CN113476317A

CN113476317A - Antioxidant liposome and preparation method thereof

Info

Publication number: CN113476317A
Application number: CN202110744890.7A
Authority: CN
Inventors: 卢永杰; 程路诗; 张炽坚; 张兵; 江桦; 关焕敏; 艾勇; 何廷刚
Original assignee: Hua An Tang Biotech Group Co ltd
Current assignee: Hua An Tang Biotech Group Co ltd
Priority date: 2021-06-30
Filing date: 2021-06-30
Publication date: 2021-10-08

Abstract

The invention relates to the technical field of liposome, in particular to an antioxidant liposome and a preparation method thereof. The invention discloses an antioxidant liposome, which uses Magnolia sieboldii extract water as a preparation solvent of the liposome, and the obtained liposome has antioxidant effect. In addition, the liposome does not use toxic and harmful organic solvents, so that the production process is safe and environment-friendly, and the liposome is high in safety. The liposome can be applied in cosmetics to impart antioxidant function or enhance antioxidant effect.

Description

Antioxidant liposome and preparation method thereof

Technical Field

The invention relates to the technical field of liposome, in particular to an antioxidant liposome and a preparation method thereof.

Background

In recent years, liposome has attracted much attention and is widely used in the fields of biomedicine, cosmetics, health foods and the like.

Liposomes are lipid bilayers or multilayers formed mainly of phospholipids, having a structure similar to a cell membrane. In the field of cosmetics, as means for delivering active ingredients having a moisturizing function, an antioxidant effect, a whitening effect, and the like to the skin, the active ingredients are often encapsulated in liposomes. However, no research on the function of the liposome carrier itself such as antioxidation exists at present.

Disclosure of Invention

In view of the above, the present invention provides an antioxidant liposome and a preparation method thereof, wherein the liposome has an antioxidant effect.

The specific technical scheme is as follows:

the invention provides an antioxidant liposome, which comprises: comprises the following components in percentage by mass:

0.01-5% of a medicament;

2-20% of liposome-forming lipid substances;

the balance of the magnolia sieboldii extract water;

the drug comprises a fat-soluble drug.

Magnolia sieboldii, a deciduous tree or shrub of Magnolia genus of Magnoliaceae family, has white petals, purple stamen, attractive fragrance, and similar shape to Magnolia sieboldii. The Magnolia sieboldii is a rare and famous flower which survives in the age of glacier in the fourth century of the Taigu, is listed in the name of endangered plants in China, is a rare plant in the world which is mainly protected in China, has the leaves as jade carving, is cut like jade, has strong fragrance, and is an important light industrial raw material for landscaping tree species and spice extraction.

The flower, leaf and stem of Magnolia sieboldii contain aromatic oil, and the red shell of flower seed can be used for extracting high-grade perfume. The natural perfume can be widely used in food, chemical industry, medicine, cigarette, candy and cosmetics, and is the best product in flavoring agents. The Magnolia sieboldii is not only specific aromatic, but also white and beautiful, and has long florescence, so the Magnolia sieboldii is a precious ornamental tree species for greening and beautifying courtyards. The woodiness of the Magnolia sieboldii is also excellent, and the Magnolia sieboldii can be used as furniture, agricultural tools and carving materials.

Magnolia sieboldii has strong skin care effect. The invention uses the magnolia sieboldii extract water as the preparation solvent of the liposome, and the obtained liposome has the anti-oxidation effect. The antioxidant liposome provided by the application is applied to cosmetics, and if the active ingredient to be encapsulated in the liposome is an antioxidant substance, the antioxidant liposome can synergistically enhance the antioxidant effect of the active ingredient; if the active ingredients are substances for whitening or moisturizing, the antioxidant liposome can endow cosmetics with antioxidant function.

In the invention, the Magnolia sieboldii extract water is obtained by distilling Magnolia sieboldii under a low-temperature vacuum condition. The distillation temperature is 30-50 ℃, the pressure is-70 to-90 kPa, and the time is 5-8 h.

In the present invention, the liposome-forming lipid material comprises, by mass: 2-15% of phospholipid and 0-5% of cholesterol.

Preferably, the amount of the surfactant is, in mass percent,

2-10% of phospholipid;

0.01-4% of a medicament;

0-4% of cholesterol;

the balance being water.

More preferably, the amount of the surfactant is, in mass percent,

2-8% of phospholipid;

0.1-3% of a medicament;

0-3% of cholesterol.

The phospholipid comprises one or more of soybean lecithin, egg yolk lecithin, hydrogenated lecithin, dilauroyl phosphatidylcholine, dimyristoyl phosphorylcholine, dipalmitoyl phosphorylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine and dioleoyl phosphatidylethanolamine.

The invention also comprises the following components in percentage by mass: 0.1 to 20% of a surfactant, preferably 0.1 to 15%, more preferably 0.1 to 8%.

The surfactant is ionic surfactant and/or nonionic surfactant, wherein the cationic surfactant comprises quaternary ammonium salt, the anionic surfactant comprises higher fatty acid salt or sulfate salt, and the nonionic surfactant comprises one or more than two of tween, span, PEG fatty acid ester and polyglycerol-10-hard fatty acid ester.

The invention also comprises the following components in percentage by mass: 5 to 60% of a polyol, preferably 5 to 45%, more preferably 5 to 30%.

The polyhydric alcohol comprises one or more of glycerol, propylene glycol, butylene glycol, pentylene glycol, and isoprene glycol.

In the present invention, the fat-soluble drugs include: one or more of glabridin, ceramide, resveratrol and fat-soluble vitamins, preferably glabridin. In the invention, the antioxidant effect of the liposome prepared by compounding the magnolia sieboldii extract water and the glabridin is obvious because the magnolia sieboldii extract water and the glabridin liposome without the magnolia sieboldii extract water are added.

The medicament further comprises: a water-soluble drug. The water-soluble medicine comprises one or more than two of sodium hyaluronate, nano metal colloid dispersion liquid and water-soluble vitamins.

When the medicament is a fat-soluble medicament and a water-soluble medicament, the dosage ratio of the fat-soluble medicament and the water-soluble medicament is not particularly limited.

The invention also provides a preparation method of the antioxidant liposome, which comprises the following steps:

step 1: mixing lipid substance and medicine for forming liposome with the extract water of Magnolia sieboldii to form suspension;

step 2: and shearing the suspension at a high speed under the heating and heat preservation state to obtain the liposome emulsion.

Lipid substances and slightly soluble medicines which form the liposome are mixed with purslane plant extract water to obtain suspension, and the suspension is dispersed by high-speed shearing at a proper temperature, so that the phospholipid and the slightly soluble medicines are crushed to form a micron scale. Under the micron scale, after long-time shearing, the insoluble drug particles and the phospholipid particles interact to emulsify, and then self-assemble to form the micron-scale liposome.

The preparation equipment and the process of the antioxidant liposome provided by the invention are simple, and the preparation method is suitable for large-scale production and improves the production efficiency.

Before the mixing in step 1, the invention further comprises: adding a surfactant. The addition of a surfactant can facilitate the solubilization of the fat-soluble drug.

Before mixing, the method also comprises the following steps: the polyol is added. The addition of the polyol can further facilitate the dissolution of a portion of the fat-soluble drug.

In step 2 of the invention, the heating temperature of the suspension liquid needs to be higher than the phase transition temperature of the phospholipid. The heating temperature is 50-100 ℃, and preferably 50-70 ℃; then, maintaining the heating temperature for shearing, wherein a shearing disperser is adopted for shearing; the shearing rate is 8000-20000 rpm (rotor diameter is 13mm), the shearing time is more than 30min, preferably more than 60min at the shearing rate of 8000-15000 rpm (rotor diameter is 13mm), more preferably 60-90 min. The liposome obtained after shearing is micron-sized.

The nano-scale liposome has better dispersibility and stability and better skin permeability. Therefore, after the high-speed shearing is performed in step 2 of the present invention, the method further comprises: homogenizing under high pressure. The high-pressure homogenization can crush and thin the micron-sized liposome to obtain the nano-sized liposome. The high-pressure homogenization specifically comprises the following steps: each cycle was carried out at a pressure of 400bar, 600bar, 800bar, 1000bar for 2 or more times.

According to the technical scheme, the invention has the following advantages:

the invention provides an antioxidant liposome, which uses Magnolia sieboldii extract water as a preparation solvent of the liposome, and the obtained liposome has antioxidant effect. In addition, the liposome does not use toxic and harmful organic solvents, so that the production process is safe and environment-friendly, and the liposome is high in safety. The liposome can be applied in cosmetics to impart antioxidant function or enhance antioxidant effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.

FIG. 1 is an optical microscope photograph (3 μm on scale) of liposomes in an emulsion prepared in step 1 of example 2 of the present invention;

FIG. 2 is an optical microscope photograph (3 μm on scale) of liposomes in the emulsion obtained in step 1 of example 3 of the present invention;

FIG. 3 is an optical microscope photograph (3 μm on scale) of liposomes in the emulsion obtained in step 1 of example 4 of the present invention;

FIG. 4 is an optical microscope photograph (3 μm on scale) of liposomes in the emulsion obtained in step 1 of example 5 of the present invention;

FIG. 5 is an optical microscope photograph (3 μm on scale) of liposomes in the emulsion obtained in step 1 of example 6 of the present invention;

FIG. 6 is an optical microscope photograph (3 μm on scale) of liposomes in an emulsion prepared in step 1 of example 7 of the present invention;

FIG. 7 is an optical microscope photograph (3 μm on scale) of liposomes in an emulsion prepared in step 1 of example 9 of the present invention;

FIG. 8 is a graph showing the results of measurement of intracellular reactive oxygen species content in test example 3 of the present invention.

Detailed Description

In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be apparent that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The reagents in the examples of the present invention are all commercially available.

Wherein, the high-speed homogeneous dispersion machine of experimental type: IKA, Germany.

Experimental high-pressure homogenizer: suzhou ATS Inc.

Zeta nanometer particle size analyzer: malvern, uk.

Reagent:

soybean lecithin: PC > 95%, Sigma-Aldrich Sigma Aldrich trade company, Shanghai.

Glabridin: HPLC purity is more than or equal to 90 percent, Qinghai lake pharmaceutical industry Co.

Cholesterol: purity > 96%, Sigma-Aldrich Sigma Aldrich trade ltd.

Polyglycerol-10-stearate: heliochemical trade (shanghai) limited.

Butanediol: purity > 98%, manufactured by Nippon Daiillo Co., Ltd.

Example 1

The embodiment is a preparation method of magnolia sieboldii extract water, and the preparation method comprises the following specific steps:

selecting fresh Magnolia sieboldii as a raw material, adding no external water and solvent, distilling the water and other volatile substances in the plant under the low-temperature vacuum condition (extraction temperature of 35 ℃, extraction pressure of-90 kPa, extraction time of 6h), and extracting to obtain Magnolia sieboldii extract water.

Example 2

The preparation formula of the antioxidant liposome is shown in table 1, and the preparation steps are as follows:

(1) adding phospholipid (PC 95%), glabridin and cholesterol into the amount of Magnolia sieboldii extract water, heating in water bath at 50 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 30min to obtain uniform emulsion.

(2) And (2) homogenizing the uniform emulsion obtained in the step (1) under high pressure, and circulating for 2 times under 400/600/800/1000bar pressure respectively to obtain uniform antioxidant liposome.

As can be seen in FIG. 1, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the emulsion in step 1.

Examples 3 to 5

(1) adding phospholipid (PC 95%), glabridin and cholesterol into the amount of Magnolia sieboldii extract water, heating in water bath at 60 deg.C, and high-speed shearing with experiment type IKA high-speed shearing disperser at 10000rpm for 60min to obtain uniform emulsion.

As can be seen from FIGS. 2 to 4, after the high-speed shearing in step 1, a multi-layer spherical structure similar to liposome can be observed in the emulsion in step 1.

Example 6

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into the amount of Magnolia sieboldii plant extraction water, heating in a water bath at 50 ℃, and high-speed shearing for 30min at 10000rpm by using an experimental IKA high-speed shearing dispersion machine to obtain uniform emulsion.

As can be seen in FIG. 5, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the emulsion in step 1.

Examples 7 to 8

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixed solution of Magnolia sieboldii extract water and butanediol, heating in a water bath at 50 ℃, and shearing at a high speed of 10000rpm for 30min by using an experimental IKA high-speed shearing dispersion machine to obtain a uniform emulsion.

As can be seen in FIG. 6, in example 7, after high shear in step 1, a multi-lamellar globular structure resembling liposomes is observed in the emulsion in step 1.

Example 9

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into the amount of Magnolia sieboldii extract water, heating in water bath at 60 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 60min to obtain uniform emulsion.

Example 10

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixed solution of Magnolia sieboldii plant extract water and butanediol, heating in a water bath at 50 ℃, and shearing at a high speed of 8000rpm for 30min by using an experimental IKA high-speed shearing dispersion machine to obtain a uniform emulsion.

(2) And (2) homogenizing the uniform emulsion obtained in the step (1) under high pressure, and circulating for 2 times under 400/600/800/1000bar pressure respectively to obtain transparent and uniform antioxidant liposome.

Example 11

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixed solution of Magnolia sieboldii plant extract water and butanediol, heating in a water bath at 60 ℃, and shearing at a high speed of 10000rpm for 30min by using an experimental IKA high-speed shearing dispersion machine to obtain a uniform emulsion.

Example 12

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixed solution of Magnolia sieboldii plant extract water and butanediol, heating in a water bath at 50 ℃, and shearing at a high speed of 10000rpm for 60min by using an experimental IKA high-speed shearing dispersion machine to obtain a uniform emulsion.

Example 13

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixed solution of Magnolia sieboldii plant extract water and butanediol, heating in a water bath at 50 ℃, and shearing at a high speed of 10000rpm for 90min by using an experimental IKA high-speed shearing dispersion machine to obtain a uniform emulsion.

Comparative example 1

The formulation of the liposome of this example is shown in table 1, and the specific preparation steps are as follows:

(1) adding phospholipid (PC 95%), glabridin and cholesterol into water of the above formula, heating in water bath at 60 deg.C, and high-speed shearing with experiment type IKA high-speed shearing disperser at 10000rpm for 60min to obtain uniform emulsion.

(2) And (2) homogenizing the uniform emulsion obtained in the step (1) under high pressure, and circulating for 2 times under 400/600/800/1000bar pressure respectively to obtain uniform liposome.

Comparative examples 2 to 3

The comparative example is the preparation of glabridin liposome, the formula is shown in table 1, and the preparation steps are as follows:

(1) dissolving phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol in 200g methanol-chloroform (mass ratio of 62:38) to obtain clear and transparent solution, and performing rotary evaporation to remove organic solvent to obtain the film.

(2) And (2) placing the film obtained in the step (1) in a water bath at 60 ℃, and adding a mixed solution of the formula amount of the magnolia sieboldii plant extraction water and butanediol while stirring to obtain a crude emulsion. The crude emulsion was homogenized under high pressure and circulated 2 times each at 400/600/800/1000bar to give liposomes.

Comparative example 4

The comparative example is the preparation of liposomes, the formulation is shown in table 1, and the specific preparation steps are as follows:

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into a mixture of water and butanediol, heating in water bath at 30 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 30min to obtain uniform emulsion.

(2) Homogenizing the uniform emulsion obtained in the step (1) under high pressure, and circulating for 2 times under 400/600/800/1000bar pressure to obtain transparent and uniform liposome.

TABLE 1 example and comparative example formulations

Test example 1

The liposomes prepared in example 9 and comparative 1 were dispersed using PBS as a dispersing agent, and the average particle diameter was measured using a Zeta sizer; separating the encapsulated and free glabridin by an ultrafiltration centrifugal tube, detecting the glabridin content by HPLC and calculating the encapsulation rate. Test results are shown in table 2.

As can be seen from the results in Table 2, the appearance, average particle size and glabridin content of the antioxidant liposomes prepared in most of the examples are substantially the same as those of the liposomes prepared in the film method of comparative examples 2 to 3, which indicates that the properties of the liposomes prepared by the preparation method of the examples of the present invention are substantially the same as those of the liposomes prepared by the film method. In example 9, compared to the glabridin liposome prepared in comparative example 1, there was substantially no difference in appearance, average particle size, and glabridin content, which indicates that the liposome prepared in the example using magnolia sieboldii plant extract water instead of pure water had substantially the same effect, and may be used as a solvent instead of pure water. The glabridin liposome prepared by the liposome preparation method of comparative example 4 is in a yellow turbid state in appearance, and insoluble particles exist; the sample has larger particle size, the encapsulation efficiency of the glabridin medicament is obviously reduced, which shows that when the temperature is insufficient, the phospholipid phase cannot be fully converted into the liquid crystal phase, and fat-soluble medicaments such as glabridin and the like are difficult to fully react with phospholipid molecules, so that the particle size is increased, the encapsulation efficiency is reduced, and the quality of a liposome preparation is reduced.

Table 2 characterization results of glabridin liposomes of examples and comparative examples

Test example 2

The liposomes prepared in the examples and comparative examples were subjected to preservative challenge tests.

Inoculating certain amount of bacteria and fungi, and detecting the change of microbial quantity according to the detection method of the preservative efficacy test of the microorganisms in United states Pharmacopeia USP32<51> at intervals of 0 day, 7 days, 14 days, 21 days and 28 days, wherein the test results are shown in Table 1.

Microorganism detection was carried out for example 9 and comparative example 1 according to the national Standard GB7918.2-87 "Standard test method for microorganisms for cosmetics

As can be seen from tables 3 and 4, the purslane plant extract aqueous glabridin liposome prepared in the examples can inhibit the growth of microorganisms and has a certain preservative effect.

Table 3 preservative challenge testing of the liposomes of the examples and comparative examples

TABLE 4

Test example 3

The test of the cellular active oxygen content was performed on the magnolia sieboldii extract water prepared in example 1, and the liposome samples prepared in example 9 and comparative example 1.

According to a cell density of 1.5X 10⁵One/well seeded in 6-well plates, CO at 37 ℃₂Culturing and incubating in an incubator overnight,when the cells fused to 40% of the total volume, the samples were treated and incubated in an incubator for 24h, and then light treatment was performed. A solvent (deionized water) control (UVA-), a solvent control (UVA +), a sample group (UVA +, samples of example 1, example 9, and comparative example 1), and an epigallocatechin gallate (EGCG) positive control group (UVA +), were set, with 3 duplicate wells per group. Wherein the concentration of the sample group is 2 percent, and the concentration of EGCG is 62.5 ug/ml. UVA induction group adopts 5J/cm²The UVA of (a) is irradiated and the solvent control (UVA-) is treated in the dark. After irradiation, the cells of the experimental group are washed by PBS, 1ml of DCFH-DA probe with the concentration of 25uM is added into each hole, the cells are incubated for 45min in a cell incubator at 37 ℃, a culture solution containing the DCFH-DA probe is discarded, the cells are washed by PBS, the cells are digested by pancreatin, then the cells are washed by PBS, and finally fresh PBS is added. The average fluorescence intensity values were calculated by flow cytometry and the results are shown in FIG. 1.

The fluorescence intensity represents the active oxygen content, and the higher the fluorescence intensity, the higher the active oxygen content. After the cells are irradiated by UVA, the content of active oxygen in the cells is obviously increased. As shown in fig. 8, after glabridin liposome or glabridin extract water is added to the experimental group, the content of active oxygen in cells can be effectively reduced, and the experiment group has obvious antioxidation effect, and the liposome obtained by compounding the glabridin extract water and the glabridin has enhanced antioxidation effect, can reduce more active oxygen content, and shows that the glabridin extract water and the glabridin have antioxidation synergistic effect.

The above-mentioned embodiments are only used for illustrating the technical solutions of the present invention, and not for limiting the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. An antioxidant liposome, comprising: comprises the following components in percentage by mass:

0.01-5% of a medicament;

2-20% of liposome-forming lipid substances;

the balance of the magnolia sieboldii extract water;

the drug comprises a fat-soluble drug.

2. The antioxidant liposome of claim 1, wherein the Magnolia sieboldii extract water is obtained by distilling Magnolia sieboldii under low temperature vacuum condition.

3. The antioxidant liposome of claim 1, wherein the liposome-forming lipid material comprises, in mass percent: 2-15% of phospholipid and 0-5% of cholesterol.

4. The antioxidant liposome of claim 1, further comprising, in mass percent: surfactants and/or polyols.

5. The antioxidant liposome of claim 1, wherein the lipid soluble drug comprises: one or more of glabridin, ceramide, resveratrol and fat-soluble vitamins.

6. The preparation method of the antioxidant liposome is characterized by comprising the following steps:

7. The method according to claim 6, wherein the shearing rate in step 2 is 8000rpm or more and the time is 30min or more.

8. The method of claim 6, wherein the temperature of the heating and incubating in step 2 is above the phase transition temperature of the phospholipid in the lipid material.

9. The method of claim 6, wherein the step 2 further comprises, before obtaining the liposome emulsion: homogenizing under high pressure;

the high-pressure homogenization specifically comprises the following steps: each cycle was carried out at a pressure of 400bar, 600bar, 800bar, 1000bar for 2 or more times.

10. The method according to claim 6, wherein the step 1 further comprises adding a surfactant and/or a polyol before the mixing.