CN106307543A - Nano-liposome with thymol and method for preparing nano-liposome - Google Patents

Abstract

The invention discloses a thymol nano-liposome and a preparation method thereof. The formula of the thymol nano-liposome is thymol, soybean lecithin, distearoylphosphatidylacetamide-methoxy Polyethylene glycol 5000, cholesterol, antioxidant and Tween 80; Simultaneously, the present invention prepares thymol nano-liposomes through magnetic stirring, rotary evaporation and ultrasonic treatment process, and the average particle size of the prepared liposomes is The liposome has a diameter of 96-105nm, a polydispersity index of 0.196-0.201, and an encapsulation efficiency of 86.1-89.3%. The liposome has a nearly spherical structure and can still maintain good stability after being stored at room temperature for 12 days. The invention can improve the water dispersibility, stability and bioavailability of thymol, and has sustained release effect.

Description

Thymol nano liposome and preparation method thereof

technical field

The invention belongs to the technical field of microcapsule embedding, and more specifically relates to a thymol nanoliposome. Simultaneously, the present invention also relates to a preparation method of thymol nano liposome.

Background technique

Thymol, also known as thymol, has a chemical name of 5-methyl-2-isopropylphenol, which exists in plant essential oils such as thyme oil, oregano oil, and clove basil oil. Antioxidant properties, often used in the preparation of flavors such as gummies, mushrooms and citrus. Thymol exerts its antibacterial properties by destroying the integrity of cell membranes, affecting the balance of inorganic ions and pH stability. The hydroxyl group on the benzene ring of thymol can provide H ⁺ to free radicals, thereby delaying the oxidation process. In addition, thymol also has potential liver protection, anticancer and antitumor effects. Although thymol is certified as a GRAS food additive by the FDA, its poor water dispersibility and stability limit its application in industry. By constructing a suitable carrier, its water solubility can be improved, the reaction interface can be expanded, and its antibacterial and antioxidant activities can be further improved.

Liposomes are closed vesicles with a bilayer structure formed by phospholipids or their analogues dispersed in an aqueous phase. At present, it is generally accepted that the formation mechanism of liposomes is the interaction between phospholipids and water molecules. The polar head of phospholipids is located on the inner and outer surfaces of the lipid bilayer, and the hydrophobic tail is located in the middle of the lipid bilayer, forming a bilayer vesicle spontaneously. structure. This structural property allows liposomes to simultaneously embed hydrophilic, hydrophobic, and amphiphilic substances. Because of its simple preparation method, targeting, sustained release, cell affinity and tissue compatibility, it has been successfully applied to the embedding and delivery of antibacterial agents, enzymes, food additives and nutrients in the food field. Controlling the particle size of liposomes at the nanometer scale can further improve their biocompatibility, penetration and in vivo targeting.

There is no report about thymol nanoliposomes at present, and traditional liposome formulas and preparation methods still have the disadvantages of using toxic organic solvents, large particle size and uneven dispersion, low stability and difficult industrialization.

In order to expand the scope of application of thymol and improve its water dispersibility, stability and physiological utilization, the present invention adopts thin film dispersion method to prepare thymol nano-liposomes, and develops a water-dispersed liposome with antioxidant and antibacterial effects simultaneously. Sexual food additives.

Contents of the invention

The object of the present invention is to provide a kind of thymol nano liposome and preparation method thereof, as a kind of novel food, cosmetic additive, to improve the dispersibility, stability and bioavailability of thymol.

To achieve the above object, the present invention provides the following technical solutions:

A thymol nanoliposome, the formula of the thymol nanoliposome is as follows: in terms of mass components: 9-25 parts of thymol, 90-200 parts of soybean lecithin, distearyl Phosphatidylacetamide-methoxypolyethylene glycol 5000 4-10 parts, cholesterol 3-8 parts, antioxidant 0.5-1.2 parts and Tween 80 0.15-0.4 parts.

Preferably, the thymol is a natural product extracted from thyme oil and oregano oil.

Preferably, the cholesterol is egg yolk cholesterol.

Preferably, the antioxidants are vitamin E and epigallocatechin gallate.

The present invention also provides a kind of preparation method of thymol nano liposome, comprises the steps:

S1. Weigh thymol, vitamin E, soybean lecithin and distearoylphosphatidylacetamide-methoxypolyethylene glycol 5000, and dissolve them in edible alcohol through magnetic stirring;

S2, the solution obtained in S1 is evaporated under reduced pressure to remove the solvent, and the solute forms a uniform film;

S3. Adding phosphate buffer solution and Tween 80 for dissolving epigallocatechin gallate into the film, and ultrasonicating in an ice bath for 8-10 minutes to obtain thymol nanoliposomes.

Preferably, in S1, the mass ratio of soybean lecithin to distearoylphosphatidylacetamide-methoxypolyethylene glycol 5000 is 10-20:1, and the mass ratio of thymol to soybean lecithin is 1:8 -14. The mass ratio of vitamin E to soybean lecithin is 0.005-0.01:1, the magnetic stirring time is 0.5-2h, and the solid-liquid ratio is 5:1 based on mg/mL.

Preferably, in S2, the decompression evaporation temperature is 40° C., and the vacuum degree is 0.05-0.1 Mpa.

Preferably, in S3, the buffer is a 0.05M phosphate buffer with pH 7.2, and according to the liquid-solid ratio in mg/ml, the volume of the buffer is 10-15 times the amount of soybean lecithin, as shown in the table The solid-liquid ratio of gallocatechin gallate to phosphate buffer solution is 0.0001-0.0002:1, and the volume ratio of Tween 80 to buffer solution is 0.002-0.005:1.

Preferably, in S3, the ice bath has an ultrasonic power of 200w, ultrasonic for 2s, and stops for 2s.

Technical effect of the present invention and advantage: a kind of thymol nanoliposome provided by the invention, compared with traditional technology, the present invention has the following characteristics:

(1) Most of the raw materials used are food-grade or natural sources, avoiding the introduction of toxic reagents, and have higher safety;

(2) Vitamin E and epigallocatechin gallate in the formula synergistically play an anti-oxidative effect, prevent phospholipid oxidation, and increase the stability of liposomes;

(3) The particle size of the prepared thymol nanoliposomes is 96-105nm, the polydispersity index is 0.196-0.201, the encapsulation efficiency is 86.1-89.3%, and the storage at room temperature for 12 days can still maintain good stability;

(4) The method is simple to operate, and it is easier to realize large-scale industrial production.

Description of drawings

Figure 1 is a transmission electron micrograph of thymol nanoliposome (×20000).

Fig. 2 is a time-varying graph of particle size and encapsulation efficiency of thymol nanoliposomes.

detailed description

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. The specific embodiments described here are only used to explain the present invention, not to limit the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Example 1

A thymol nanoliposome, the formula of the thymol nanoliposome is as follows: according to mass components: its thymol 10mg, soybean lecithin 100mg, distearoylphosphatidyl acetamide-formazine Oxypolyethylene glycol 5000 5mg, cholesterol 3mg, vitamin E 0.5mg, epigallocatechin gallate 0.2mg, Tween 80 0.16ml.

Weigh 10mg thymol, 100mg soybean lecithin, 5mg distearoylphosphatidylacetamide-methoxy polyethylene glycol 5000 and 0.5mg vitamin E, and dissolve them in 23ml edible alcohol after 1h magnetic stirring; 0.1Mpa, the temperature is 40 ℃, and the solvent is evaporated under reduced pressure, and the solute forms a uniform film; 80ml of phosphate buffer solution with 0.2mg of epigallocatechin gallate and 0.16ml of Tween 80 are added to the film , ultrasonication in an ice bath for 10min (200W, 2s on, 2s off), to obtain thymol nanoliposomes.

The prepared thymol nanoliposomes had an average particle diameter of 96.39nm, a PDI of 0.201, an average encapsulation efficiency of 88.2%, and a specific shape of a nearly spherical structure.

Example 2

A thymol nanoliposome, the formula of the thymol nanoliposome is as follows: according to mass components: its thymol 10mg, soybean lecithin 80mg, distearoylphosphatidylacetamide-formazine Oxy-polyethylene glycol 5000 4mg, cholesterol 4mg, vitamin E 0.4mg, epigallocatechin gallate 0.25mg, Tween 80 0.2ml.

Take by weighing 10mg thymol, 80mg soybean lecithin, 4mg distearoylphosphatidylacetamide-methoxy polyethylene glycol 5000 and 0.4mg vitamin E, dissolve in 18.8ml edible alcohol through 1.5h magnetic stirring; The vacuum degree is 0.08Mpa, and the solvent is evaporated under reduced pressure at a temperature of 40°C, and the solute forms a uniform film; 100ml phosphate buffer solution and 0.2ml Tween 80 dissolved in 0.25mg epigallocatechin gallate are added to Thymol nanoliposomes were obtained in the thin film by ultrasonication in an ice bath for 8 minutes (200W, 2s on, 2s off).

The prepared thymol nanoliposomes had an average particle diameter of 98.12nm, a PDI of 0.196, an average encapsulation efficiency of 89.3%, and a specific shape of a nearly spherical structure.

Example 3

A thymol nanoliposome, the formula of the thymol nanoliposome is as follows: according to mass components: its thymol 20mg, soybean lecithin 180mg, distearoylphosphatidyl acetamide-formazine Oxy-polyethylene glycol 5000 9mg, cholesterol 3mg, vitamin E 0.9mg, epigallocatechin gallate 0.25mg, Tween 80 0.16ml.

Weigh 20mg thymol, 180mg soybean lecithin, 9mg distearoylphosphatidylacetamide-methoxypolyethylene glycol 5000 and 0.9mg vitamin E, and dissolve them in 41.8ml edible alcohol after 2h magnetic stirring; The solvent is evaporated under reduced pressure at a temperature of 1Mpa and a temperature of 40°C, and the solute forms a uniform film; 180ml of phosphate buffer solution dissolved with 0.25mg of epigallocatechin gallate and 0.36ml of Tween 80 are added to the film , ultrasonication in an ice bath for 10min (200W, 2s on, 2s off), to obtain thymol nanoliposomes.

The prepared thymol nanoliposomes had an average particle diameter of 102.88nm, a PDI of 0.208, an average encapsulation efficiency of 86.1%, and a specific shape of a nearly spherical structure.

Example 4 Determination of particle size and polydispersity index of thymol nanoliposomes

The liposomes were appropriately diluted with PBS buffer solution, added to a special cuvette, and the average particle size and polydispersity index were measured by a nanometer particle size analyzer.

Example 5 Determination of Thymol Nanoliposome Encapsulation Efficiency

Pour the thymol nanoliposome suspension into a 125 mL pear-shaped separating funnel, add 20 mL of petroleum ether (30-60°C), shake to make it fully mix with the thymol nanoliposome, and statically Separate the layers, remove petroleum ether by rotary evaporation, dissolve the residue in the round-bottomed flask with ethyl acetate and dilute to 25 mL, then take 1 mL into a 10 mL volumetric flask, and dilute to the mark with ethyl acetate;

The ethyl acetate solution prepared by the same method with blank liposomes was used as a blank control solution, and the absorbance value A was measured, and the content of unembedded thymol was calculated according to the standard curve equation;

The calculation formula is: encapsulation efficiency=[(total thymol amount - unembedded thymol amount)/total thymol amount]×100%

Example 6 Morphological observation of thymol nanoliposomes

After diluting thymol nanoliposomes with PBS buffer to an appropriate multiple, take an appropriate amount and drop it on the front of the copper grid, use filter paper to blot excess samples, add 2% phosphotungstic acid solution for negative staining for 4 minutes, and then use filter paper to blot dry The excess dye solution was dried at room temperature and observed with a transmission electron microscope (TEM).

The measurement results are shown in Figure 1-2.

Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, it still The technical solutions recorded in the foregoing embodiments may be modified, or some of the technical features may be replaced equivalently, and any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the within the protection scope of the present invention.

Claims (9)

1. a thymol nano-liposome, is characterized in that: the prescription of described thymol nano-liposome is as follows: according to mass components: its thymol 9-25 parts, soybean lecithin 90-200 4-10 parts of distearoylphosphatidylacetamide-methoxy polyethylene glycol 5000, 3-8 parts of cholesterol, 0.5-1.2 parts of antioxidant and 0.15-0.4 parts of Tween 80. 2. a kind of thymol nano liposome according to claim 1, is characterized in that: described thymol is the natural product that extracts from thyme oil, oregano oil. 3. a kind of thymol nanoliposome according to claim 1, is characterized in that: described cholesterol is egg yolk cholesterol. 4. a kind of thymol nanoliposome according to claim 1, is characterized in that: described antioxidant is vitamin E and epigallocatechin gallate. 5. a preparation method of thymol nano liposomes as claimed in claim 1, is characterized in that: comprises the steps: S1. Weigh thymol, vitamin E, soybean lecithin and distearoylphosphatidylacetamide-methoxypolyethylene glycol 5000, and dissolve them in edible alcohol through magnetic stirring; S2, the solution obtained in S1 is evaporated under reduced pressure to remove the solvent, and the solute forms a uniform film; S3. Adding phosphate buffer solution and Tween 80 for dissolving epigallocatechin gallate into the film, and ultrasonicating in an ice bath for 8-10 minutes to obtain thymol nanoliposomes. 6. the preparation method of a kind of thymol nano liposome according to claim 5 is characterized in that: in S1, soybean lecithin and distearoylphosphatidylacetamide-methoxy polyethylene glycol The mass ratio of 5000 is 10-20:1, the mass ratio of thymol to soybean lecithin is 1:8-14, the mass ratio of vitamin E to soybean lecithin is 0.005-0.01:1, and the magnetic stirring time is 0.5-2h. The unit is mg/mL, and the solid-liquid ratio is 5:1. 7. The preparation method of a thymol nanoliposome according to claim 5, characterized in that: in S2, the decompression evaporation temperature is 40°C, and the vacuum degree is 0.05-0.1Mpa. 8. the preparation method of a kind of thymol nanoliposome according to claim 5 is characterized in that: in S3, buffer is the phosphate buffer of 0.05M of pH7.2, according to mg/ml The liquid-solid ratio is the unit, the buffer volume is 10-15 times the amount of soybean lecithin, the solid-liquid ratio of epigallocatechin gallate and phosphate buffer is 0.0001-0.0002:1, Tween 80 and The volume ratio of the buffer is 0.002-0.005:1. 9. The preparation method of a kind of thymol nanoliposome according to claim 5, characterized in that: in S3, the ultrasonic power of the ice bath is 200w, the ultrasound is 2s, and the suspension is 2s.