CN113456592A

CN113456592A - Antiseptic and anti-inflammatory liposome and preparation method thereof

Info

Publication number: CN113456592A
Application number: CN202110741846.0A
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: 2021-10-01

Abstract

The invention relates to the technical field of liposome, in particular to an antiseptic and anti-inflammatory liposome and a preparation method thereof. The invention discloses a preservative and anti-inflammatory liposome, which uses purslane plant extract water as one of raw materials for preparing the liposome, so that the obtained liposome has natural preservative and anti-inflammatory effects, and no preservative is required to be additionally added for preserving the liposome. 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.

Description

Antiseptic and anti-inflammatory liposome and preparation method thereof

Technical Field

The invention relates to the technical field of liposome, in particular to an antiseptic and anti-inflammatory 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. However, the liposome is not easy to store, and a chemical preservative is mostly added to store the liposome, so that the safety risk of the product is increased.

Disclosure of Invention

In view of the above, the invention provides an antiseptic and anti-inflammatory liposome and a preparation method thereof, the liposome has a natural antiseptic effect, no preservative is required to be additionally added, and the product safety is high.

The invention provides an antiseptic and anti-inflammatory liposome which comprises the following components in percentage by mass:

2-15% of phospholipid;

0.01-5% of a medicament;

0-5% of cholesterol;

the balance of purslane plant extract water;

the drug comprises a fat-soluble drug.

Purslane (Portulaca oleracea Linn.) is a succulent herbaceous plant of Portulaca genus of Portulacaceae family, namely Portulaca oleracea, Gypsophila japonica, longmingcoloid, Changshoucai and the like, is one of 78 medicinal and edible wild plants defined by China ministry of health in temperate and tropical areas all over the world, and is collected in Chinese pharmacopoeia. According to the invention, the purslane plant extract water is used as one of raw materials for preparing the liposome, so that the obtained liposome has natural preservative and anti-inflammatory effects, and no preservative is required to be additionally added for preserving the liposome.

The antiseptic anti-inflammatory liposome provided by the invention 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.

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.

In the invention, the purslane plant extract water is obtained by distilling purslane under the 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 invention, the preparation raw materials of the antiseptic and anti-inflammatory liposome also comprise 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.

In the invention, the preparation raw materials of the antiseptic and anti-inflammatory liposome also comprise 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.

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 fat-soluble medicine comprises: one or more of glabridin, ceramide, resveratrol and fat-soluble vitamins, preferably glabridin. The invention discovers that the anti-inflammatory effect of the liposome can be synergistically enhanced by compounding the glabridin and the purslane plant extract water.

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 preservative and anti-inflammatory liposome, which comprises the following steps:

step 1: mixing phospholipid, cholesterol, medicine and herba Portulacae plant extract water to form suspension;

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

The preparation method comprises the steps of mixing phospholipid, insoluble drugs and purslane plant extract water to obtain suspension, dispersing the suspension at a proper temperature through high-speed shearing, and crushing the phospholipid and the insoluble drugs 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 antiseptic and anti-inflammatory liposome provided by the invention are simple, and the antiseptic and anti-inflammatory liposome 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 a preservative and anti-inflammatory liposome, which uses purslane plant extract water as one of raw materials for preparing the liposome, so that the obtained liposome has natural preservative and anti-inflammatory effects, and no preservative is required to be additionally added for preserving the liposome. 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.

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 the IL-1 content test provided in test example 3 of the present invention;

FIG. 9 is a graph showing the results of the FLG content test provided 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 purslane extraction water, and the preparation method comprises the following specific steps:

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

Example 2

The preparation formula of the antiseptic and anti-inflammatory liposome is shown in table 1, and the preparation steps are as follows:

(1) adding phospholipid (PC 95%), glabridin and cholesterol into herba Portulacae extract water, heating in water bath at 50 deg.C, and high-speed shearing with experiment type 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 to obtain the uniform antiseptic and anti-inflammatory 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 herba Portulacae 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 herba Portulacae plant extract water, heating in water bath at 50 deg.C, and high-speed shearing with experiment type IKA high-speed shearing disperser at 10000rpm for 30min 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 the mixture of herba Portulacae extract water and butanediol, 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.

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 herba Portulacae 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.

Example 10

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into the mixture of purslane plant extract water and butanediol, heating in water bath at 50 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 8000rpm 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 to obtain the transparent and uniform antiseptic and anti-inflammatory liposome.

Example 11

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into the mixed solution of purslane plant extract water and butanediol, heating in water bath at 60 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 30min to obtain uniform emulsion.

Example 12

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into the mixture of purslane plant extract water and butanediol, heating in water bath at 50 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 60min to obtain uniform emulsion.

Example 13

(1) adding phospholipid (PC 95%), polyglycerol-10-stearate, glabridin and cholesterol into the mixture of purslane plant extract water and butanediol, heating in water bath at 50 deg.C, and high-speed shearing with an experimental IKA high-speed shearing disperser at 10000rpm for 90min to obtain 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, 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 liposomes, the formulation is shown in table 1, and the specific 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 purslane plant extraction water and butanediol in a prescribed amount 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

(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 antiseptic and anti-inflammatory liposomes prepared in most of the examples have substantially no difference in appearance, average particle size and glabridin content from the liposomes prepared by the thin film method in the 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 thin film method. Example 9 compared with 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 effect of liposome prepared by using purslane plant extract water instead of pure water was substantially uniform, and the liposome was able to replace pure water as a solvent. 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 data 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 3.

The microbial detection was carried out according to the national standard GB7918.2-87 "cosmetic microbial Standard test method", and the test results are shown in Table 4.

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 test results for liposomes of examples and comparative examples

TABLE 4

Test example 3

The liposomes prepared in example 9 and comparative example 1 were evaluated for anti-inflammatory ability of the samples in an animal skin allergy model.

The experimental method comprises the following steps:

SPF mice 5 weeks old were acclimatized for 1 week, and the skin on the back of the mice was removed with depilatory cream. Mice were divided into blank group, model group, positive group, glabridin liposome group (comparative example 1), glabridin liposome complex purslane extraction water group (example 9), and purslane extraction water group.

The administration period is once a day, and the blank group and the model group are smeared with propylene glycol (200 uL/mouse) on the back of the mouse; positive group mice were smeared on their backs with 0.5% sodium hyaluronate in propylene glycol (200 uL/mouse); the glabridin liposome group was smeared on the back of mice with the sample (200 uL/mouse) prepared in comparative example 1; the glabridin liposome-compounded purslane extraction water group is smeared on the back of a mouse with the sample (200 uL/mouse) prepared in the example 9; purslane extract water group purslane extract water (200 uL/mouse) was smeared on the back of mice. The above groups were administered once a day for seven days.

On day seven of dosing, a mouse model of histamine-induced acute pruritus was established. All groups were administered first, and the positive group was given diphenhydramine hydrochloride (physiological saline solution, dose 2mg/ml/10g) by intraperitoneal injection. After all groups had finished administration for 30min, 100uL of histamine (physiological saline solution, 1mg/ml dose) was administered to all groups except the blank group by intraperitoneal injection, and then mice were sacrificed by cervical dislocation, and dorsal skin tissues were fixed in paraformaldehyde and cryopreserved. Skin tissues collected by dissecting each group of mice are prepared into homogenate, and the contents of IL-1 (interleukin-1) and FLG (filaggrin) are detected according to the method of an ELISA kit instruction.

Filaggrin (FLG) and interleukin 1(IL-1) play an important role in the pathological mechanism of skin inflammation, the Filaggrin is an important component of the horny layer, the horny substance forming cells are synthesized and distributed in different parts of the horny layer, and are gradually degraded by enzyme along with the process of the horny substance forming cell migration, so that the Filaggrin is degraded into small molecular substances required by the horny layer of natural moisturizing factors, and the Filaggrin plays an important role in the aspects of moisturizing and barrier integrity. Interleukins play an important role in transmitting information, activating and regulating immune cells, mediating T, B cell activation, proliferation and differentiation, and in inflammatory responses.

As shown in FIG. 8, the model group showed a significant increase in IL-1 content compared to the blank group, indicating that inflammation of the skin occurred after histamine drug injection, resulting in an increase in IL-1 content. The IL-1 content can be reduced in the positive group and the sample group, so that a certain anti-inflammatory effect is achieved, and the glabridin liposome compounded with the purslane extracted water has a more obvious anti-inflammatory effect. As shown in fig. 9, the model group showed a significant reduction in FLG protein content compared to the blank group, indicating that the reduction in FLG is caused by the generation of skin inflammation following histamine drug injection. The positive group and the sample group can resist the reduction of FLG protein content caused by inflammation, which shows that the anti-inflammatory effect is achieved, and the glabridin liposome prepared by compounding purslane extracted water has better anti-inflammatory 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. The preservative and anti-inflammatory liposome is characterized by comprising the following components in percentage by mass:

2-15% of phospholipid;

0.01-5% of a medicament;

0-5% of cholesterol;

the balance of purslane plant extract water;

the drug comprises a fat-soluble drug.

2. The preservative anti-inflammatory liposome of claim 1, further comprising, in mass percent: 0.1-20% of a surfactant.

3. The preservative anti-inflammatory liposome according to claim 1 or 2, further comprising, in mass percent: 5-60% of polyhydric alcohol.

4. The preserved anti-inflammatory liposome of claim 1, wherein the purslane plant extract water is obtained from distillation of purslane under low temperature vacuum conditions.

5. The preserved anti-inflammatory liposome of claim 1, wherein the drug further comprises a water soluble drug.

6. The preparation method of the preservative and anti-inflammatory 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 according to claim 6, wherein the temperature for the heating and holding in step 2 is not lower than the phase transition temperature of the phospholipid.

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.