ratio of 2:1, and 3mmol / L cholesterol, 3mmol / L oleic acid, 1.5mmol / L glyceride, etc. were added to obtain a lutein lipid solution. The organic solvent was blown dry by a nitrogen purge instrument to form a uniform film at the bottom of the test tube. 15mmol / L sodium phosphate solution was added to the composite film for hydration, the hydration temperature was 60℃, and the time was 120min; ultrasonication was performed under stirring conditions in an ice-water bath, the ultrasonic power was 800W, the pulse was 5s / 5s, and the treatment time was 20min. After stirring for 3h at 30℃, the stirring speed was 1000rpm / min; the dispersion was centrifuged at a speed of 10000rpm / min for 10min to remove the unencapsulated lutein. The obtained dispersion was extruded 10 times through a 200nm polycarbonate membrane to obtain lutein liposomes, which were used as the control group.

[0037] The lipid hydrolysis rate and bioaccessibility of lutein liposomes were measured in an in vitro digestion system. After polyphenol modification, the lipid hydrolysis rate increased by 11-45%, and the bioaccessibility of lutein increased by 2.3-8.5 times. C57BL / 6 mice were gavaged with liposomes once a day, 20 mg / kg each time, and the control group was given lutein liposomes in the same way. The mouse feces were taken every day for flora analysis, and the mouse tissues were taken after 7 days to extract the lutein in the mouse tissues. The results showed that compared with the control group, the experimental group significantly increased the abundance of bifidobacteria and lactobacilli in the mouse intestine, and the cumulative content of lutein in the liver increased by 5 times.

Claims

1. A method for preparing composite polyphenol-modified lutein liposomes, characterized in that: (1) dissolving lutein and lipid phase in a chloroform-methanol solution with a volume ratio of 2:1 to obtain a lutein lipid solution; (2) removing the organic solvent by rotary evaporation, so that the composite forms a uniform film at the bottom of the round-bottom flask, and increasing the vacuum degree to 0.1 MPa and continuing to pump dry for 30 min; (3) dissolving the composite polyphenol in a hot phosphate solution to obtain a composite polyphenol solution, and dripping the composite polyphenol solution into the film obtained in step (2) at a hydration temperature of 35 to 65° C. for 30 to 150 min, and removing the organic solvent by rotary evaporation to form a uniform film of the composite at the bottom of the round-bottom flask; (4) adding bile salt solution to dissolve the film obtained in step (3), ultrasonically treating the film in an ice-water bath with stirring, the ultrasonic power is 800 W, the pulse is 5 s / 5 s, the treatment time is 10 to 30 min, and stirring is continued for 4 to 6 h at a temperature of 35 to 40° C. and a stirring speed of 800 to 1200 rpm / min; (5) centrifuging the dispersion obtained in step (4) at 12000 rpm / min for 15 min to remove unencapsulated lutein; (6) The dispersion obtained in step (5) is extruded through a 200 nm polycarbonate membrane for 10 to 20 times, and excess composite polyphenols are removed through a G200 polysaccharide gel column to obtain composite polyphenol-lutein liposomes.

2. The method for preparing a composite polyphenol-modified lutein liposome according to claim 1, characterized in that: In step (1), the lipid phase is composed of cholesterol, monoacylglycerol and oleic acid, the mass ratio of cholesterol, monoacylglycerol and oleic acid is 2:2:1, and the lutein concentration in the lutein lipid solution is 0.1-2.0%.

3. The method for preparing a composite polyphenol-modified lutein liposome according to claim 1, characterized in that: The composite polyphenol in step (3) is one or more of epigallocatechin gallate (EGCG), xanthohumol, anthocyanin, curcumin or quercetin, the molar ratio of lutein to the composite polyphenol is 1:2 to 1:10, the dropping speed is 10 to 30 drops per minute, and the stirring speed is 100 to 700 rpm / min.

4. The method for preparing a composite polyphenol-modified lutein liposome according to claim 1, characterized in that: In step (4), the molar ratio of bile salt to lipid phase is 2:1 to 5:1.

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