CN111281851A

CN111281851A - PH-targeted flexible nanoliposome with acne removing effect and preparation method thereof

Info

Publication number: CN111281851A
Application number: CN201911333830.5A
Authority: CN
Inventors: 王立强; 张嘉颖
Original assignee: Cheng Dingyi
Current assignee: Cheng Dingyi
Priority date: 2019-12-10
Filing date: 2019-12-10
Publication date: 2020-06-16

Abstract

The invention relates to a pH-targeted flexible nano liposome with an acne-removing effect and a preparation method thereof. The flexible nano liposome can be added into an external preparation, the difficulties of low skin transmittance, no targeting property and poor safety of effective components in the external preparation are improved by utilizing the deformability of the flexible nano liposome, and the preparation method is simple and convenient and is suitable for preparing similar flexible nano liposomes.

Description

PH-targeted flexible nanoliposome with acne removing effect and preparation method thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a pH-targeted flexible nanoliposome with an acne removing effect and a preparation method thereof.

Background

Acne is a chronic inflammatory disease involving skin hair follicles and sebaceous glands, is mainly related to hyperseborrhea, follicular sebaceous gland duct blockage, bacterial infection and inflammatory reaction, and is clinically manifested as acne, papule, pustule and nodule in the parts with much sebum such as face. The cells in the stratum corneum, the outermost layer of the skin, are tightly connected and form a "brick wall structure" with intercellular lipids, providing a tight and effective physical barrier to the skin. The traditional skin care product has complex active ingredients, many of which are macromolecules, can only stay on the surface or epidermal layer of skin, are difficult to enter the dermal layer, and the skin transmittance is very low and is only 5%. Common acne inflammation (such as pimple, cyst and lump) mostly erodes to the dermis, components with low permeability are difficult to reach the inflammation part, and the treatment and skin care effects are poor. Therefore, there is a need in the art for a new technique for delivering effective ingredients to the dermis and inflammation sites with high efficiency.

Disclosure of Invention

The invention aims to provide a pH targeted flexible nano liposome with an acne removing effect, and solves the problems of low skin transmittance and no targeting of an acne removing active ingredient.

The invention also aims to provide a preparation method of the pH targeted flexible nano liposome with the acne removing effect, which improves the safety, effectiveness and targeting property of the coated active ingredients.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a pH targeted flexible nano liposome with acne removing effect contains N-caprylyl-D-sphingosine (10-30 wt.%), β -sitosterol (1.0-2.5 wt.%), membrane softener (1.0-5.5 wt.%), pH sensitive material (1.0-3.0 wt.%), PEG-4005.0-15.0 wt.%), acne removing active component (2.0-15.0 wt.%) and hydrating solution (rest) for the rest, wherein N-caprylyl-D-sphingosine is the main membrane material of flexible nano liposome, β -sitosterol is the stable supporting material of flexible nano liposome, the flexible nano liposome has deformability due to the addition of membrane softener, the modification of pH targeted material makes the flexible nano liposome release more active components in acidic environment, and PEG-400 can increase the solubility of active components during hydration and increase the encapsulation rate.

After the technical scheme is adopted, the pH targeted flexible nano liposome with the acne removing effect has the following beneficial effects: the flexible nano liposome is used as a transdermal carrier, and the membrane material is added with a membrane softener, so that the flexible nano liposome has self-deformation capability; after the flexible nano liposome is applied to the skin, the stratum corneum can be hydrated firstly to open a channel to promote permeation, lipid components in the flexible liposome have the tendency of avoiding a dry environment, and the water gradient of the surface layer and the deep layer of the skin can promote the permeation, so that the flexible nano liposome can penetrate through the skin barrier more easily and can penetrate into the epidermis layer and the dermis layer to exert the maximum effect. After the active ingredients are wrapped by the flexible nano liposome and penetrate through the horny layer, reservoirs can be formed in epidermis and dermis, so that the active ingredients are slowly released, the acne part is durably treated, the growth of related flora such as propionibacterium acnes and the like in the skin and excessive secretion of grease are inhibited, the inflammation is relieved and the regeneration of tissues at scar positions is promoted; meanwhile, as the inflammation part and the tissue are in an acidic environment, the flexible nano liposome modified by the pH sensitive material can be enriched and released more at the acne part, so that the targeting effect is achieved, the distribution and release of effective components at the healthy part are reduced, and the safety and the effectiveness are greatly improved. After the active ingredients are coated, the solubility and the stability of the active ingredients are improved, and the irritation to direct external application of skin is reduced; the flexible nano liposome is used as a carrier, has no side effect on a human body after metabolism, and is safe and effective.

The film softener comprises one or two of poloxamer 124, fatty amine polyoxyethylene ether, ethoxylated-C12-18-alcohol and cetyl palmitate.

The hydration solution is a composition of potassium dihydrogen phosphate and 0.1mol/L sodium hydroxide, and the pH value is 6.5-8.0.

The pH sensitive material is one or two of poly (N-N-butylacrylamide), diethylhexyl maleate and polyethylene glycol-hydrazone bond-polystyrene copolymer.

The adding ratio of the N-octanoyl-D-sphingosine to the pH sensitive material is 9: 1.

The active ingredients include centella asiatica extract, neem leaf extract, hops extract, polygonum cuspidatum root extract, purslane extract, scutellaria baicalensis root extract, phloretin.

After the technical scheme is adopted, the pH targeted flexible nano liposome with the acne removing effect has the following beneficial effects: according to the mechanism of different occurrence and development stages of the acnes, the active ingredients are screened in a targeted manner, so that the skin can be nursed, the acnes can be prevented, the acnes at different stages can be relieved and treated, and the broad-spectrum effectiveness is realized. The phloretin can inhibit symbiotic bacteria such as Propionibacterium acnes in acne, and also has effect of inhibiting excessive sebum secretion; the hop extract contains xanthohumol and lupulone, and can inhibit inflammation medium and eliminate inflammation; the herba Portulacae extract and the folium Azadirachta indica extract have effects of scavenging free radicals, shrinking pore, resisting bacteria and diminishing inflammation; the centella asiatica extract has the effect of promoting the activity of scar fibroblasts, and also has the antibacterial and antioxidant effects; the polygonum cuspidatum root extract has antibacterial and antioxidant effects, has a good inhibition effect on the activity of melanocytes, and has the effect of fading acne marks.

The invention relates to a pH targeted flexible nano liposome with acne removing efficacy and a preparation method thereof, wherein the pH targeted flexible nano liposome comprises the following steps:

(1) weighing quantitative N-octanoyl-D-sphingosine, β -sitosterol, membrane softener, pH targeting material and acne-removing active ingredients, and dissolving in organic solvent which is mixed solution of methanol and chloroform;

(2) performing rotary evaporation at 30 deg.C under reduced pressure for 20-30min to remove organic solvent, and forming uniform film on the wall of the container;

(3) then placing the mixture in a vacuum drying oven, and carrying out vacuum drying for 2.0-3.5h at normal temperature to remove residual organic solvent;

(4) adding a hydration solution containing PEG-400, stirring and hydrating for 1-3 h;

(5) after the probe type ultrasonic work is carried out for 1min, the pH targeted flexible nano liposome coated with the acne removing active ingredients can be obtained by extruding the mixture through a 0.45 mu m filter membrane.

After the technical scheme is adopted, the invention relates to a preparation method of pH-targeted flexible nanoliposomes, which has the following beneficial effects: the method is simple to operate, and the prepared pH-targeted flexible nano-liposome with the acne removing effect has the average particle size of less than 100nm, can penetrate through pores with the size of 1/5-1/10 per se due to the self deformability, and is combined with the hydration effect of the stratum corneum, so that the problem of low skin transmittance is solved. The flexible liposome wraps the effective components to penetrate into the epidermis layer and the dermis layer and stay for a long time, so that the active components are efficiently released, and the effects of really controlling oil, diminishing inflammation, resisting bacteria and removing acnes are achieved. The pH sensitive material is added to modify the flexible nano liposome in the process of preparing the flexible nano liposome, so that the prepared pH targeted flexible nano liposome can more release active ingredients coated on inflammatory parts (acidic environment), reduces the distribution on healthy parts, and achieves the effect of real effectiveness and high targeting. The flexible nano liposome is used as a carrier, has no side effect on a human body after metabolism, and is safe and effective.

The pH targeting flexible nano-liposome prepared according to the prescription and the method can be added into an external preparation.

Drawings

Fig. 1 is a graph showing the particle size determination of the phloretin-coated pH-targeted flexible nanoliposome.

FIG. 2 is a graph showing the particle size determination of pH-targeted flexible nanoliposomes coated with centella asiatica extract.

FIG. 3 is a graph showing the particle size measurement of pH-targeted flexible nanoliposomes coated with extracts of rhizoma Polygoni Cuspidati and hops.

FIG. 4 is a transmission electron microscope image of the coated phloretin pH targeted flexible nanoliposome.

Fig. 5 is a release curve diagram of the phloretin-coated flexible nanoliposome and the pH-targeted flexible nanoliposome in an acidic environment.

Fig. 6 is a release curve diagram of the flexible nanoliposome coated with the centella asiatica extract and the pH-targeted flexible nanoliposome in an acidic environment.

Fig. 7 is a release curve diagram of the flexible nanoliposome coated with the polygonum cuspidatum root extract and the hops extract and the polygonum cuspidatum root extract in the pH-targeted flexible nanoliposome in an acidic environment.

Fig. 8 is a release profile of the hop extract in acidic environment from the flexible nanoliposomes coated with the polygonum cuspidatum root extract and the hop extract and the pH-targeted flexible nanoliposomes.

FIG. 9 is a histological section of rabbit right ear. a is a model control group; b is a vitamin A acid cream positive control group; c is a flexible nano liposome group coated with phloretin; d is a pH targeted flexible liposome group coated with phloretin.

FIG. 10a is a graph of ten days on trial for male volunteer A; b is a five-day effect chart of a female volunteer B trial.

Detailed Description

In order to further explain the technical solution of the present invention, the present invention is explained in detail by the following specific examples.

Example 1

In this embodiment, the pH-targeted flexible nanoliposome coated with the anti-acne active ingredient is a flexible nanoliposome coated with phloretin, that is, the pH-targeted flexible nanoliposome is coated with phloretin, and the phloretin is the anti-acne active ingredient.

The pH-targeted flexible nanoliposome coated with phloretin comprises the following raw materials:

14.4 wt% of N-octanoyl-D-sphingosine, 1.8 wt% of β -sitosterol, 1241.5 wt% of poloxamer, 1.6 wt% of poly (N-N-butyl acrylamide), 7.6 wt% of phloretin and 7.26 wt% of PEG-40011.2, and the balance of hydration solution.

(II) a preparation method of the phloretin-coated pH targeted flexible nanoliposome, which comprises the following steps:

(1) quantitatively weighing N-octanoyl-D-sphingosine, β -sitosterol, poloxamer 124, poly (N-N-butyl acrylamide) and phloretin according to the formula, and dissolving in an organic solvent, wherein the organic solvent is a mixed solution of methanol and trichloromethane (v: v ═ 3: 1);

(2) performing rotary evaporation at 30 deg.C under reduced pressure for 30min to remove organic solvent, and forming uniform film on wall of eggplant-shaped bottle (container);

(3) placing in a vacuum drying oven, and vacuum drying at normal temperature for 3.5h to remove residual organic solvent;

(4) adding PEG-400 into the hydration solution with the pH value of 7.0, pouring into a bottle, and hydrating for 3h, namely adding the hydration solution containing the PEG-400 into the bottle, wherein the hydration solution is a composition of potassium dihydrogen phosphate and 0.1mol/L sodium hydroxide (V: V is 70: 30);

(5) after the probe type ultrasonic work is carried out for 1min, the pH targeted flexible nano liposome coated with the phloretin can be obtained by extruding the solution through a 0.45 mu m filter membrane.

Example 2

In this embodiment, the pH-targeted flexible nanoliposomes coated with the anti-acne active ingredients are flexible nanoliposomes coated with an asiatic pennywort herb extract, that is, the pH-targeted flexible nanoliposomes are coated with an asiatic pennywort herb extract, and the asiatic pennywort herb extract is an anti-acne active ingredient.

The coated centella asiatica extract pH targeted flexible nano liposome comprises the following raw materials:

18.9 wt% of N-octanoyl-D-sphingosine, 1.8 wt% of β -sitosterol, 2.9 wt% of hexadecyl palmitate, 2.1 wt% of polyethylene glycol-hydrazone bond-polystyrene copolymer, 10.6 wt% of centella asiatica extract, PEG-40012.4 wt% and the balance of a hydration solution.

(II) a preparation method of a pH-targeted flexible nano liposome coated with a centella asiatica extract, which comprises the following steps:

(1) quantitatively weighing N-octanoyl-D-sphingosine, β -sitosterol, cetyl palmitate, polyethylene glycol-hydrazone bond-polystyrene copolymer and centella asiatica extract according to the formula, and dissolving in an organic solvent, wherein the organic solvent is a mixed solution of methanol and chloroform (v: v ═ 5: 1);

(3) placing in a vacuum drying oven, and vacuum drying at normal temperature for 3.0h to remove residual organic solvent;

(4) adding PEG-400 into a hydration solution with the pH value of 7.5, pouring the hydration solution into a bottle, and hydrating for 2h, wherein the hydration solution is a composition of potassium dihydrogen phosphate and 0.1mol/L sodium hydroxide (V: V is 60: 40);

(5) after the probe type ultrasonic work is carried out for 1min, the pH targeted flexible nano liposome coated with the asiatic pennywort herb extract can be obtained by extruding the asiatic pennywort herb extract through a filter membrane of 0.45 mu m.

Example 3

In this embodiment, the pH-targeted flexible nanoliposome coated with the acne-removing active ingredient is a flexible nanoliposome coated with the hops extract and the polygonum cuspidatum root extract, that is, the pH-targeted flexible nanoliposome is coated with the hops extract and the polygonum cuspidatum root extract, and the hops extract and the polygonum cuspidatum root extract are the acne-removing active ingredients together.

The composition of the pH targeted flexible nanoliposome coating the hop extract and the giant knotweed root extract is as follows:

21.6 wt% of N-octanoyl-D-sphingosine, 2.2 wt% of β -sitosterol, 1243.6 wt% of poloxamer, ethoxylated-C_12-181.2 wt% of alcohol, 1.6 wt% of poly (N-N-butylacrylamide), 0.8 wt% of diethylhexyl maleate, 6.9 wt% of hop extract, 3.4 wt% of polygonum cuspidatum root extract, PEG-4009.8 wt% and the balance of hydration solution.

(II) a preparation method of pH targeted flexible nanoliposome coating hop extract and giant knotweed root extract, which comprises the following steps:

(1) quantitatively weighing N-octanoyl-D-sphingosine, β -sitosterol, poloxamer 124, and ethoxylated-C_12-18-alcohol, poly (N-butylacrylamide), diethylhexyl maleate, polygonum cuspidatum root extract and hop extract are dissolved in an organic solvent which is a mixed solution of methanol and chloroform (v: v ═ 1.5: 1);

(2) performing rotary evaporation at 35 deg.C under reduced pressure for 30min to remove organic solvent, and forming uniform film on wall of eggplant-shaped bottle (container);

(3) placing in a vacuum drying oven, and vacuum drying at normal temperature for 2.5h to remove residual organic solvent;

(4) adding the PEG-400 with the amount of the prescription into a hydration solution with the pH value of 7.5, pouring the hydration solution into a bottle, and hydrating for 3h, wherein the hydration solution is a composition of potassium dihydrogen phosphate and 0.1mol/L sodium hydroxide (V: V is 60: 40);

(5) after the probe type ultrasonic work is carried out for 1min, the pH targeted flexible nano liposome coated with the polygonum cuspidatum root extract and the hop extract can be obtained by extruding through a 0.45-micron filter membrane.

In the present invention, the plant extracts in the active ingredient are all self-extracted substances. Wherein the content of the first and second substances,

a. grinding rhizoma Polygoni Cuspidati into powder with a high-speed pulverizer, adding ethanol, performing ultrasonic extraction for 20min, performing reflux extraction for 3h, extracting for three times, mixing extractive solutions, concentrating under reduced pressure, sequentially extracting with chloroform and n-butanol for three times, mixing extractive solutions, and vacuum drying to obtain rhizoma Polygoni Cuspidati extract with effective component content of 74.2%;

b. crushing dried purslane, sieving with a 60-mesh sieve, adding 90% ethanol, heating, refluxing, extracting, performing suction filtration after extraction, extracting for 3 times, combining filtrates of each time, adjusting the pH value with hydrochloric acid, extracting for 3 times with ethyl acetate, taking a water phase, adjusting the pH value with ammonia water, extracting for 3 times with dichloromethane, combining dichloromethane layers of each time, performing rotary evaporation and drying to obtain a purslane extract, wherein the content of active ingredients is 64.8% when the purslane extract is obtained;

c. crushing and grinding centella into powder, drying to constant weight, adding an ethanol solution 8 times of the raw materials, carrying out ultrasonic extraction for 30min, and filtering to obtain a filtrate, wherein the content of the effective components is 34.6%;

d. oven drying and grinding Azadirachta indica folium, soaking in 75% methanol, ultrasonic treating for 30min, heating and reflux extracting for 2 hr for 3 times, mixing extractive solutions, and rotary steaming and drying to obtain effective component content of 49.4%;

e. oven drying Scutellariae radix to constant weight, grinding into powder, sieving with 40 mesh sieve, extracting with 80% ethanol for 6 hr, performing enzymolysis with polygalacturonase for 5 hr, performing ultrasonic treatment for 30min, centrifuging, collecting supernatant, and vacuum rotary steaming to remove solvent to obtain 58.5% effective component.

f. Drying flos Lupuli to constant weight, pulverizing, sieving with 40 mesh sieve, adding 80% ethanol solution, performing ultrasonic treatment for 10min, heating and reflux extracting for 80min, extracting twice, mixing extractive solutions, storing in dark place, separating and purifying with resin chromatography column, and evaporating to remove solvent to obtain flos Lupuli extract with effective component content of 61.3%.

In addition, according to mechanisms of different occurrence and development stages of the acnes, the active ingredients are screened in a targeted manner, so that the skin can be nursed, the acnes can be prevented, the acnes at different stages can be relieved and treated, and the broad-spectrum effectiveness is achieved. The phloretin can inhibit symbiotic bacteria such as Propionibacterium acnes in acne, and also has effect of inhibiting excessive sebum secretion; the hop extract contains xanthohumol and lupulone, and can inhibit inflammation medium and eliminate inflammation; the herba Portulacae extract and the folium Azadirachta indica extract have effects of scavenging free radicals, shrinking pore, resisting bacteria and diminishing inflammation; the centella asiatica extract has the effect of promoting the activity of scar fibroblasts, and also has the antibacterial and antioxidant effects; the polygonum cuspidatum root extract has antibacterial and antioxidant effects, has a good inhibition effect on the activity of melanocytes, and has the effect of fading acne marks.

Performance test of nano-liposome

(one) particle diameter

The instrument comprises the following steps: NanoBrook Omni type nanometer particle size and ZETA potential analyzer

The test method comprises the following steps: 3mL of the prepared pH targeted flexible nano liposome coated with the active ingredient is put in a plastic cuvette, and the particle size is measured.

As a result:

as shown in fig. 1. The particle size of the phloretin-coated flexible nanoliposome of example 1 was determined to be 76.32 ± 4.30 nm.

As shown in FIG. 2, the particle size of the flexible nanoliposome coated with centella asiatica extract in example 2 was determined to be 79.62 + -2.69 nm.

As shown in FIG. 3, the particle size of the flexible nanoliposome coated with the root extract of Polygonum cuspidatum and the extract of Humulus lupulus in example 3 was measured to be 92.35 + -1.24 nm.

(II) encapsulation efficiency

The instrument comprises the following steps: model 1200 high performance liquid chromatography (DAD detector, Agilent, USA)

The experimental method comprises the following steps:

the liquid phase detection conditions in example 1 were column: eilit Sinochrom ODS-BP 5 μm, 4.6mm × 250 mm; mobile phase: 0.04% phosphoric acid acetonitrile 58: 42 (V: V); flow rate: 1.0 mL/min; detection wavelength: 285 nm; sample introduction amount: 10 μ L.

The liquid phase detection conditions in example 2 were chromatographic column: eilit Sinochrom ODS-BP 5 μm, 4.6mm × 200 mm; mobile phase: water acetonitrile 70: 30 (V: V); flow rate: 1.0 mL/min; detection wavelength: 205 nm; sample introduction amount: 10 μ L.

In example 3, the conditions for detecting the liquid phase of the polygonum cuspidatum root extract are as follows: elite Hypersil ODS 25 μm, 4.6 mm. times.250 mm; mobile phase: water acetonitrile 70: 30 (V: V); flow rate: 1.0 mL/min; detection wavelength: 306 nm; sample introduction amount: 10 μ L. The liquid phase detection conditions of the hop extract are as follows: elite Hypersil ODS 25 μm, 4.6 mm. times.250 mm; mobile phase: 0.1% phosphoric acid acetonitrile 17: 83 (V: V); flow rate: 1.0 mL/min; detection wavelength: 370 nm; sample introduction amount: 10 μ L.

Taking 1mL of the prepared pH-targeted flexible nanoliposome coated with active ingredients in a 10mL volumetric flask, adding methanol for demulsification and volume fixing, performing ultrasonic treatment for 15min, filtering with 0.45 μm filter membrane, and measuring liquid phase to obtain W_{General assembly}. Taking 1mL of prepared pH-targeted flexible nano liposome coated with active ingredients in a 30kDa ultrafiltration centrifuge tube, centrifuging at 4000rpm for 20min, taking the filtrate in a 10mL volumetric flask, demulsifying with methanol to a constant volume, performing ultrasonic treatment for 15min, filtering with a 0.45 μm filter membrane, and measuring the liquid phase to obtain W_{Swimming device}。

Encapsulation ratio (%) - (W)_{General assembly}-W_{Swimming device})/W_{General assembly}×100％

As a result:

the encapsulation efficiency of the active ingredient in the pH-targeted flexible nanoliposome coated with phloretin in example 1 is (88.94 +/-4.62)%.

In example 2, the encapsulation efficiency of the active ingredient in the pH-targeted flexible nanoliposome coated with centella asiatica extract is (72.34 ± 6.43)%.

In example 3, the encapsulation rates of the active ingredients in the pH-targeted flexible nanoliposome coated with the polygonum cuspidatum root extract and the hops extract are (65.72 ± 0.49)%, and (74.19 ± 2.56)%, respectively.

(III) skin permeability and retention

The instrument comprises the following steps: RYJ-6B transdermal diffusion tester (Shanghai yellow sea medical testing apparatus Co., Ltd.)

The experimental method comprises the following steps:

after the cervical dislocation of SD rats (purchased from animal experiments of Fujian medical university with center license number: SCXK (Min) 2016-.

Clamping the skin of the rat in the middle of a vertical diffusion pool, wherein the horny layer faces a supply pool, and the dermal layer faces a receiving pool; respectively adding 1.5mL of active ingredient, liposome coated with the active ingredient (membrane material does not contain membrane softener) and pH targeted flexible nano liposome coated with the active ingredient into a supply pool, and adding 6mL of 20% methanol solution into a receiving pool to ensure that the drug is dissolved in a leakage groove condition in the transdermal process; the temperature of the diffusion cell is kept constant (32 +/-0.5) DEG C, and the magnetic stirring speed is 300 rpm. Taking 1.0mL of receiving solution at 0h, 1h, 2h, 4h, 6h, 8h, 10h, 12h and 24h, supplementing an equal amount of blank receiving solution, and exhausting air in the receiving solution to prevent air bubbles from contacting the lower part of the isolated skin of the rat. The content of the received solution was measured by high performance liquid chromatography after passing through a 0.45 μm filter membrane, and the cumulative permeation amount Q per unit was calculated_n. Taking off the in vitro skin, soaking in methanol for 30s, wiping off the residual active ingredients on the surface, cutting into small pieces in a 2mL centrifuge tube, adding 1mL methanol, vortexing for 2min, performing ultrasound for 15min, centrifuging at 12000rpm for 15min, taking the supernatant, and performing content determination by using high performance liquid chromatography to obtain the skin retention for 24 h.

Specific cumulative permeation volume Q_nThe calculation was performed as follows:

Q_n＝(C_nV_r+∑(n-1)/(i＝1)C_iV_s)/A

wherein C is_nIs the active ingredient concentration of the receiving liquid, C, at the sampling time t_iIs the active ingredient concentration of the receiving liquid at a time point preceding the sampling time point t, V_rIs the volume of the receiving cell, i.e. 6mL, V_sIs the volume of the receiving liquid sampled, i.e. 1mL, A is the effective permeation area of the diffusion cell, i.e. 2.12cm²。

As a result:

example 1

TABLE 124 h cumulative penetration and retention of phloretin in each group

Example 2

TABLE 2 24h cumulative permeation and retention of centella asiatica extracts in each group

Example 3

TABLE 3 24h cumulative permeation and retention of Polygonum cuspidatum root extract in each group

TABLE 4 24h cumulative penetration and retention of hop extracts in each group

(IV) comparative release experiment in vitro acidic Environment

The method comprises the following steps: and determining the in vitro release degree of the flexible nano liposome and the pH targeted flexible nano liposome by a dialysis method. Phosphate buffer solution containing 0.5 percent of Tween-80 and pH of 6.0 is selected as a release medium, 1.0mL of the flexible nano liposome solution coated with the acne-removing active ingredients and 1.0mL of the pH-targeted flexible nano liposome solution with the same concentration are respectively absorbed and injected into a dialysis bag (MWCO: 8000-. Taking 1mL of release solution at 0, 0.5, 1, 2, 4, 6, 8, 10, 12, 24 and 48h respectively, simultaneously supplementing 1mL of blank buffer solution, measuring the content by using high performance liquid chromatography, and calculating the cumulative release percentage of each time point according to the following formula:

Q_n％＝[C_nV_o+(C₁+C₂+C₃+…+C_n-1)×V]/C_oV_o×100％

wherein Q_n% of the cumulative percentage of active ingredient released at the nth sampling point, C_oIs the initial concentration of the active ingredient, C_nIs the concentration of the active ingredient at the nth sampling point, V_oTo release the medium volume, V is the volume per sample.

As a result: the release curves are shown in figures 5, 6, 7 and 8, and the release curves show that the cumulative release amount of active ingredients in the pH targeted flexible nano liposome is higher than that of the flexible nano liposome under the acidic environment. This demonstrates that the flexible nanoliposome modified with pH sensitive material can release the loaded active ingredient better in acidic environment.

(V) animal experiment is adopted to carry out preliminary efficacy evaluation

The instrument comprises the following steps: JS-750T microscope

The method comprises the following steps: adult rabbits are randomly grouped into a model control group, an external vitamin A acid cream positive control group, a phloretin-coated flexible nano liposome group and a phloretin-coated pH targeted flexible nano liposome group respectively. Dipping 2% coal tar with a cotton swab, smearing the coal tar on the opening of the ear canal at the inner side of the right ear of the rabbit within the range of 2cm multiplied by 2cm, 0.5 mL/day, 1 time/day, and performing molding after 21 days of administration, wherein 3 rabbits are randomly selected from each group, and performing histological observation under a microscope, and after the molding is successful, the external application administration can be performed. The vitamin A acid cream positive control group is administrated according to the instruction, the phloretin-coated flexible nano liposome group and the phloretin-coated pH targeted flexible liposome group are administrated according to the conversion formula of the dosage between human and rabbit, 1 time/day and 15 days of continuous administration. After 24h of the last administration, the right auricle was excised, the tissue block was fixed with 10% formalin, then paraffin-embedded, sectioned, stained with hematoxylin-eosin (HE), and finally examined for histological changes under a microscope.

As a result: after the model is successfully made, the cuticle of the epidermis of the right ear of the rabbit is obviously thickened, hyperkeratosis is caused, the acanthocyte layer is thickened, hair follicles are moderately expanded, keratinocytes are severely proliferated, the volume of sebaceous glands is increased, hair follicle keratoses are appeared, hair follicle walls are thickened, inflammation is obvious, and a histological picture is shown in an attached figure 9 a; the vitamin A acid cream positive control group can observe that the tissue structure is complete, the epidermis layer and the hair follicle are normal, the inflammation is subsided, and the details are shown in figure 9 b; the phloretin-coated flexible nanoliposome group can observe that the tissue structure is complete, hair follicles on the epidermal layer are still slightly dilated, mild keratinocyte hyperplasia and slight inflammation are realized, and the details are shown in figure 9 c; the phloretin-coated pH-targeted flexible liposome group was observed to have a complete tissue structure, normal epidermis and hair follicles, and a substantial regression of inflammation, as shown in fig. 9d for details.

(VI) smearing test on human body

The method comprises the following steps: 30 volunteers (20-40 years old, male and female halves, unlimited skin types, no skin diseases and the exclusion of pregnant people) are selected and divided into two groups (the sex, age and the like in the two groups have no significant difference), the control group is self-made blank emulsion, the test group is emulsion added with the pH targeted flexible liposome coated with phloretin, and the appearance is similar. During the test period, the volunteers are ordered to keep light diet and sufficient sleep, other skin care products are stopped, the lotion is smeared according to the acne-removing parts per se, the lotion is smeared twice after the face is cleaned by warm water in the morning and at night every day, and the acne-removing effect is fed back by self evaluation after the lotion is continuously smeared for 14 days.

As a result: after the two groups of volunteers use the lotion for 14 days, 2 of 15 control groups fed back the lotion by self evaluation and have acne removing effect, and the acne removing rate is 13.3%; in 15 persons in the test group, 12 persons in the test group have the acne removing effect after feeding back the emulsion by self evaluation, and the acne removing rate is 80 percent. The effect details of one male volunteer on trial for ten days are shown in figure 10a, and the effect details of one female volunteer on trial for five days are shown in figure 10b

In summary, the invention relates to a pH-targeted flexible nanoliposome coated with an acne-removing active ingredient, which has higher retention and transmittance compared with liposome; animal experiments and human trial experiment results show that the pH targeted flexible nano liposome can coat active ingredients to be efficiently released at an inflammation part (acidic environment), and the effect of targeted acne removal is really achieved.

The above examples are not intended to limit the products and methods of the present invention, and any suitable changes or modifications made by one of ordinary skill in the art should be considered without departing from the scope of the present invention.

Claims

1. A pH targeted flexible nano liposome with an acne removing effect is characterized by comprising, by weight, 10% -30% of N-caprylyl-D-sphingosine, 1.0% -2.5% of β -sitosterol, 1.0% -5.5% of a membrane softener, 1.0% -3.0% of a pH sensitive material, PEG-4004.0% -15.0% of an acne removing active ingredient, 2.0% -15.0% of an acne removing active ingredient and the balance of a hydration solution.

2. The pH-targeted flexible nanoliposome with acne removing efficacy according to claim 1, wherein: the film softener comprises poloxamer 124, fatty amine polyoxyethylene ether and ethoxylation-C_12-18-one or both of an alcohol and cetyl palmitate.

3. The pH-targeted flexible nanoliposome with acne removing efficacy according to claim 1, wherein: the pH sensitive material comprises one or two of poly (N-N-butylacrylamide), diethylhexyl maleate and polyethylene glycol-hydrazone bond-polystyrene copolymer.

4. The pH-targeted flexible nanoliposome with acne removing efficacy according to claim 1, wherein: the ratio of N-octanoyl-D-sphingosine to pH sensitive material is 9: 1.

5. The pH-targeted flexible nanoliposome with acne removing efficacy according to claim 1, wherein: the hydration solution is a potassium dihydrogen phosphate and 0.1mol/L sodium hydroxide composition with the pH value of 6.5-8.0.

6. The pH-targeted flexible nanoliposome with the effect of removing acne as claimed in claim 1, wherein the active ingredients for removing acne comprise one or more of centella asiatica extract, neem leaf extract, hops extract, polygonum cuspidatum root extract, purslane extract, scutellaria baicalensis root extract, and phloretin.

7. A preparation method of pH targeted flexible nanoliposomes with acne removing efficacy is characterized in that:

(1) weighing quantitative N-octanoyl-D-sphingosine, β -sitosterol, membrane softener, pH sensitive material and acne-removing active ingredients, and dissolving in organic solvent which is a mixed solution of methanol and chloroform;

(2) performing rotary evaporation at 30 deg.C under reduced pressure for 20-30min to remove organic solvent until a uniform film is formed on the wall of the container;