Preparation process of lutein liposome for improving bioavailability

Abstract

The invention provides a lutein liposome preparation process for improving bioavailability. The lutein liposome preparation process comprises the following steps: S1, preparing raw materials; s2, preparing a lutein lipid solution: dissolving lutein and a lipid phase in a trichloromethane methanol solution to obtain the lutein lipid solution; s3, forming a thin film: carrying out rotary evaporation on the lutein lipid solution, and removing the organic solvent to form a uniform thin film; s4, preparation and hydration of a composite polyphenol solution: dissolving composite polyphenol in a thermal phosphate solution to obtain the composite polyphenol solution, dropwise adding the composite polyphenol solution into the film for hydration, and performing rotary evaporation to remove an organic solvent to form a uniform film; s5, redissolution and ultrasonic treatment: adding the film into a bile salt solution for redissolution, performing ultrasonic treatment under the ice-water bath stirring condition, and continuing stirring after ultrasonic treatment; and S6, centrifuging and purifying: centrifuging the dispersion liquid to remove unembedded xanthophyll, extruding the dispersion liquid through a polycarbonate film, and removing excessive composite polyphenol through a sephadex column to obtain the composite polyphenol xanthophyll liposome. By adopting the preparation process, the bioavailability of the xanthophyll can be improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of lutein liposome, and particularly relates to a lutein liposome manufacturing process for improving bioavailability. BACKGROUND

[0002] Lutein is a natural pigment widely existing in foods such as fruits and vegetables, and as one of the main components of macular pigment of the eye, it has the functions of absorbing blue light and enhancing retinal tissue, and has significant effects in protecting vision and preventing cataracts, which has attracted widespread attention. However, the molecular structure of lutein has a long carbon chain and contains multiple hydrophobic groups, which makes it poorly soluble in water and has low bioavailability in the human body, which greatly limits its application in the fields of food, medicine and the like.

[0003] At present, the method for improving the bioavailability of lutein mainly improves its solubility, such as the Chinese patent (CN111903846A) discloses a stable and high-bioavailability lutein microcapsule and a preparation method thereof; and the Chinese patent (CN111714466A) discloses a preparation method of a lutein preparation with high bioavailability and high stability.

[0004] However, the above-mentioned patents only develop from the single angle of improving solubility, which has certain limitations, because the absorption of lutein in the human body is a complex process and is affected by many factors.

[0005] Lutein is mixed with lipids in the body and wrapped in lipid droplets, which are released from the lipid droplets in the small intestine by the action of lipase, and exist in the form of liposomes in the gastrointestinal environment, and then are absorbed by the intestinal tract through the pathway mediated by lipid transport-related proteins. Therefore, it is of great practical significance to develop a process capable of effectively improving the bioavailability of lutein. SUMMARY

[0006] The present application provides a lutein liposome manufacturing process for improving bioavailability to solve the technical problems proposed in the background art.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a lutein liposome manufacturing process for improving bioavailability, characterized by comprising the following steps:

[0008] S1 raw material preparation: weighing lutein, lipid phase, complex polyphenol and cholate, preparing chloroform-methanol solution, hot phosphate solution and physiological saline;

[0009] S2 preparation of lutein lipid solution: dissolving the lutein and the lipid phase in the chloroform-methanol solution to obtain a lutein lipid solution;

[0010] S3 forming thin film: the lutein liposome solution is subjected to rotary evaporation to remove the organic solvent and form a uniform thin film, and the vacuum degree is increased to continue to dry;

[0011] S4 preparation and hydration of the complex polyphenol solution: the complex polyphenol is dissolved in the hot phosphate solution to obtain a complex polyphenol solution, and the thin film is added dropwise with the complex polyphenol solution for hydration, followed by rotary evaporation to remove the organic solvent and form a uniform thin film again;

[0012] S5 resolubilization and ultrasonic treatment: the thin film is added to a cholate solution for resolubilization, and ultrasonic treatment is performed under ice water bath stirring, followed by continued stirring after ultrasonic treatment;

[0013] S6 centrifugation and purification: the dispersion is centrifuged to remove un-embedded lutein, and then the dispersion is extruded through a polycarbonate membrane and then passed through a dextran gel column to remove excess complex polyphenol, thereby obtaining complex polyphenol lutein liposomes.

[0014] Further, in S1, 0.5 g of lutein is weighed, the lipid phase includes 2 g of cholesterol, 2 g of monoglyceride, and 1 g of oleic acid, the complex polyphenol is 2 g of EGCG, and the cholate is 3 g; the volume of the chloroform-methanol solution is 100 ml, the volume ratio is 2:1, the volume of the hot phosphate solution is 50 ml, and the specific amount of each raw material, the volume of the solution, and the ratio are specified, which standardizes the raw material preparation link, avoids the problem of unstable product quality caused by raw material dosage errors, ensures the accuracy of the raw material ratio of each batch of lutein liposomes, thereby ensuring the consistency and stability of product quality, and is conducive to large-scale industrial production.

[0015] Further, in S3, the vacuum degree is increased to 0.1 MPa to continue to dry for 30 min; in S4, the hydration temperature is 40℃, the time is 60 min, the dropwise addition speed is 15 drops per min, and the stirring speed is 300 r / min; in S5, the ultrasonic power is 800 W, the pulse is 5 s / 5 s, the treatment time is 15 min, the temperature for continued stirring is 37℃, the stirring speed is 1000 r / min, and the stirring time is 5 h; the temperature, time, speed, and other parameters in each step are specified in detail, which provides precise operation specifications for the entire production process. Suitable drying vacuum degree and time can effectively remove organic solvent residues; accurate hydration, ultrasonic, and stirring parameters can make the liposome formation process more complete and stable, improve the encapsulation efficiency and structural integrity of the liposomes, and further improve the quality and efficacy of the product.

[0016] Furthermore, in step S6, the centrifugation speed is 12000 r / min, and the centrifugation time is 15 min; the pore size of the polycarbonate membrane is 200 nm, and the number of compressions is 12; the dextran gel column is a G200 dextran gel column. Key parameters for centrifugation, polycarbonate membrane compression, and the dextran gel column were determined. The centrifugation parameters can efficiently separate unencapsulated lutein; the pore size and number of compressions of the polycarbonate membrane can precisely control the liposome particle size, making it more suitable for application requirements; the specific dextran gel column can effectively remove excess complex polyphenols, resulting in better purification and ultimately obtaining high-purity, uniformly sized complex polyphenol-lutein liposomes, enhancing the product's bioavailability and stability.

[0017] Furthermore, in step S4, when the composite polyphenol is dissolved in the hot phosphate solution, the temperature of the hot phosphate solution is controlled at 50-60°C, the stirring speed is 200-300 r / min, and the dissolution time is 10-15 min, so as to ensure that the composite polyphenol is completely dissolved and a clear and transparent composite polyphenol solution is obtained.

[0018] In step S4, when adding the composite polyphenol solution to the film for hydration, the dropping rate is 1-2 ml / min. After the dropping is complete, hydration is carried out at 40-50℃ for 30-40 minutes, and a low-speed stirring speed of 150-200 r / min is maintained during the hydration process to promote uniform hydration of the film. Refining the dissolution conditions of the composite polyphenol ensures that the composite polyphenol is fully dissolved in the hot phosphate solution, avoiding problems such as turbidity or uneven composition caused by insufficient dissolution. This makes the subsequent hydration process smoother, helps to form a film of uniform quality, lays a good foundation for the subsequent formation of liposomes, and thus improves the quality and stability of liposome production. The specific operating parameters for the hydration of the composite polyphenol solution are clearly defined. Appropriate dropping rate, hydration temperature, time, and stirring speed can ensure uniform hydration of the film, avoid local over- or under-hydration, promote more uniform and stable formation of liposomes, effectively improve the encapsulation efficiency and structural stability of liposomes, and enhance product quality.

[0019] Furthermore, before centrifugation and purification in step S6, the dispersion is subjected to high-pressure homogenization at a pressure of 80-120 MPa for 3-5 cycles. The high-pressure shear force is used to homogenize the liposome particle size, thereby further improving the stability and encapsulation efficiency of the liposomes.

[0020] In step S6, after removing excess complex polyphenols using a dextran gel column, a freeze-drying process is added. The resulting complex polyphenol-lutein liposome suspension is pre-frozen at -40℃ for 2-3 hours, and then sublimated at a vacuum of 0.01-0.05 mbar with a heating rate of 0.5-1℃ / min for 12-18 hours to obtain freeze-dried liposome powder, extending the product's shelf life and facilitating storage and transportation. High-pressure homogenization before centrifugation purification utilizes high-pressure shear force to significantly reduce liposome particle size and make its particle size distribution more uniform. Uniform particle size helps improve the absorption efficiency of liposomes in vivo, reduces instability caused by particle size differences, enhances the stability and encapsulation efficiency of liposomes, thereby improving the bioavailability and product performance of lutein liposomes. The freeze-drying process after purification, which converts the liposomes into freeze-dried powder, greatly reduces the product's water content, inhibits microbial growth and liposome oxidation, and significantly extends the product's shelf life. At the same time, freeze-dried powder is easy to store and transport, reduces the requirements for storage and transportation conditions, and improves the market adaptability and commercial value of the product. Detailed Implementation

[0021] The technical solutions in the embodiments of the invention will be clearly and completely described below with reference to the embodiments of the invention. Obviously, the described embodiments are only some embodiments of the invention, and not all embodiments. Based on the embodiments of the invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the invention.

[0022] Example

[0023] Step 1: Raw Material Preparation

[0024] Lutein selection: Select lutein raw materials with a purity of ≥95%. Its appearance should be a yellow to orange-yellow crystalline powder, without obvious lumps or impurities. Store in a cool (temperature ≤20℃), dry (relative humidity ≤60%), and light-proof environment to avoid deterioration of the raw materials due to oxidation, moisture absorption, etc.

[0025] lipid phase formulation

[0026] Cholesterol: Pharmaceutical grade cholesterol is used, with a melting point of 147-150℃, acid value ≤1.0, and saponification value ≤5.0, to ensure its purity and stability.

[0027] Monoacylglycerols: Food-grade monoacylglycerols are selected, with a monoglyceride content of ≥90% and an iodine value of ≤3.0, to ensure good compatibility with other ingredients.

[0028] Oleic acid: Chemically pure oleic acid (food grade) is used, with a purity of ≥98%, an acid value of 190-202, and a color (APHA) of ≤200.

[0029] Weigh the cholesterol, monoglycerides, and oleic acid accurately in a mass ratio of 2:2:1 using an electronic balance with an accuracy of 0.0001g. After weighing, place the three components in a clean container and mix thoroughly.

[0030] Preparation of compound polyphenols: If choosing a single polyphenol, such as EGCG (epigallocatechin gallate), the purity must be ≥98%; xanthohumol purity ≥95%; anthocyanin purity ≥90%; curcumin purity ≥95%; quercetin purity ≥98%. If choosing a combination of multiple polyphenols, the above purity requirements must also be met. Check the appearance of the polyphenol raw materials; there should be no discoloration, off-odor, etc. Storage conditions are the same as for lutein.

[0031] Bile salt preparation: Use pharmaceutical grade bile salts such as sodium taurocholate or sodium glycocholate, with a purity ≥98% and a melting point of 200-205℃. Store in a dry environment at room temperature and check for deliquescence before use.

[0032] Solution preparation: Chloroform-methanol solution: Prepare at a volume ratio of 2:1 using a Grade A volumetric flask for precise measurement. The preparation process should be carried out in a fume hood to avoid inhaling organic solvent vapors. After preparation, seal and store in a brown reagent bottle to prevent evaporation and contamination. Hot phosphate solution: Dissolve sodium dihydrogen phosphate and disodium hydrogen phosphate in deionized water at a specific ratio (e.g., 0.05 mol / L, pH 7.4). Adjust the pH of the solution precisely using a pH meter. Heat the solution to 60-70°C for later use, using a magnetic stirrer to ensure uniform heating during the heating process.

[0033] Physiological saline: Use commercially available 0.9% sodium chloride solution that meets pharmacopoeia standards. Before use, check whether the packaging is intact and whether the solution is cloudy or has any sediment.

[0034] Step 2: Preparation of lutein lipid solution

[0035] Accurately weigh the lutein and lipid components, and add them sequentially to a round-bottom flask containing an appropriate amount of chloroform-methanol solution. The volume of the round-bottom flask should be 2-3 times the total volume of the solution to ensure thorough stirring and dissolution.

[0036] Place the round-bottom flask in a constant-temperature water bath at 35-40℃. Stir with an electric stirrer at 150-250 rpm until the lutein and lipid phase are completely dissolved, forming a clear and transparent solution. During this process, closely observe the solution. If insoluble matter or turbidity appears, extend the stirring time or appropriately increase the temperature (but not exceeding 45℃) to ensure the lutein concentration reaches 0.1-2.0%.

[0037] Step 3: Forming a thin film

[0038] Install the round-bottom flask containing the lutein lipid solution onto a rotary evaporator, and connect the condenser and vacuum pump. Set the rotary evaporator speed to 60-80 rpm, the water bath temperature to 40-45°C, and gradually adjust the vacuum to 0.08-0.1 MPa to begin rotary evaporation. Observe the inner wall of the round-bottom flask. When most of the organic solvent has evaporated, the solution gradually thickens, and a thin film begins to form at the bottom of the flask, continue evaporation for 5-10 minutes to ensure complete removal of the organic solvent. Then, increase the vacuum to 0.1 MPa and continue evaporation for 30 minutes to further remove residual organic solvent, allowing the complex to form a uniform and dense thin film at the bottom of the round-bottom flask.

[0039] Step 4: Preparation and hydration of the composite polyphenol solution

[0040] Preparation of the compound polyphenol solution: Accurately weigh the compound polyphenols according to the molar ratio of lutein to compound polyphenols (1:2-1:10) and dissolve them using a hot phosphate solution. Slowly add the weighed compound polyphenols to the preheated phosphate solution at 60-70℃ while stirring at a speed of 200-300 rpm until the compound polyphenols are completely dissolved, forming a homogeneous compound polyphenol solution.

[0041] Hydration Procedure: Place the round-bottom flask containing the membrane in a constant-temperature water bath, maintaining the water bath temperature at 35-65℃ (select the appropriate temperature based on the specific polyphenol type and experimental requirements). Using a constant-pressure dropping funnel, slowly add the composite polyphenol solution dropwise into the round-bottom flask at a rate of 10-30 drops per minute, while simultaneously stirring with an electric stirrer at 100-700 rpm to ensure thorough contact and hydration of the composite polyphenol solution with the membrane. The hydration time is 30-150 minutes; closely observe the dissolution and dispersion of the membrane during this period.

[0042] After hydration, the round-bottom flask is placed back on the rotary evaporator and rotary evaporation is carried out according to the operating conditions of step three (rotation speed 60-80 r, water bath temperature 40-45℃, vacuum degree 0.08-0.1 MPa) to remove the organic solvent and allow the composite to form a uniform film again at the bottom of the round-bottom flask.

[0043] Step 5: Reconstitution and Ultrasonic Treatment

[0044] Reconstitution: Accurately weigh the bile salts according to the molar ratio of bile salts to lipid phase (2:1-5:1), dissolve them in an appropriate amount of physiological saline to prepare a bile salt solution. Add the bile salt solution to a round-bottom flask containing the above-mentioned film, and stir at 300-500 rpm using an electric stirrer at room temperature to fully reconstitute the film and form a uniform dispersion.

[0045] Ultrasonic treatment: Place the round-bottom flask containing the dispersion in an ice-water bath, maintaining the temperature at 0-5℃. Use an ultrasonic cell disruptor to perform ultrasonic treatment, setting the ultrasonic power to 800W, pulses to 5s / 5s, and treatment time to 10-30 minutes. During ultrasonication, continuously stir the dispersion at a speed of 100-200 rpm to ensure uniform ultrasonication. After ultrasonic treatment, transfer the round-bottom flask to a constant-temperature water bath and continue stirring at 35-40℃ for 4-6 hours at a stirring speed of 800-1200 rpm to allow for complete liposome formation and stabilization.

[0046] Step Six: Centrifugation and Purification Centrifugation: The dispersion obtained in Step Five is first homogenized using a high-pressure homogenizer. The homogenized liquid is then transferred to centrifuge tubes and centrifuged using a high-speed refrigerated centrifuge at a speed of 12000 r / min, a temperature of 4℃, and a time of 15 min. After centrifugation, the supernatant is carefully aspirated, and the precipitate (unencapsulated lutein and impurities) is discarded, retaining the supernatant containing liposomes.

[0047] Extrusion: The supernatant is extruded through a 200nm polycarbonate membrane using a liposome extruder, with 10-20 extrusions per cycle. During extrusion, it is crucial to maintain stable pressure (generally controlled at 5-10 MPa) to ensure uniform liposome particle size.

[0048] Gel column purification: Prepare a G200 dextran gel column and pack and equilibrate it according to standard methods. Slowly load the extruded dispersion into the gel column, using physiological saline as the eluent, and control the elution flow rate at 0.5-1.0 ml / min. Collect the eluent containing liposomes, and confirm by detection (e.g., high performance liquid chromatography) that excess polyphenols in the eluent have been removed, obtaining polyphenol-lutein liposomes.

[0049] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A process for manufacturing lutein liposomes with improved bioavailability, characterized in that: Includes the following steps: S1 Raw Material Preparation: Weigh lutein, lipid phase, complex polyphenols, and bile salts; prepare chloroform methanol solution, hot phosphate solution, and physiological saline. S2 Preparation of lutein lipid solution: The lutein and lipid phase are dissolved in the chloroform-methanol solution to obtain a lutein lipid solution; S3 Thin film formation: The lutein lipid solution is rotary evaporated to remove organic solvents and form a uniform thin film. The vacuum degree is increased and the film is further dried. S4 Composite Polyphenol Solution Preparation and Hydration: The composite polyphenol is dissolved in the hot phosphate solution to obtain a composite polyphenol solution. The composite polyphenol solution is added dropwise to the film for hydration. Then, the organic solvent is removed by rotary evaporation to form a uniform film again. S5 Resolution and Ultrasonic Treatment: The film is resolution in bile salt solution, ultrasonicated under ice-water bath stirring conditions, and stirred again after ultrasonic treatment; S6 Centrifugation and Purification: The dispersion was centrifuged to remove unencapsulated lutein, then the dispersion was squeezed through a polycarbonate membrane and then passed through a dextran gel column to remove excess complex polyphenols, yielding complex polyphenol-lutein liposomes.

2. The process for producing lutein liposomes with improved bioavailability according to claim 1, characterized in that: In step S1, 0.5g of lutein is weighed out; the lipid phase includes 2g of cholesterol, 2g of monoacylglycerol, and 1g of oleic acid; the complex polyphenol is 2g of EGCG and 3g of bile salts; the volume of the chloroform-methanol solution is 100ml, the volume ratio of chloroform to methanol is 2:1; the volume of the hot phosphate solution is 50ml, and the hot phosphate solution is a mixture of sodium dihydrogen phosphate (NaH2PO4) and disodium hydrogen phosphate (Na2HPO4).

3. The process for producing lutein liposomes with improved bioavailability according to claim 1, characterized in that, In step S3, the vacuum level is increased to 0.1 MPa and the pumping continues for 30 minutes. In step S4, the hydration temperature is 40℃, the time is 60min, the dropping rate is 15 drops per minute, and the stirring speed is 300r / min; in step S5, the ultrasonic power is 800W, the pulse is 5s / 5s, the processing time is 15min, the temperature for continued stirring is 37℃, the stirring speed is 1000r / min, and the stirring time is 5h.

4. The process for producing lutein liposomes with improved bioavailability according to claim 1, characterized in that, In step S6, the centrifugation speed is 12000 r / min and the centrifugation time is 15 min; the pore size of the polycarbonate membrane is 200 nm and the number of compressions is 12; the dextran gel column is a G200 dextran gel column.

5. The process for producing lutein liposomes with improved bioavailability according to claim 1, characterized in that: In step S4, when the composite polyphenol is dissolved in the hot phosphate solution, the temperature of the hot phosphate solution is controlled at 50-60℃, the stirring speed is 200-300 r / min, and the dissolution time is 10-15 min, so as to ensure that the composite polyphenol is completely dissolved and a clear and transparent composite polyphenol solution is obtained.

6. The process for producing lutein liposomes with improved bioavailability according to claim 1, characterized in that, In step S4, when the composite polyphenol solution is added to the film for hydration, the dropping rate is 1-2 ml / min. After the addition is complete, the film is hydrated at 40-50°C for 30-40 minutes. During the hydration process, the film is stirred at a low speed of 150-200 r / min to promote uniform hydration.

7. The process for producing lutein liposomes with improved bioavailability according to claim 1, characterized in that, Before centrifugation and purification in step S6, the dispersion is subjected to high-pressure homogenization at a pressure of 80-120 MPa for 3-5 cycles. The high-pressure shear force is used to homogenize the liposome particle size, thereby further improving the stability and encapsulation efficiency of the liposomes.

8. The process for producing lutein liposomes with improved bioavailability according to claim 1, characterized in that, In step S6, after removing excess complex polyphenols using a dextran gel column, a freeze-drying process is added. The resulting complex polyphenol-lutein liposome suspension is pre-frozen at -40℃ for 2-3 hours, and then sublimated and dried at a heating rate of 0.5-1℃ / min under a vacuum of 0.01-0.05 mbar for 12-18 hours to obtain freeze-dried liposome powder, which extends the product's shelf life and facilitates storage and transportation.