KR20150008825A - Cosmetic composition for reinforcing skin barrier containing isoquercitrin, and the liposome for enhancing transdermal delivery of isoquercitrin - Google Patents

Abstract

The present invention relates to a cosmetic composition containing an extract of Persicaria hydropiper and a skin delivery system for promoting skin permeability of the extract of Persicaria hydropiper and, more specifically, to a cosmetic composition which contains an extract of Persicaria hydropiper or an effective ingredient thereof to promote a skin protection function from oxidative damage due to oxidative stress, thereby reinforcing the skin barrier, and to an ethosome or elastic liposome for maximizing the skin permeability of an extract of Persicaria hydropiper or an effective ingredient thereof.

Description

TECHNICAL FIELD The present invention relates to a cosmetic composition for skin barrier strengthening containing isoquoisolin and a liposome for enhancing transdermal absorption of isosquimolene, and a liposome for enhancing transdermal delivery of isoquercitrin,

The present invention relates to a skin delivery system for enhancing the skin absorption ability of a cosmetic composition and an extract of Fusarium oxysporum containing an Fusarium exudate, and more particularly, A cosmetic composition capable of enhancing the skin barrier by enhancing the protective function, and an ethosomal or elastic liposome for maximizing the skin absorption ability of the extract or the active ingredient thereof.

Many skin diseases are associated with exposure to sunlight. Ultraviolet rays cause skin cancer, photoaging and various skin diseases associated with them. Reactive oxygen species (ROS) are produced in skin exposed to ultraviolet light. Active oxygen acts as a causative agent of aging, especially skin aging. They are also produced by normal metabolic processes, disease states, and stress. There is an antioxidant defense system that removes free radicals against the active oxygen. However, these defensive systems can be overwhelmed if the amount of ultraviolet light is large. The most reactive reactive oxygen species that lead to skin cell and tissue damage are known as ¹ O ₂ and OH. They break down the antioxidant defense system, which consists of antioxidant enzymes and non-enzymatic antioxidants, to tilt the oxidant / antioxidant balance towards the oxidized state and ultimately accelerate skin aging, such as reduced elasticity, wrinkles and spots and freckles. In order to delay and inhibit skin aging, it is necessary to construct an antioxidant defense system capable of efficiently removing active oxygen generated and protecting cells and tissues from active oxygen (L. Packer, UVA, UVB) and skin Antioxidants, In: Free radical damage and its control, (CA Rice-Evans and RH Burdon, eds), Elsevier Science BV, 239 (1994); K. Scharffetter-Kochanek, , In: antioxidants in disease mechanisms and therapy, Advances Pharmacology, (H. Sies, eds) (1997) and JJ Thiele, CO Barland, R. Ghadially, PM Elias, Permeability and antioxidant Barriers in aged epidermis, Skin Aging BA Gilchrest, J. Krutman, eds), Springer-Verlag Berlin Heidelberg, Germany, 65 (2006))

Recently, researches using liposomes have been attracting much attention in research for improving the skin absorption ability of physiologically active ingredients. Liposomes have high biocompatibility and are capable of delivering a variety of active substances in vivo, thus being widely used in the field of pharmaceuticals and cosmetics (EJ An, CK Kang, JW Kim, and BS Jin, Lipid-based vesicles as transdermal delivery system, KIC News , 13 (4), 24 (2010); and MMA Elsayed, OY Abdallah, VF Naggar, and NM Khalafallah, Lipid vesicles for skin delivery of drugs: Int . J. Pharm . , 332, 1 (2007)).

However, recently, problems have been raised regarding the physical instability of the lipid bilayer membrane, low collection efficiency, and low emulsion stability within the formulation (MMA Elsayed, OY Abdallah, VF Naggar, and NM Khalafallah, Deformable liposomes and ethosomes : E. Touitou, N. Dayan, L. Bergelson, B. Godin, and M. Eliaz, Ethosomes - Novel Vesicular Carriers for Enhanced Skin Delivery, Int . J. Pharm . , 332, delivery: characterization and skin penetration properties, J. Control. Release , 65, 403 (2000)), and various attempts have been made to solve this problem.

For example, new flexible and resilient vesicles have been developed for the purpose of improving the skin permeability of functional materials, allowing the film to flexibly deform so as to better pass through the narrow gaps between keratinocytes, Ethosome, and elastic liposome.

The etosome is a vesicle made by dissolving phospholipid in ethanol. Since it is made of phospholipid, which is in vivo substance, it has high biocompatibility and has the advantage that various active substances can be delivered in vivo.

In addition, the elastic liposome has a lipid bilayer structure composed of a phospholipid and a surfactant, and the lipid bilayer membrane of the vesicle has a variable formation due to the action of the surfactant, so that it can be easily deformed when the external stress is applied.

The inventors have found that Persicaria hydropiper L.) extracts showed excellent cytoprotective effect on the extracts and their active ingredients. In addition, when the extracts or their active ingredients were carried by preparing the etosome and elastic liposome under specific conditions The present invention has been accomplished based on the discovery that it has a very excellent transdermal absorption rate and can maximize its efficacy.

Accordingly, it is an object of the present invention to provide a cosmetic composition for enhancing skin barrier containing a dried extract or an effective ingredient thereof.

It is another object of the present invention to provide a system which can more effectively transfer the extract or its effective ingredient to the skin.

In order to accomplish the above object, the present invention provides a cosmetic composition for skin barrier enhancement containing a crude extract or an effective ingredient thereof.

The present invention also provides an etosome and an elastic liposome capturing the extract or its effective ingredient.

INDUSTRIAL APPLICABILITY The cosmetic composition of the present invention is excellent in protecting skin from oxidative stress caused by ultraviolet rays or the like and is effective in restoring and strengthening the skin barrier function. When the transdermal delivery system according to the present invention is used, And the active ingredient thereof can be absorbed into the skin with higher efficiency, so that even when the same amount of the active ingredient is used, it is possible to provide an effect such as a higher antioxidative effect and a whitening effect.

Figure 1 is a schematic representation of the process of preparing the extract.
FIG. 2 shows the TLC of isozykosinate from the extract. As the eluant, ethyl acetate: chloroform: formic acid: distilled water was mixed at a ratio of 8: 1: 1: 1 (v / v), where ① represents the extract (ethyl acetate fraction), ② represents isoquosityrin .
3 is enough water pepper extract of human erythrocyte hemolysis in the presence of Rose Bengal (rose-bengal) (ethyl acetate fraction), iso-site query and lean (+) - shows the cytoprotective effect of α- tocopherol (τ ₅₀ in the control group Lt; / RTI >
Fig. 4 shows the cell survival rate of the extract and the isoquosityrin.
Fig. 5 shows the survival rate of HaCaT cells according to the UVB irradiation dose. Cells were exposed to UVB irradiation and cytotoxicity levels were measured by MTT (control: no UVB exposure).
FIG. 6 shows the cell survival effect of the extract (ethyl acetate fraction) and isoquosityrin on UVB-induced cell damage in the HaCaT cell system.
FIG. 7 shows the size of the etosome according to the concentration of the extract.
Figure 8 shows the size of the etosome according to the concentration of isosquatolines.
Figure 9 shows the capture efficiency of ethosomes treated with different concentrations of isoquosityrin in a system consisting of 2% lecithin and 20% ethanol.
FIG. 10 shows the size of the etosome according to the concentration of isoquositrin after 2 weeks of the production of the etosome.
FIG. 11 shows the efficiency of collecting the etosome according to the concentration of isoquositrin after 2 weeks of the production of the etosome.
FIG. 12 is a graph showing cumulative permeation amount of skin extracts on the skin over time when using ethosomes, ethanol solutions, liposomes, and distilled water.
FIG. 13 shows the amount of foliar extracts remaining in the stratum corneum after 24 hours of use of ethosomes, ethanol solutions, liposomes and distilled water.
FIG. 14 shows the total skin permeation amount of the extracts of extracts in the use of ethosomes, ethanol solutions, liposomes and distilled water.
Figure 15 shows the amount of isosquimolene cumulatively transmitted to the skin over time when using ethosomes, ethanol solutions, liposomes, and distilled water.
Figure 16 shows the amount of isoquosityrin remaining in the stratum corneum 24 hours after the use of ethosomes, ethanol solutions, liposomes and distilled water.
Figure 17 shows the total skin permeation of isosquimolene when using ethosomes, ethanol solutions, liposomes and distilled water.
Figure 18 shows the size change of the elastic liposome formulation containing the extracts for one week.
Fig. 19 shows the collection efficiency of the extract obtained from the elastic liposome.
Fig. 20 shows the collection efficiency of isosquatolines captured in the elastic liposome.
FIG. 21 shows the permeation amount of the extract from the skin over a certain area of skin.
FIG. 22 shows the contents of the extracts (Tape) present in the stratum corneum after 24 hours, the skin extracts existing in the epidermis and dermis, excluding the stratum corneum, and the transdermal components present in the skin through the skin will be.
FIG. 23 shows the permeation amount of isosquatolines over time for a fixed skin area.
FIG. 24 is a graph showing the results of isoquaternary (Tape) present in the stratum corneum after 24 hours, isoquercitrin present in the epidermis and dermis, excluding the stratum corneum, and isoquercitrin Transdermal.

The present invention provides a cosmetic composition capable of enhancing a skin barrier function by containing an extract of Aspergillus or an effective ingredient thereof as an active ingredient. In particular, the active ingredient is preferably isoquaternary.

In addition, the present invention provides an etosome and an elastic liposome capable of effectively delivering the extract or its effective ingredient to the skin.

The skin barrier is collapsed by oxidative stress induced from ultraviolet rays and the like, and as a result, skin aging is accelerated. Therefore, the composition of the present invention restores skin barrier function from oxidative damage caused by oxidative stress, .

The term " Persicaria " hydropiper L.) is a one-year-old herb with a pine tree spruce, distributed throughout temperate and temperate regions of the Northern Hemisphere, and is often wild in wetlands or waterfronts. The stem grows to about 40 ~ 100cm and the branches are split a lot. The leaves have no hairs. The alternate leaves are lanceolate with no petiole. Both ends are narrow and there are few hairs on the surface. The edges are flat and the prefix is scattered on the back side. It grows from June to September, but a white-red flower with a reddish tint at the end of the stem runs on the flower. Achene fruit is covered with calyx and ripened black. Flowers, leaves and fruits can be used for food. It is used for hemorrhage of uterus, hemorrhoids and other internal bleeding due to hemostatic effect. Leaves and stems have excellent antimicrobial action, lower blood pressure and strengthen intestinal and uterine tension.

On the other hand, flavonoids are polyphenols which are widely present in plants and have diverse physiological activities such as antiviral, anti-inflammatory and antioxidative effects. They are generally present in plant tissues in the form of glycosides. Typical flavonoids include quisotriene , 3 ', 4', 5,7-penta-hydroxyflavone). It is known that it has strong antioxidative, anti-inflammatory, anti-cancer, anti-aging, and alleviating atherosclerosis. Quercetin 3-Ο-β-glucopyranoside is a glycoside of quasitoline (Chemical Formula 1). It is known that isozycositrin has biological activities such as antiallergic, anti-inflammatory and antioxidative activities. The present inventors have found that isozy-quinolin and fructose extract isolated from corn steep liquor have remarkable antioxidant activity and that HaCaT cells [JE Kim, HJ Lee, MS Lim, MA Park, and Park PAR, Cellular protective effect and liposome formulation for enhanced transdermal delivery of Persicaria hydropiper L. Extract, J. Soc . Cosmet . Scientists Kroea , 38 (1), 15 (2012)].

The extracts of the present invention can be prepared by conventional plant extraction methods. Extraction sites are not particularly limited, and they can be extracted from various parts such as outposts, leaves, stems, flowers, roots, and the like. In addition, it is preferable that extraction is carried out by using water, an organic solvent or a mixed solvent thereof as an extraction solvent. The organic solvent may be at least one solvent selected from the group consisting of methanol, ethanol, glycerin, propylene glycol, butylene glycol, ethyl acetate and hexane.

In the present invention, the extraction method using the extraction solvent may be a general plant extraction method such as a hot water extraction method or an organic solvent extraction method. Specifically, the dried plant material or the pulverized material thereof may be added to the extraction solvent It is suitable to immerse at 4 to 30 ° C. for 0.5 to 30 days while stirring as necessary, or to extract by heating at 30 to 70 ° C. for 2 to 72 hours. The amount of the solvent used for the extraction is preferably 5 to 20 times (weight ratio) to the raw material to be extracted.

In addition, the liposome of the present invention may contain a crude extract extracted using the above-described solvent as an active ingredient, and the extract is further subjected to complete decompression concentration by recovering a solvent evaporated using a distillation apparatus equipped with a cooling condenser, It may also be contained in powder form.

In addition, the liposome according to the present invention may contain, as an active ingredient, fractions obtained by stepwise treating hexane, ethyl acetate, butanol and water in a volume of 1 to 10 times the dry powder obtained by concentrating the above extracts under reduced pressure. For example, the dried extract obtained by concentrating the extract of the above extracts is dispersed in water and then fractionated by treating with the same amount or 1-10 times volume of n-hexane, and the obtained water layer is again the same as that obtained by treating n-hexane Ethyl acetate and butanol are fractionated by stepwise treatment. At this time, each of the n-hexane layer, the ethyl acetate layer, the butanol layer and the finally obtained water layer was completely concentrated under reduced pressure while recovering the solvent evaporated by using a distillation apparatus equipped with a cooling condenser, It can be used as an active ingredient.

In addition, the present invention can utilize an efficacious ingredient contained in the dried extract, which can be separated from the dried extract using a known method or a commercially available one. Preferably, the active ingredient contained in the extract is isoquercitrin.

In addition, the present invention provides a liposome carrying an efficacy component contained in a dried or extract extract, as a system for enhancing the transdermal absorption rate of a dried extract or an efficacious ingredient contained therein, and more particularly, it provides an etosome and an elastic liposome do.

Ethanol used in the manufacture of ethosomes is well known to have a skin permeation enhancing effect, and ethosomes have been reported to have higher skin affinity than beesuckle liposomes. This feature allows the etosome to be more effective in delivering the active substance to the skin and delivering the active to the deeper part of the skin (Dubey, D. Mishra, T. Dutta, M. Nahar, DK Saraf, and NK Jain, Dermal and transdermal delivery of an ethanolic liposomes, J. Control. Release , 123, 148 (2007), and V. Dubey, D. Mishra, and NK Jain, Melatonin loaded ethanolic liposomes, physicochemical characterization and enhanced transdermal delivery, Eur . J. Pharm . Biopharm . , 67, 398 (2007)).

Considering the ability of the extract of the present invention to deliver the extract and the isoquosityrin to the skin, the concentration of the ethosomal extract in the case of the extract is 0.04% (w / v) , And in the case of isosquitolines, it is preferable to support those having a concentration of 0.03% (w / v) based on the volume of the solvent in which the ethosomes are dispersed.

In addition, elastic liposomes can be more easily deformed when externally stressed, allowing them to penetrate the stratum corneum better than ordinary liposomes and to penetrate more active material into the skin (G. Ceve, A. Schatzlein, and H. Richardsen, Ultradeformable lipid vesicles can penetrate the skin and other semipermeable barriers unfragmented. Evidence from double lable CLSM experiments and direct size measurements, Biochim . Biophys . Acta , 1546 , 21 (2002). Twenty 80, Span 80, and dipotassium glycyrrhizinate have been commonly used as surfactants in surfactant formulations. Recently, however, there has been a growing interest in the safety of cosmetic raw materials, such as PEG (polyethylene glycol ) Is used as a surfactant. Examples of such surfactants include Tego ^® care 450 (polyglyceryl-3 methylglucose distearate) and the like, It is not.

In the elastic liposome of the present invention, the weight ratio of the phospholipid to the surfactant is not limited thereto. Preferably, the weight ratio of the phospholipid to the surfactant is 95: 5, 15.

Considering the ability of the elastic liposome of the present invention to deliver the extract and the isoquosityrin to the skin, the concentration of the extract or the isoquitoline is preferably 0.1% (w / v) relative to the volume of the solvent in which the elastic liposome is dispersed ). ≪ / RTI >

Liposomes such as the above-described ethosomes and elastic liposomes increase the skin permeability, so that even when the extracts or their active ingredients are used in the same amount, the skin absorption rate is improved and the liposomes can more effectively act on the skin.

The cosmetic composition of the present invention contains an ethoxylate or an elastomeric liposome containing an extract or an effective ingredient thereof in an amount of 0.5 to 10% by weight based on the total weight of the composition.

The cosmetic composition according to the present invention may further contain additives or additives commonly used in the art in addition to the above-mentioned effective ingredients to form skin, skin toner, skin softener, skin lotion, astringent, lotion, Lotion, Nourishing Cream, Massage Cream, Moisture Cream, Hand Cream, Essence, Nutrition Essence, Serum, Concentrate, Pack, Mask, Soap, Cleansing Foam, Cleansing Lotion, Cleansing Cream, Body Cleanser, Body Lotion, Body Oil, Shampoo, Rinse , Foundation, makeup base, fact, loose powder, pressed powder, compact, eye shadow, eyeliner or lipstick.

Hereinafter, the present invention will be described more specifically with reference to examples and test examples. It is to be understood that the scope of the present invention is not limited to these embodiments and that variations, substitutions, and insertions conventionally known in the art can be carried out, And this is included in the scope of the present invention.

[Referential Example 1] Preparation of extract

100 g of the dried leaves (purchased from Kyungdong Market, collected from Hongcheon County, Gangwon Province) were finely cut and then immersed in 2 L of 50% ethanol while mixing at room temperature for one week and then filtered. The extract was concentrated under reduced pressure, and then the non-polar component was removed using hexane. Ethyl acetate was added to the residue, and the ethyl acetate fraction was concentrated under reduced pressure to obtain a powder (see FIG. 1).

[Referential Example 2] Isocyanurate Isolation

(TLC) was used to isolate the extract from the extract prepared in Reference Example 1, and confirmed by high performance liquid chromatography (HPLC).

Separation of isoquosityrin from the crude extract was carried out using preparative TLC. TLC eluted with ethyl acetate: chloroform: formic acid: distilled water at a ratio of 8: 1: 1: 1.

The extracts from TLC showed a total of 6 bands, which are shown in Fig. In Fig. 2, (1) represents the ethyl acetate fraction of the extract, and (2) represents isoquinolin.

HPLC was used for the determination of isoquosityrin. The HPLC was separated by gradient elution with 2% acetic acid and 50% acetonitrile at a concentration of 0.5% acetic acid. The R _f value of the flavonoid standard and the color development Respectively. Among the isolated components, isoquosityrin with an R _f value of 0.27 was the most abundant component in the extracts. The UV-visible spectrophotometer, NP-2-aminoethyl diphenylborinate PEG-Polyethylene glycol) colorimetric method and IR spectroscopic method.

Separated isoquinolines were further purified and used in the experiments.

[Test Example 1] Measurement of cytoprotective effect by photohemolysis

Red blood cells from healthy adults were immediately drawn into heparin-added test tubes and centrifuged at 3,000 rpm for 5 minutes to separate red blood cells and plasma. The separated red blood cells were washed with 0.9% phosphate buffer saline (PBS), centrifuged and the white blood cell layer was removed. RBCs were washed and separated 3 times, and red blood cells were stored in a refrigerator at 4 ° C. All experiments were performed within 12 hours after blood collection. The light hemolysis method was carried out according to the already established method. The erythrocyte suspension used in the experiment was 0.6 OD at 700 nm, and the number of erythrocytes was 1.5 × 10 ⁷ cells / mL.

1.5 x 10 ⁷ cells / mL 3.5 mL of erythrocyte suspension was placed in a Pyrex test tube (No. 9820), and then 50 μL of isoquositrin or test extract was added as a test substance, Respectively corresponded to 5 μg / mL. Also, for comparison, (+) -? - tocopherol treated in the same amount as the positive control group was used. The red cell suspensions to which the test substance was added were pre-incubated in a dark place for 30 minutes, 0.5 mL of rose-bengal (12 μM) was added as a photosensitizer, the entrance was sealed with a parafilm, Light irradiation.

Light irradiation for light hemolysis was performed by placing a 20 W fluorescent lamp in a 50 cm x 20 cm x 25 cm box filled with black inside and arranging a pyrex test tube containing a red cell suspension at a distance of 5 cm from a fluorescent lamp so as to be parallel to fluorescent lamps, Respectively. After irradiation, the degree of destruction of erythrocytes induced by dark reaction (post-incubation) ¹ O ₂ over time was determined from the transmittance (% transmittance) at 700 nm every 15 minutes. At this wavelength, the increase in red cell suspension light transmittance is proportional to the degree of red blood cell hemolysis. All experiments were carried out in a constant temperature chamber at 20 ° C. The effect of isosquamolyne on photohemolysis was calculated by plotting τ ₅₀ , the time at which 50% of the red blood cells were hemolyzed from the graph consisting of the dark time and the degree of hemolysis. The time required for 50% destruction of red blood cells (τ ₅₀ ) is greater as the cell protection activity of active oxygen is greater. At this time, red blood cells not subjected to light irradiation and not treated with the test substance were used as a negative control. The results are shown in FIG. 3, and all experiments were repeated three times and averaged.

In the negative control group, τ ₅₀ was 31.0 ± 0.30 min, and the error range was within ± 1 minute. In all cases, reproducibility was good. Hemolysis did not occur until 120 minutes after the dark reaction, except when the rose bengal was added and the light was not irradiated and only the light irradiation was performed without addition of rose bengal.

Figure 3 looking, isopropyl query site Lin minutes in 5μg / mL τ ₅₀ is 351.5, water pepper extract is measured by the τ ₅₀ 250.9 minutes in 5μg / mL, with a fat-soluble antioxidant and a blocking agent in the radical chain reaction of lipid peroxidation It can be confirmed that it exhibits much greater cytoprotective activity at a lower concentration than the known (+) -? - tocopherol (10 μg / mL, τ ₅₀ = 34.0 min). In addition, it can be confirmed that isoquatolin exhibits greater protective activity than the ethyl acetate fraction of the extract.

[Test Example 2] Cytotoxicity measurement of the extract and the isoquosityrin

The concentrations of cytotoxic and isoquinolines were determined and MTT assay was performed to determine the range of concentrations to be used in the experiment. MTT assays are based on the ability of mitochondrial dehydrogenase in living cells to convert MTT, a yellow, soluble substrate, to dark blue, water-insoluble formazan, and the amount of formazan produced is proportional to the number of living cells.

The concrete method is as follows.

In this test, human horny cell line HaCaT cells were used. It was sold from Fusenig (German Cancer Research Center, DKFZ). DMEM supplemented with 10% fetal bovine serum (PAA, Austria) and 1% penicillin-streptomycin (PAA, Austria) was used as a culture medium for DMEM (Dulbecco's modified Eagle's medium).

HaCaT cells were incubated at 37 ° C and 5% CO ₂ for 24 hours in a 96-well plate. The cells were incubated with either 3.125 μg / ml, 6.25 μg / ml, 12.5 μg / ml, 25 μg / mL, 50 μg / mL and 100 μg / mL, respectively. Cells were cultured for 24 hours and washed with PBS. Cell viability was measured by MTT (3- (4,5-dimethythiazol-2-yl) -2,5-diphenytetrazolium bromide (MTT), Sigma, USA ) Analysis method. Specifically, the MTT solution (2 μg / mL) was added to the cells washed with PBS and reacted for 3 hours, and the formazan produced was dissolved in DMSO and measured at 570 nm.

Cells not treated with the sample were used as a negative control, and the cell viability was determined based on the 100% standard. Cell viability was calculated according to the following formula 1, and the results are shown in FIG. At this time, the experiment was repeated three times and averaged.

As shown in FIG. 4, the cell extract of HaecaT cells showed a cell viability of 90% or higher up to a concentration of 50 μg / mL, In the case of isozy- cytolin, the active ingredient isolated from the extracts, the cell viability was up to 90% at concentrations up to 25 μg / mL, but cytotoxicity was observed at concentrations above 50 μg / mL.

Therefore, in the subsequent experiments, the extract and the isoquosityrin were used to a concentration of 50 μg / mL.

[Test Example 3] Cell survival rate according to UVB irradiation dose

In this test, the same HaCaT cells as in Test Example 2 were used. After incubation at 37 ° C and 5% CO ₂ for 24 hours in a 96-well plate using DMEM containing 10% fetal bovine serum (PAA, Austria) and 1% penicillin-streptomycin (PAA, Austria) HaCaT cells adhered at 5 × 10 ⁴ cells / well in a 96-well plate were washed with PBS, and UVB was irradiated to HaCaT cells using an ultraviolet irradiator (CL-1000 Ultraviolet Crosslinker, UVP, USA) 100 ~ 500 mJ / cm ² ) and the effect on cell viability was observed. The results are shown in Fig. At this time, the experiment was repeated three times and averaged.

5, when irradiated with UVB of 100 mJ / cm ^2, the cell viability was not greatly affected as in the case of not irradiating ultraviolet rays, but the cell viability was 80% when irradiated with UVB of 200 mJ / cm ² , in 300mJ / cm ² 63%, the 400mJ / cm ² 36% and the 500mJ / cm ² can be seen that represents the survival rate of 33%.

On the basis of the above results, in the test for measuring the cytoprotective effect from the following ultraviolet rays, 400 mJ / cm ² was used as the ultraviolet light quantity.

* [Test Example 4] HaCaT cell protection effect against ultraviolet light

In this test, the same HaCaT cells as in Test Example 2 were used. After incubation at 37 ° C and 5% CO ₂ for 24 hours in a 96-well plate using DMEM containing 10% fetal bovine serum (PAA, Austria) and 1% penicillin-streptomycin (PAA, Austria) HaCaT cells adhered at 5 × 10 ⁴ cells / well to a 96-well plate were washed with PBS and irradiated with UVB 400 mJ / cm ² using an ultraviolet irradiator (CL-1000 Ultraviolet Crosslinker, UVP, USA).

Prior to UVB irradiation, the extracts or isozy- cytolines were treated at concentrations of 12.5 μg / mL, 25 μg / mL and 50 μg / mL. After the sample treatment, the cells were cultured for 24 hours, washed with PBS, and the cell viability was measured by MTT assay. Specifically, the MTT solution (2 μg / mL) was added to the cells washed with PBS and reacted for 3 hours, and the formazan produced was dissolved in DMSO and measured at 570 nm.

As a negative control group, the sample and the untreated group without UV treatment were taken as a 100% standard and the relative cell viability was determined. The cell viability was calculated according to the above formula (1), and the results are shown in FIG. At this time, the experiment was repeated three times and averaged.

After irradiation of 400 mJ / cm ² UVB, the cell survival rate was 36% as compared with the control group without UVB irradiation. However, as can be seen from FIG. 6, cell viability was 85%, 90% and 92% when HaCaT cells treated with 12.5 μg / mL, 25 μg / mL and 50 μg / Showed a very high effect of inhibiting apoptosis in ultraviolet irradiation. The cell survival rate was 87%, 90% and 84% at the concentrations of 12.5 μg / mL, 25 μg / mL and 50 μg / mL, respectively, even when treated with isozycositrin, Able to know. As a result, the extract and the isoquosityrin showed a large cytoprotective activity against ultraviolet light when they were treated before irradiation with ultraviolet light.

On the other hand, in the case of isoquosityrin, the cell survival rate at the concentration of 50 μg / mL was 8% lower than that of the same concentration of the Fusarium oxysporum extract, probably as a result of the toxicity of isoquosityrin itself to cells at 50 μg / mL.

[Reference Example 3] Preparation of ethosome

1. Preparation of ethosomes carrying the extract

(YP Fang, YH Tsai, PC Wu, and YB Huang, Comparison of 5-aminolevulinic acid-encapsulated liposome versus ethosome for skin delivery for photodynamic therapy, Int . J. Pharm. , 356, 144 (2008); D. Paolino, G. Lucania, D. Mardente, F. Alhaique, and M. Fresta, Ethosomes for skin delivery of ammonium glycyrrhizinate: in vitro percutaneous permeation through human skin and in vivo anti-inflammatory . activity on human volunteers, J. Control Release, 106, 99 (2005); and Fang YP, YB Huang, Wu PC, and YH Tsai, Topical delivery of 5-aminolevulinic acid-encapsulated ethosomes in a hyperproliferative skin animal model using the CLSM technique to evaluate the penetration behavior, Eur . J. Pharm . Biopharm . , 73 , 391 (2009)). (2% w / v based on a final 20% ethanol solution of a solvent in which ethosomes are dispersed)) and L-α-phosphatidylcholine (about 60%, Sigma Was dissolved in chloroform (10 mL) as an organic solvent in a 50 mL round bottom flask. At this time, the extract was dissolved in an amount of 0.03% ~ 0.1% (w / v) based on the volume of the organic solvent. The organic solvent was then evaporated using a rotary evaporator (BUCHI, Switzerland). The organic solvent was evaporated and the same amount (10 mL) of 20% ethanol solution as that of the above organic solvent was added to the thin film formed on the wall of the flask and hydrated by stirring at 500 rpm above the lipid transition temperature. The beads were placed in an ultrasonic shredder (BRANSON, USA) for 30 minutes to give a more homogeneous size of the obtained etosome.

2. Preparation of isosquatoline-bearing ethosomes

In the same manner as in the above-described method for producing etosome of the invention, an isosquimolin-bearing etosome was prepared using isoquosityrin in place of the extract.

However, the used isoquositrin was dissolved in an amount of 0.01% to 0.05% (w / v) based on the volume of an organic solvent (10 mL of chloroform).

[Reference Example 4] Preparation of elastic liposome

1. Preparation of Elastic Liposomes Containing Fructus Extract

The production of elastic liposomes was carried out by the thin film hydration method, which is the same method as the preparation of the etosome formulation, as follows. After adding 0.1% of the extracted extract to a 50 mL round bottom flask, the egg PC (phosphatidylcholine) and the surfactant Tego ^® care 450 were mixed at different ratios (100: 0, 95: 5, 90:10, 85:15, 80:20 ) And dissolved in 15 mL of chloroform. When the organic solvent was removed using a rotary evaporator (BUCHI, Switzerland), a lipid membrane was formed on the wall of the flask. Phosphate buffer, a thin lipid membrane generated _{(1.6mM NaH 2 PO 4 · 2H} 2 O, 9.6 mM Na ₂ HPO ₄ .12H ₂ O, pH 7.4) to form an elastic liposome. In order to uniformize the particle size of the elastic liposome thus obtained, a glass bead was added and an ultrasonic shredder (BRANSON, USA) was added for 30 minutes. Ethyl acetate extract of the extract of Fusarium oxysporum were not removed by the elastic liposome. The final lipid concentration of the elastic liposome formulation was 0.5 wt% (w / v) The concentration was 0.1 wt% (w / v) based on the volume of the phosphate buffer solution.

2. Preparation of elastic liposome carrying isoquosityrin

An elastic liposome carrying isoquosityrin was prepared in the same manner as in the production of the elastic liposome carrying the prototype extract described in the above item 1, using isoquosityrin instead of the extract.

[Test Example 5] Measurement of particle size of the ethosomes carrying the dried extract or isoquositrin

1. Measurement method

In order to prepare the ethos according to the concentration of the solvent used and to determine the particle size of the ethosomes, 2% lecithin was used and the concentration of ethanol was varied to adjust the particle size and stability Respectively. The results are shown in Table 1 below.

2% lecithin 20% EtOH 143.7 nm 30% EtOH 192.3 nm 40% EtOH 209.1 nm

As can be seen in Table 1, when the concentration of ethanol exceeds 20%, the particle size increases with increasing concentration.

Therefore, 20% ethanol with a small particle size was selected and applied to the following experiment. Based on this, 20% ethanol containing ethosomes with various concentrations of extract or isoquositrin were prepared.

Particles dispersed in solution undergo Brownian motion depending on their size. When irradiated with light, large particles slowly move and small particles move fast. By analyzing this motion by photoelectron correlation method, the particle size is obtained by Einstein-Stokes equation. The size of the liposome formulation in the solvent was measured using a particle size analyzer, Otsuka ELS-Z (OTSUKA, Japan), which analyzes the particle size using light scattering intensity. He-Ne laser was used and the particle size was measured by cumulative analysis. In addition, the particle size distribution was determined using Contin.

All experiments were repeated 3 times and statistical analysis was performed with Student's t- test at the 5% significance level.

2. The size of the ethosomes carrying the extract

The particle size was measured for the ethosomes carrying the extracts of the extracts prepared by the method of Reference Example 3 at 0.03%, 0.04%, 0.05% and 0.10%. The results are shown in FIG.

7, when 0.03%, 0.04%, 0.05%, and 0.10% of the ethosomes containing the extracts of the extracts were prepared, 0.04% of the extracts containing ethosomes had a particle size of about 173.0 nm And it shows a stable particle size distribution. On the other hand, in the case of the ethosomes containing 0.10% of the extracts, the particle size was 572.0 nm as monodispersed form, but the layer separation occurred the next day after the preparation and it was unstable. In addition, 0.03% and 0.05% showed a polydisperse shape immediately after the production of ethosomes and no stable ethosomes were formed.

3. Size of ethosomes carrying isoquosityrin

The content of isoquosityrin prepared by the method shown in Reference Example 3 was 0.01%, 0.02%, 0.03%. 0.04% and 0.05%, respectively. The results are shown in FIG.

8, 0.01%, 0.02%, 0.03%. 0.04% and 0.05% of isozy- cinolin-containing ethosomes were produced, all of which showed a monodisperse shape with a particle size of less than 250 nm.

[Experimental Example 6] Measurement of collecting efficiency of ethosomes carrying an aspartic acid or isosquatolin

1. Measurement of collecting efficiency of ethosomes carrying an extract

1 mL of the completed ethosomal suspension containing 0.04% of the extract prepared according to the method of Reference Example 3 was taken and the uncoloured extract was removed using a 1.2 μm syringe filter (Minisart CA 26 mm). After that, 15 ~ 30 mL of ethanol was used to destroy the membrane of the etosome. The ethanol was evaporated using a rotary evaporator and then 1 mL of ethanol was added. In order to compensate the amount of ethanol extract in ethosomal suspension, the same amount of ethanol extract was dissolved in 20% ethanol and the solution was treated in the same manner as above. The samples thus prepared were measured at the maximum absorption wavelength of 360 nm using UV. The value obtained using UV was substituted into the following equation (2) to calculate the collecting efficiency of the etosome.

C _P : Concentration of the extract solution through a 1.2 μm syringe filter

C _E : Concentration of extracts in 20% ethanol

C ₀ : Concentration of first extract

All experiments were repeated 3 times and statistical analysis was performed at Student's t- test at the 5% significance level. The results are shown in Table 2.

0.04% EtO2 loaded with extracts Particle Size (nm) Immediately after manufacture 173.0 + - 103.4 One week after manufacturing 190.1 ± 136.9 Collection efficiency (%) 55.58 + - 7.33

Table 2 shows the results of measuring the particle size change and stability with time of the capturing efficiency of the ethosome carrying the 0.04% Fructus extract and the particle size immediately after the preparation and the one week after the Fructus extract-containing ethosomes Giving. It was confirmed that the particle size of the ethosome carrying 0.04% extracts was 173.0 ± 103.4 nm and the collection efficiency was 55.58 ± 7.33%.

After one week, the particle size of the ethosomes carrying 0.04% extracts was 190.1 ± 136.9 nm and the particle size was increased by 9.8%. However, even after one week, the particle distribution remained monodispersed, and no precipitation or segregation was observed, indicating that a stable etosome was formed.

Therefore, it was confirmed that the stable particle size distribution and proper particle size were maintained. Therefore, the ethosomes collected with 0.04%

2. Measurement of the collection efficiency of isosquatoline-carrying ethosomes

The content of isoquosityrin prepared by the method shown in Reference Example 3 was 0.01%, 0.02%, 0.03%. 0.0 > mL < / RTI > of the completed ethosomal suspension carried at 0.04% and 0.05%, and the uncoloured isoquosityrin was removed using a 1.2 m syringe filter (Minisart CA 26 mm). After that, 15 ~ 30 mL of ethanol was used to destroy the membrane of the etosome. The ethanol was evaporated using a rotary evaporator and then 1 mL of ethanol was added. The thus prepared sample was subjected to HPLC to measure the concentration of isosquinoline. The value obtained by HPLC was substituted into the following equation (3) to calculate the collecting efficiency of the etosome.

C _F : Isosquat concentration not passed through 1.2 μm syringe filter

C ₀ : Initial isoquositrin concentration

All experiments were repeated 3 times and statistical analysis was performed at Student's t- test at the 5% significance level. The results are shown in FIG.

FIG. 9 shows that the collection efficiency is high at a concentration of 0.03% or less, whereas the collection efficiency is decreased at a concentration of 0.04% or more.

In order to confirm the stability of isozy-cytoline, the particle size and the collection efficiency were measured for two weeks at intervals of one week, and the results are shown in FIGS. 10 and 11.

10, it can be seen that the isoquosityrin concentration is 0.04% or more, the particle size becomes larger or the unstable multi-phase form appears over time. As shown in FIG. 11, It is possible to confirm that the collection efficiency tends to decrease at concentrations other than those of the above-mentioned concentrations.

In addition, the ethosomes carrying the 0.03% isoquosityine showed 222.85 ± 0.85 nm particle size and 82.26% collection efficiency. Therefore, it was confirmed that the ethosome containing 0.03% isoquositrin had no significant change in particle size and collection efficiency and was stable.

Therefore, it was confirmed that the particle size distribution, the collection efficiency and the stability to maintain the proper particle size were confirmed. Therefore, the ethosomes capturing 0.03% isoquosityine were applied to the skin permeation experiment.

[Test Example 8] Experimental skin permeation experiment of ethosomes (Franz diffusion cell)

1. Measurement method

The skin permeation experiment was carried out using Franz diffusion cells and ethosomes prepared by the method of Reference Example 3 in order to examine the effect of the ethosomes on skin permeation enhancement of the extract or isosquatolines. The rat skin used in the skin permeation experiment was extracted from ICR non-muscular albino mice (8-week-old female), which had been sacrificed by cervical dislocation. The extracted skin was used after removing subcutaneous fat and tissue. After 5 mL of receptor phase (HCO-60: ethanol: PBS = 2:20:78 (w / w / w%)) was added to the receptor chamber, the donor The skin was fixed between the aqueous phases. During the experiment, the temperature was maintained at 37 ± 1 ° C using a constant-temperature water bath. 0.2 mL of each sample was applied to the surface of the skin through the donor, and 0.5 mL of the receiving phase was collected through a sampling port each time according to time. Immediately after harvesting, the same amount of water phase was replenished in the receiving chamber. The amount of the extracted extract in the collected samples was measured by an appropriate method.

After 24 hours, the skin of the rats was washed three times with PBS to determine the amount of the extract or the isoquosityrin remaining on the stratum corneum and skin. After washing, the portion not in contact with the aqueous phase was cut out, and the amount of the remaining extract or isoquositolin remaining in the stratum corneum was measured separately using a tape stripping method. To measure the amount of extracts or isoquoisolin remaining in the stratum corneum, the stratum corneum part of the skin was peeled off three times. To the tape thus obtained, 10 mL of ethanol was added, and the resulting extract was extracted with an ultrasonic washing machine for 1 hour Or isoquosityrin. The ethanol was then evaporated using a rotary evaporator and the extracted extract or isoquosityine was dissolved in 0.5 mL of water. After the tape stripping, the skin with the stratum corneum removed was cut with surgical scissors, and the treatment of the fine skin was performed in the same manner as the tape. The amount of the extracted extract or isoquosityine in the sample thus obtained was measured by an appropriate method.

The experiment was repeated 3 times and statistical analysis was performed with Student's t- test at the 5% significance level.

*2. Experimental skin permeation experiment of ethosomes carrying the extract

The skin permeation experiment described in the above 1. was carried out using 20% ethanol-containing ethosomes carrying 0.04% extracts. As a control, liposomes containing only 0.04% of the extracts, 0.04% of ethanol extract, and 0.04% of the extract were dissolved in distilled water.

In addition, the amount of the extracted extract in the sample was measured at 360 nm using UV, and the results are shown in FIG. 12 to FIG.

FIG. 12 shows that the amount of Fusarium oxysporum extract, cumulatively transmitted through the skin over time, tends to increase in a time-dependent manner in the ethanol solution used as an etosome and a control for 24 hours. The cumulative transdermal permeability of the skin after 24 hours was highest at 50.08 ± 1.01 μg / cm ² in the ethosomes, followed by ethanol solution (42.82 ± 1.40 μg / cm ² ), liposomes (23.91 ± 0.93 μg / cm ² ) (11.07 ± 0.46 μg / cm ² ).

Further amounts (Tape) of water pepper extract remaining in the horny layer] Turning now to FIG 13, after 24 hours, a little ^{(54.07 ± 0.21μg / cm 2)} > liposomes ^{(41.11 ± 0.15μg / cm 2)} > ethanol solution (29.59 ± 0.07 Eto μg / cm ^2)> distilled water (2.67 ± 0.04μg / cm ²⁾ showed a net, the amount of water pepper extract that remains on the skin, remove the stratum corneum (skin) Eto'o also get ^{(13.26 ± 0.11μg / cm 2)} > liposomes ( 8.42 ± 0.74μg / cm ^2)> in ethanol ^{(6.94 ± 0.06μg / cm 2)} > of distilled water (3.76 ± 0.06μg / cm ²⁾ can be seen to appear in this order.

Also, looking at Figure 14, it was 125.75μg / cm ² in all formulations the amount of water pepper extract used in the skin permeation experiments, the total skin permeation amount of water pepper extract is a little ETO ^{(117.41 ± 1.28μg / cm 2)} > ethanol solution (79.34 ± 1.43μg / cm ^2)> liposomes ^{(73.44 ± 0.40μg / cm 2)} > distilled water (17.50 ± 0.49μg / cm ²⁾ can be seen to appear in this order.

Therefore, the permeation efficiency of ethosomes was 93.37%, and the permeation efficiencies of ethanol extracts, liposomes and distilled water were 63.10%, 58.40% and 13.92%, respectively, and 0.04% The ability to deliver the extract to the skin is the most excellent of the etosome.

3. In vitro skin permeation experiment of isosquatoline-carrying ethosomes

The skin permeation experiment described in 1. above was carried out using 20% ethanol-containing ethosomes loaded with 0.03% isosquimolin. At this time, as a control group, liposome carrying only 0.03% isoquitoline, ethanol solution in which 0.03% isoquoisolin was dissolved, and 0.03% isoquosityrin dissolved in distilled water were used.

In addition, the amount of isoquositolin in the sample was measured using HPLC, and the results are shown in FIG. 15 to FIG.

15, the cumulative amount of isozy- cytolin permeated to the skin with time tends to increase in a time-dependent manner in both the ethosomes used as an etosome and the control group for 24 hours. Skin accumulated permeation amount (Transdermal) after 24 hours is higher with the little 65.48 ± 0.95μg / cm ² etoposide, and then the liposomes ^{(57.69 ± 0.91μg / cm 2)} , an ethanol solution ^{(39.1 ± 1.26μg / cm 2)} , distilled water (19.80 ± 0.34 μg / cm ² ).

In addition, Referring to FIG. 16, the amount of iso-site query Lin remaining in the horny layer after 24 hours (Tape) is etoposide little ^{(4.18 ± 1.49 μg / cm 2} )> in ethanol ^{(2.31 ± 1.3μg / cm 2)} > of distilled water (1.98 ± 0.05μg / cm ^2)> liposomes (1.53 ± 0.06μg / cm ²⁾ showed a net, sheep (skin) of iso-site queries lean left on the skin, remove the stratum corneum (1.92 ± 0.46μg some Eto / cm ² ), Ethanol (1.82 ± 0.81 μg / cm ² ), liposomes (1.50 ± 0.04 μg / cm ² ) and distilled water (1.44 ± 0.08 μg / cm ² )

17, the amount of isoquosityrin used in the skin permeation test was 125.75 μg / cm ² in all formulations, and the total skin permeation amount of isosquamolyne was determined as follows: ethosomia (72.96 ± 0.99 μg / cm ² ) (66.93 ± 0.75 μg / cm ² )> ethanol solution (41.98 ± 1.75 μg / cm ² )> distilled water (23.22 ± 0.47 μg / cm ² ) in that order.

Therefore, the permeation efficiency of ethosomes was 77.37%, and the permeation efficiencies of ethanol solutions, liposomes and distilled water were 70.97%, 44.51% and 24.62%, respectively. Therefore, when the amount and time of permeation to the skin are examined, it can be confirmed that the ethosome containing 0.03% isoquosityine can deliver faster and more amount to the skin than liposome.

[Test Example 9] Measurement of particle size of an elastic liposome carrying a crude extract or isoquosityine

1. Measurement method

An elastic liposome was prepared by the method shown in Reference Example 4, and the prepared liposome suspension was extruded using a mini extruder (having a polycarbonate membrane having a pore size of 80 nm).

The size of the liposome formulation in the solvent was measured using a particle size analyzer, Otsuka ELS-Z (OTSUKA, Japan), which analyzes the particle size using light scattering intensity. He-Ne laser was used and the particle size was measured by cumulative analysis. In addition, the particle size distribution was determined using Contin.

All experiments were repeated 3 times and statistical analysis was performed with Student's t- test at the 5% significance level.

2. Size of Elastic Liposome Carrying Extracts

The composition and composition of the elastic liposome and the average particle size before and after extrusion are shown in Table 3 below.

Formulation Number PC ^{a )} : S ^{b )}
(% w / w) Extract
(% (w / v)) Size before extrusion
(nm) Size after extrusion
(nm) EL ^{c )} -1 100: 0 - 161.3 ± 1.1 114.5 ± 9.1 EL ^{c )} -2 95: 5 - 183.2 ± 6.2 185.4 ± 5.1 EL ^{c )} -3 90: 10 - 121.6 ± 5.1 125.4 ± 5.1 EL ^{c )} -4 85: 15 - 95.4 ± 6.1 96.7 ± 3.3 EL ^{c )} -5 80: 20 - 137.3 ± 4.2 136.1 ± 1.6 ELP ^{d )} -1 100: 0 0.1 178.6 ± 1.6 178.9 + - 10.0 ELP ^{d )} -2 95: 5 0.1 176.5 ± 6.2 178.4 ± 0.1 ELP ^{d )} -3 90: 10 0.1 172.9 ± 1.4 162.6 ± 3.1 ELP ^{d )} -4 85: 15 0.1 105.5 ± 0.7 104.6 ± 1.2 ELP ^{d )} -5 80: 20 0.1 107.1 ± 0.2 104.9 ± 2.6

a) PC: Egg phosphatidylcholine

b) S : Surfactant (Tego ^® care 450)

c) EL: Elastic liposome formulation (not carrying any extracts)

d) ELP: Formulation of elastic liposome carrying a morpholino extract

Values are expressed as means ± SD (n = 3).

As shown in Table 3, the mean particle size of EL-1 was 161.3 nm and EL-2 was 183.2 nm in the case of viscoelastic liposome (EL-), and then the particle size increased with increasing surfactant concentration . In the case of EL-5 with a ratio of 80:20 of phospholipid to surfactant, the particle size was 137.3 nm. Elastic liposomes (ELP-) containing extracts showed a particle size distribution of 172.9 ~ 178.9nm up to 10% (w / w) of the surfactant, and showed a uniform size distribution in monodisperse form. When the concentration of the surfactant was 15% and 20%, the particle size tended to decrease from 105.5 to 107.1 nm.

Changes in particle size with storage time of elastic liposomes are important for evaluating the physical stability of liposome membranes. In general, it has been reported that when the liposome membrane becomes unstable, the gap between the liposome particles narrows and the particle size tends to increase. We investigated whether there was a change in the particle size immediately after and after one week (storage at 4 ℃) of the elastic liposome containing L. extract prepared in this study. The results are shown in Fig.

As shown in Fig. 18, the particle size of the elastic liposome after one week of production was measured, and the particle size was not significantly different from that immediately after the preparation, and the film was not broken or the layer separation phenomenon was observed after one week. Therefore, it was confirmed that the elastic liposomes prepared were kept stable for one week.

3. Size of elastic liposome carrying isoquosityrin

The composition and composition of the elastic liposome and the average particle size before and after extrusion are shown in Table 4 below.

Formulation Number PC ^{a )} : S ^{b )}
(% w / w) Isoquinolin
(% (w / v)) Size before extrusion
(nm) Size after extrusion
(nm) EL ^{c )} -1 100: 0 - 161.3 ± 1.1 114.5 ± 9.1 EL ^{c )} -2 95: 5 - 183.2 ± 6.2 185.4 ± 5.1 EL ^{c )} -3 90: 10 - 121.6 ± 5.1 125.4 ± 5.1 EL ^{c )} -4 85: 15 - 95.4 ± 6.1 96.7 ± 3.3 EL ^{c )} -5 80: 20 - 137.3 ± 4.2 136.1 ± 1.6 ELI ^{d )} -1 100: 0 0.1 195.75 ± 15.75 182.15 + - 3.45 ELI ^{d )} -2 95: 5 0.1 290.85 ± 1.35 261.15 + 0.45 ELI ^{d )} -3 90: 10 0.1 303.05 ± 1.15 343.65 + - 11.15 ELI ^{d )} -4 85: 15 0.1 341.2 ± 1.5 340.75 ± 13.95 ELI ^{d )} -5 80: 20 0.1 251.65 ± 17.65 193.85 + - 7.05

a) PC: Egg phosphatidylcholine

b) S : Surfactant (Tego ^® care 450)

c) EL: Elastic liposome formulation (not carrying any extracts)

d) ELI: Formulation of elastic liposome carrying an extract

Values are expressed as means ± SD (n = 3).

As shown in Table 4, the particle size of the elastic liposome (ELI-) containing isoquosityine increased from 195.75 nm to 341.2 nm as the surfactant concentration increased from 0 to 15% (w / w%) Respectively. This is because Tego ^® care 450 used in the experiment has a negative zeta potential as a nonionic surfactant. This negative zeta potential is thought to be due to the increase of the repulsive force between the lipid bilayers to increase the particle size.

However, the particle size of ELI-5 with a surfactant concentration of 20% (w / w%) tended to decrease again to 251.65 nm. This is because when the ratio of the surfactant is increased beyond a predetermined concentration, micelles are formed in addition to the elastic liposome and the average particle size is measured to be small.

[Test Example 10] Variable formation measurement of an elastic liposome carrying an aspartic acid or an isosquatolin

1. Measurement method

Variable formation of liposome membranes by surfactants is unique to elastic liposomes and is also different from other liposome formulations. These elastic liposomes have an advantage that the fluidity of the lipid membrane is increased and the lipid membrane can pass through the stratum corneum easily even though the particle size is larger than that of the general liposome. Therefore, in this study, the variable formation index of elastic liposome formulation was measured while increasing surfactant content.

For evaluation of the variable formability of the manufactured elastic liposome, the degree of the elastic liposome passing through the artificial permeation barrier was measured using a mini extruder. When the elastic liposome suspension was applied at a pressure of 0.2 MPa for 1 minute, the amount of the liposome suspension passed through the polycarbonate membrane having the pore size of 0.08 mu m was measured and the size of the liposome particles passed through the membrane was measured.

The elasticity value of the elastic liposome membrane was calculated according to the following equation (4).

Where J _flux is the amount of liposome that has passed through the membrane, r _v is the particle size of the liposome after extrusion, and r _p is the pore size of the membrane.

All experiments were repeated 3 times and statistical analysis was performed with Student's t- test at the 5% significance level.

2. Variable Formation of Elastic Liposome Supported with Extracts

The formulations of the elastic liposomes carrying the extracts of Experimental Example 9 were evaluated for variable formability. The results of the variable formulations of the elastic liposome formulations are shown in Table 5 below.

Formulation Variable shaping value ELP-1 15.0 ± 1.2 ELP-2 16.4 ± 0.8 ELP-3 13.6 ± 0.4 ELP-4 5.3 ± 0.1 ELP-5 5.6 ± 0.2

Comparing the ELP-2 and ELP-4 carrying the extracts of the present invention, the particle size of the ELP-2 having a surfactant content of 5% was 176.5 nm, which was smaller than that of ELP-4 (105.5 nm) having a surfactant content of 15% . However, the variable formability index of ELP-2 is 16.4 and the amount of surfactant is 5.3, indicating that the variable index of ELP-2 is much larger. Therefore, it can be seen that the variable index decreases when the amount of the surfactant is increased at a certain concentration.

This is thought to be due to the fact that, as the concentration of the surfactant increases, more micelles are formed in the solution besides the surfactant present in the elastic liposome. Therefore, the particle size of ELP-4 was measured to be small, and it was judged that the result of the variable formation decreased. These results are in agreement with previous reports that the surfactant is not only elastic liposomes but also micelles, resulting in a decrease in variable formation when the ratio of surfactant is more than a certain level (S. Jain, N Jain, D. Bhadra, AK Tiwary, and NK Jain, Transdermal delivery of an analgesic agent using elastic liposomes: preparation, characterization and performance evaluation, Curr Drug Deliv ., 2 , 223 (2005)).

From these results, it can be seen that the variable formation index of the elastic liposome carrying the extract is affected by the concentration of the surfactant.

3. Variable Formation of Elastic Liposome Containing Isoquocitrin

The formulations of the elastic liposome carrying the isoquosityine tested in Test Example 9 were evaluated for variable formability and the results for the variable formulations of the elastic liposome formulation are shown in Table 6 below.

Formulation Variable shaping value ELI-1 16.51 + - 0.63 ELI-2 34.05 0.12 ELI-3 59.47 ± 3.86 ELI-4 59.89 + - 4.86 ELI-5 18.65 + 1.35

Comparing ELI-1 and ELI-4, it can be seen that the particle size of ELI-4 (340.75 nm) with a surfactant content of 15% is much greater than ELI-1 (182.15 nm) without surfactant have. However, when the variable formability index is compared, ELI-1 is 16.51 and ELI-4 is 59.89, indicating that ELI-4 having a large amount of surfactant has a much higher variable-formation index.

This is because as the surfactant content is increased, the fluidity of the lipid membrane is increased and the variable formation of the elastic liposome is increased. However, it can be seen that the ELI-5 with a surfactant ratio of 20% shows a sharp decrease in the formation of the variable. It is considered that when the ratio of the surfactant is increased above a certain level, micelles as well as elastic liposomes are formed in the solution. Therefore, in the case of ELI-5, it can be seen that the variable formation index is greatly reduced due to the formation of micelles.

From these results, it can be seen that the variable formation index of the elastic liposome carrying isoquosityine is influenced by the concentration of the surfactant.

[Test Example 11] Measurement of collecting efficiency of elastic liposome carrying a crude extract or isoquosityine

1. Measurement method

A predetermined amount of the completed elastic liposome suspension was taken and a non-collected extract or isoquosityrin in the elastic liposome was removed using a 1.2 μm syringe filter (Minisart CA 26 mm). Thereafter, 15 to 30 mL of ethanol was used to destroy the membrane of the elastic liposome. The ethanol was evaporated using a rotary evaporator and then 1 mL of ethanol was added. The extracts or isosquatolines collected on elastic liposomes were quantified by appropriate methods.

The concentrations of the extracts and isozy-cytolines were calculated, and the concentration of the extracts was calculated by the following equation (5).

T: Concentration of first-introduced extract or isoquositrin

P: Concentration of the extract or isoquositrin which did not pass through the 1.2 μm syringe filter

All experiments were repeated 3 times and statistical analysis was performed with Student's t- test at the 5% significance level.

2. Elucidation efficiency of elastic liposome carrying the extract

The collection efficiency of the elastic liposome loaded with the extract was investigated by the above method. The absorbance at 360 nm, which is the maximum absorption wavelength of the extract, was determined by UV spectroscopy. The evaluation results are shown in Fig.

As shown in FIG. 19, ELP-1, a surfactant-free formulation, exhibited a collection efficiency of 38.6%, and the ELP-2 formulation containing 5% surfactant had a collection efficiency of 68.8% The highest collection efficiency was obtained. However, ELP-3 and ELP-4, which had surfactant concentrations of 10% and 15%, respectively, showed a collection efficiency of 56.6% and 54.1%, respectively, which is lower than that of the ELP-2 formulation. This suggests that the formation of mixed micelles affects the collection efficiency as well as the variable formation of the elastic liposome described above.

In other words, since micelles are formed when a surfactant having a specific concentration or higher is used and micelles and liposomes are mixed in the formulation, liposomal collection efficiency may be reduced, which may cause skin irritation and lower the efficiency of the skin delivery system (J Jain, N. Jain, D. Bhadra, AK Tiwary, and NK Jain, Transdermal delivery of analgesic agents using elastic liposomes: preparation, characterization and performance evaluation, Curr Drug Deliv ., 2 , 223 (2005); and C. Hofer, R. Hartung, R. Gobel, P. Deering, A. Lehmer, and J. Breul, New ultradeformable drug carriers for interleukin-2 and interferon- -alpha: theoretic and practical aspects, World J. Surg ., 24, 1187 (2000)).

3. Capture efficiency of elastic liposome carrying isoquosityrin

The collection efficiency of elastic liposome carrying isoquosityrin was evaluated by the above method, and the amount of collected extract was determined by HPLC. The evaluation results are shown in Fig.

As shown in FIG. 20, ELI-1 containing no surfactant showed a collection efficiency of 67.8%, and ELI-3 and ELI-4 had a collection efficiency of 57.50% and 54.30% Indicating that the physical properties (particle size, variable formation index, and collection efficiency) of ELI-3 and ELI-4 are very similar.

On the other hand, ELI-2, 3, 4, and 5 containing surfactant showed lower collection efficiency than ELI-1. This is consistent with the report that as the concentration of the surfactant increases, the absorption efficiency decreases due to the coexistence of the elastic liposome and the mixed micelle. This indicates that when the concentration of the surfactant is higher than a certain level, the absorption efficiency of the elastic liposome is decreased and thus it can not be used as a useful skin delivery system (SD Maurya, S. Aggarwal, VK Tilak, RC Dhakar, A. Singh, and G. Maurya, Enhanced Transdermal Delivery of Indinavir Sulfate via Transfersome, Pharmacie Globale , 1 (06) (2010)]

[Test Example 12] Experimental skin permeation test of elastic liposome (Franz diffusion cell)

1. Measurement method

In order to examine the effect of the elastic liposome on the skin permeation enhancement of the extract or isoquosityrin, Franz diffusion cell and the elastin liposome prepared according to the method of Reference Example 4, which was highly optimized and highly variable, and used in Test Examples 9 to 11 To conduct skin permeation experiments. In addition, the skin permeation of the elastic liposome was penetrated into the skin by the hydration gradient, and the drug was delivered to the skin, so that the sample administration site was performed in a non-occluded condition.

The rat skin used in the skin permeation experiment was extracted from ICR non-muscular albino mice (8-week-old female), which had been sacrificed by cervical dislocation. The extracted skin was used after removing subcutaneous fat and tissue. After 5 mL of receptor phase (HCO-60: ethanol: PBS = 2:20:78 (w / w / w%)) was added to the receptor chamber, the donor The skin was fixed between the aqueous phases. During the experiment, the temperature was maintained at 37 ± 1 ° C using a constant-temperature water bath. 0.2 mL of each sample was applied to the surface of the skin through the donor, and 0.5 mL of the receiving phase was collected through a sampling port each time according to time. Immediately after harvesting, the same amount of water phase was replenished in the receiving chamber. The amount of the extracted extract in the collected samples was measured by an appropriate method.

After 24 hours, the skin of the rats was washed three times with PBS to determine the amount of the extract or the isoquosityrin remaining on the stratum corneum and skin. After washing, the portion not in contact with the aqueous phase was cut out, and the amount of the remaining extract or isoquositolin remaining in the stratum corneum was measured separately using a tape stripping method. To measure the amount of extracts or isoquoisolin remaining in the stratum corneum, the stratum corneum part of the skin was peeled off three times. To the tape thus obtained, 10 mL of ethanol was added, and the resulting extract was extracted with an ultrasonic washing machine for 1 hour Or isoquosityrin. The ethanol was then evaporated using a rotary evaporator and the extracted extract or isoquosityine was dissolved in 0.5 mL of water. After the tape stripping, the skin with the stratum corneum removed was cut with surgical scissors, and the treatment of the fine skin was performed in the same manner as the tape. The amount of the extracted extract or isoquosityine in the sample thus obtained was measured by an appropriate method.

The experiment was repeated 3 times and statistical analysis was performed with Student's t- test at the 5% significance level.

2. Experimental skin penetration test of elastic liposome carrying a dried extract

The skin permeation experiment described in 1. above was conducted using the elastic liposome of ELP-2 of Test Example 7, which is a formulation containing 5% (w / w) of a surfactant. At this time, ELP-1, which is a general liposome without a surfactant, and 1,3-butylene glycol which has a moisturizing effect but hardly skin irritation, were used as a control group.

In addition, the amount of the extracted extract in the sample was measured at 360 nm using UV, and the results are shown in FIG. 21 and FIG.

21, ELP-2, an elastic liposome carrying an extract of Lycopersicon esculentum, showed a significantly larger amount of penetrated extracts in comparison with the control group. The extracts of ELP-2, ELP-1 and ELP-1, which are elastic liposomes carrying the extracts, and the control, 1,3-butylene glycol (stock solution in the stock solution) Respectively. In addition, the cumulative permeation amount (Transdermal) of water pepper extract after 24 hours in the case of 1,3-butylene glycol solution was 61.8μg / cm ^2, the ELP-1 was 72.5μg / cm ^{2, ELP-2} was 77.6μg / cm ² The transmittance was 19.7%, 23.0% and 24.7% for the initial loading of 314.4 μg / cm ² , respectively.

FIG. 22 shows the contents of the extracts (Tape) present in the stratum corneum after 24 hours, the skin extracts existing in the epidermis and dermis excluding the stratum corneum, and the transdermal components present in the water phase through the skin . The content of TSP in the stratum corneum was 5.1% in the case of 1,3-butylene glycol solution, and 11.1% in ELP-1 and 12.9% in ELP-2. There were a few more cases in the stratum corneum. Except the stratum corneum skin content (Skin) of the water pepper extract penetrate into (epidermis and dermis) is, 1,3-butylene glycol and ELP-1, respectively, ^{37.4μg / cm 2, 26.6μg / cm} 2 in the ELP-3, The ELP-2 (95: 5) elastic liposome formulation as in the case of the stratum corneum was 11.8%, 8.5%, and 12.9%, respectively, when converted into the penetration rate of the extract according to the initial drop amount by 46.2 μg / cm ² The amount of extracts in the epidermis and dermis was much higher. The content of transdermal in the aqueous phase through the skin was 19.7%, 23.0% and 24.7% for 1,3-butylene glycol, ELP-1 and ELP-2, respectively. (Fig. 22B). The total amount of the extracts penetrated into the skin was 115.3 μg / cm ² , 133.9 μg / cm ² and 164.5 μg / cm ² in the case of 1,3-butylene glycol and ELP-1 and ELP- ² for the initial drop of 314.4 μg / mu] g / cm < ² >. These results were 36.7%, 42.6% and 52.3%, respectively, when the skin absorption rate of the extract was compared with the initial dose. This indicates that when the elastic liposome formulation is used, a larger amount of the extract can be delivered to the skin. As a result of measuring the total content of the extracts, the extracts of ELP-1 and ELP-2 were transferred to the skin by about 18.6% by surfactant.

3. Experimental skin penetration test of elastic liposome carrying isoquosityrin

The skin permeation experiments described in 1. above were carried out using elastic liposomes of ELI-3 and ELI-4, which exhibited a high degree of variable formation index as well as physical properties such as particle size, variable formation index and collection efficiency. At this time, ELI-1, which is a general liposome without a surfactant, was used as a control group, and 1,3-butylene glycol having a moisturizing effect but little skin irritation was used as a control solution.

Also, the amount of isoquosityrin in the sample was measured by HPLC, and the results are shown in FIG. 23 and FIG.

FIG. 23, which shows the permeation amount of isoquercitrin over time for a certain area of skin (0.6362 cm ² ), shows that the amount of skin permeation increases with time in all four experimental formulations carrying isoquosityine. 1,3-butylene glycol and 24 for cumulative permeation amount after the time ^{35.51μg / cm 2, 50.82μg / cm} 2 , respectively 11.29% and a transmittance of 16.17% based on the initial drop quantity 314.4μg / cm ² in the ELI-1 . In particular, in the case of ELI-4, the amount of skin permeation rapidly increased in a time-dependent manner, and the cumulative permeation amount after 24 hours was 118.48 μg / cm ² , And the transmittance was 37.69% in terms of the initial drop amount. In the case of ELI-3, which showed similar physical properties as ELI-4, the cumulative permeation amount after 24 hours was 59.33 μg / cm ^{2, which} was 18.87% of the initial drop amount. In the case of ELI-3, the particle size increased from 303.05 nm to 343.65 nm before and after extrusion. This increase in particle size indicates that the lipid membrane of ELI-3 is unstable. This instability of the lipid film explains why the ELI-3 skin permeability is much lower than that of ELI-4 with similar physical properties.

FIG. 24 shows isoquercitrin (Tape) existing in the stratum corneum after 24 hours, isoquercitrin present in the epidermis and dermis excluding the stratum corneum, and isoquercitrin Transdermal) content of the cells. The content of isoquitolines present in the stratum corneum was 0.59% and 4.19% for 1,3-butylene glycol solution and ELI-1, respectively. For ELI-3 and ELI-4, %, And 1.12%, respectively. It was confirmed that a relatively small amount of isoquitolines remained in the stratum corneum. Skin (skin) permeated into the skin (epidermis and dermis) except the stratum corneum was 2.49 μg / cm ² in 1,3-butylene glycol and ELI-1, ELI-3 and ELI-4, ^{2.59μg / cm 2, 3.13μg / cm} 2, respectively, 0.79%, 0.82% when converted to iso query transmission site lean on the initial drop quantity by 19.33μg / cm ^2, 1.0%, in a ratio to optimize 6.15% ELI-4 showed more isoquinolines in epidermis and dermis. The proportion of isoquercitrin present in the aqueous phase through the skin was 11.29%, 16.17%, 18.87%, respectively, for 1,3-butylene glycol and ELI-1, ELI-3 and ELI- 37.69%, and the permeation amount of ELI-4 was very large. The total amount of isozy-quinolin penetrated into the skin was as follows: ELI-4 (54.40%)> ELI-3 (22.25%)> ELI-1 (21.18%)> 1,3-butylene glycol %). In particular, it can be seen that ELI-4 delivered about 31.72% more isoquosityrin to skin than ELI-1 due to the increase of variable formation due to the addition of surfactant.

Claims (8)

Claims 1. An elastic liposome carrying isosquimolin, the liposome being formed by dissolving isosquimolene in a solvent containing a phospholipid and a surfactant in a weight ratio of 85:15, and hydrating the lipid film with a phosphate buffer solution. . The elastic liposome according to claim 1, wherein the solvent is chloroform. The elastic liposome according to claim 1, wherein the phospholipid is phosphatidylcholine. The elastic liposome according to claim 1, wherein the surfactant is free of polyethylene glycol. The elastic liposome according to claim 4, wherein the surfactant is polyglyceryl-3 methyl glucose distearate. A cosmetic composition containing the elastic liposome according to any one of claims 1 to 5. The cosmetic composition according to claim 6, wherein the composition is for enhancing skin barrier. The cosmetic composition according to claim 6, wherein the elastic liposome is contained in an amount of 0.5 to 10% by weight based on the total weight of the composition.

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