KR101415222B1 - Topical Composition For Promoting Lipid Biosynthesis Comprising Forsythia Suspensa Fruit Extract As Active Ingredient - Google Patents

Abstract

The present invention relates to a composition for external application for skin containing a tangle extract as an active ingredient. The composition for external application for skin of the present invention can be used for promoting biosynthesis of triglyceride, cholesterol or ceramide by controlling the transcription factors of sterol regulatory factor binding protein (SREBP-1c), peroxisome proliferation-activating receptors (PPARs) Thereby maintaining skin homeostasis and preventing skin aging.

Description

[Technical Field] The present invention relates to a topical composition for promoting lipid biosynthesis,

The present invention relates to a composition for external application for skin, which comprises a tandem extract as an active ingredient.

The lipids that make up the skin barrier are composed of sebaceous lipid and epidermal lipid, which are produced in Sebocyte and keratinocyte, respectively. Sebum produced in Sebocyte is secreted through hair follicle and lipid produced in keratinocyte is stored in the lamellar body of S. spinosum and S.granulosum, Respectively. There are about one million sebaceous glands in the human body, and they are mainly in the face, head, breast, armpit, navel, and neck. The sebaceous gland is a holocrine gland that secretes sebum by the stem of hair and is known to secrete about 2g per day. The ingredients of sebum are known as triglyceride, free fatty acid, wax ester, squalene, cholesteryl ester, cholesterol and the like. The sebaceous glands are responsible for the regulation of cutaneous steroidogenesis, the regulation of local androgen synthesis, the interaction of neuropeptides, lipid production with antimicrobial activity, pro-inflammatory properties, And is responsible for the expression of anti-inflammatory properties. The keratinocyte lipid is composed of cholesterol, fatty acid, ceramide, and sphingolipid, and exists in a lamellar structure. In addition, ceramide is known to contain 50% of the intercellular lipid in the structure with the hydroxy group attached to the backbone composed of sphingosine, phytosphingosine and 6-hydroxysingin.

The distribution ratios and the mass of these compositions contribute to skin barrier homeostasis and provide a quick resilience to external physical and chemical damage. The epidermal lipid in the aging process, unlike the epidermal tissue, is generally reduced and the function of the skin barrier is deteriorated.

In addition, the total epidermal lipid content of elderly people is reduced to about 30% of the younger age, but the ratio of each lipid content is maintained.

It has been reported in the literature that lipids of the skin keratin are formed through the lipid biosynthetic pathway and the ceramide biosynthesis pathway and actually lipids of the skin horn are derived from phospholipids and free fatty acids of the dermis [Hamanaka et al., 2002 , J. Invsti. Dermatol. 119: 416-423. It is also known that as the ratio of free fatty acids changes, it can progress to aging skin [Rogers et al., 1996, Arch. Dermatol. Res., 288: 765-770).

Forsythia Suspensa Fruit Extract is extracted from the fruit of the allium. It is a vine bush that has a yellowish foliage in early spring. Generic forsythia has almost no fruit. The forsythia which opens its fruit is called three-leaf forsythia. It is dried in the sun after it is dried in autumn. There are oleanolic acid, tannic acid, phyllin, monoterpene glycoside (procidide, procidide monoester) in the skin, and fruits include sterols, sterol compounds, sapinin (no hemolytic) Synocide and the like. The water extract of this plant is known to have antimicrobial action against Staphylococcus aureus, Paratyphoid, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae, and Pertussis bacteria.

The inventors of the present invention have studied the applicability of cosmetics among various natural products that have not been known so far. As a result, it has been found that the extract of Yeast Extract has sterol regulatory element-binding proteins (hereinafter referred to as "SREBP-1c" Promotes the transcription factors of peroxisome proliferator-activated receptors (PPARs) and fatty acid synthase (FAS), promotes biosynthesis of intercellular proteins, It is intended to treat and prevent the lipid homeostasis of a skin epidermal layer to maintain the skin barrier effect of the epidermis and to maintain the homeostatic function and anti-aging function of the whole skin.

Accordingly, the object of the present invention as described above is achieved by a composition containing a tangle extract as an active ingredient. The composition has a skin homeostasis-maintaining effect and an anti-aging effect by increasing skin lipid biosynthesis

Preferably, the composition is characterized by promoting transcription factors of sterol regulatory factor binding protein (SREBP-1c), peroxisome proliferation-activating receptors (PPARs) and fatty acid synthase (FAS).

Also preferably, the composition is characterized by increasing the amount of cholesterol, ceramide or triglyceride expressed.

Preferably, the tangle extract is contained in an amount of 0.00001 to 40.0% by weight based on the total weight of the composition.

Also preferably, the composition is in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream and spray Lt; RTI ID = 0.0 > formulations < / RTI >

The combined extract of the present invention has increased expression of SREBP-1c, PPARs and FAS, lipid increase through nile red staining & flow cytometry, increased cholesterol expression, increased triglyceride expression and increased ceramide expression.

Figure 1 shows lipid production when keratinocyte extract and linolenic acid were applied to keratinocytes.

The present invention relates to a composition for promoting biosynthesis of a skin epidermal lipid. The present invention relates to a composition for external application for skin, which contains an alginate extract as an active ingredient, and maintains skin homeostasis by increasing lipid biosynthesis. The composition can be used to increase the biosynthesis of triglyceride, cholesterol or ceramide by controlling the transcription factors of sterol regulatory factor binding protein (SREBP-1c), peroxisome proliferation-activating receptors (PPARs) or fatty acid synthetase (FAS) To treat and prevent the disruption of the homeostasis of skin epidermal lipids due to lipid degradation such as ceramide.

Hereinafter, a method of preparing a dried extract of the present invention will be described with reference to the following specific examples of the present invention. The dried fruit of the dried fruit is washed with purified water, dried and crushed into small pieces, Of lower alcohol is added. It is extracted at a constant temperature for a certain period, filtered, and then dried with a rotary evaporator.

According to the present invention, the tangle extract is contained in an amount of 0.00001 to 40.0 wt% with respect to the total weight of the composition, and more preferably, the tangle extract is contained in an amount of 0.001 to 10 wt% with respect to the total weight of the composition. When the content of the extract is less than 0.00001% by weight, no effective effect is exhibited. When the content of the extract is more than 40.0% by weight, the improvement effect on the increase of the content is insignificant and there is a problem in safety and stability of the form and is not economical .

The composition of the present invention comprising the extract of the present invention is characterized by having an effect of increasing the expression of SREBP-1c, PPARs, FAS, increasing lipid, increasing cholesterol, increasing ceramide expression, and increasing triglyceride expression.

The components included in the composition of the present invention may contain, in addition to the above-mentioned effective components, the components conventionally used in the composition for external application for skin, such as conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, Carrier.

In the case of application to medicines, an inorganic or organic carrier, which is used as an active ingredient of the extract of Yeast extract according to the present invention, may be added to formulate it into an oral or parenteral dosage form in the form of solid, semi-solid or liquid.

Examples of the agent for oral administration include tablets, pills, tablets, soft capsules, powders, fine granules, powders, emulsions, syrups, fillets and the like. For parenteral administration, injections, drops, ointments, lotions, sprays, suspensions, emulsions, suppositories, and the like may be mentioned. In order to formulate the active ingredient of the present invention, it can be easily formulated according to a conventional method. Surfactants, excipients, coloring agents, spices, preservatives, stabilizers, buffers, suspending agents and other adjuvants for commercialization can be suitably used.

The compositions of the present invention may be prepared in any of the formulations conventionally manufactured in the art and may be in the form of solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, Oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream and spray, but are not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

When the formulation of the present invention is a paste, cream or gel, an animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as the carrier component .

When the formulation of the present invention is a solution or an emulsion, a solvent, a dissolving agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid esters.

In the case where the formulation of the present invention is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In the case of a spray, in particular, / Propane or dimethyl ether.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

When the composition of the present invention is a soap, a surfactant-containing cleansing formulation or a surfactant-free cleansing formulation, it may be applied to the skin and then wiped off, washed or rinsed with water. The surfactant-containing cleansing formulation may be a cleansing foam, a cleansing water, a cleansing towel and a cleansing pack. The surfactant-free cleansing formulation may be a cleansing cream, , Cleansing lotion, cleansing water and cleansing gel, but is not limited thereto.

The composition of the present invention may be used alone or in combination, or may be used in combination with other compositions than the present invention. In addition, the composition according to the present invention can be used according to a conventional method of use, and the number of times of use may be varied depending on the skin condition or taste of the user.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Manufacturing example  One: Almond extract  Produce

The method for preparing the extract of Ankylosing Pigment used in the present invention is as follows. 100 g of fruit of the allium is washed with purified water, dried and crushed into small pieces, to which 70% lower alcohol of 1-10 times its dry weight is added. After 5 days of extraction at 15-35 ° C, the mixture was filtered through a 300-mesh filter cloth, filtered through filter paper No. 5, and dried with a rotary evaporator to obtain 29.5 g of dry weight.

Experimental Example  1: Cell culture

The cells used in the experimental examples were cultured in the following manner.

① keratinocyte: Keratinocyte

Human normal skin cells, keratinocytes (Korean Cell Line Bank, Korea), were inoculated into 48-well microplates (Nunc, Denmark) at a density of 1 × 10 ⁶ cells per well. DMEM medium (Sigma, USA) For 24 hours. Then, the cells were further cultured in serum-free DMEM medium supplemented with the yeast extract of Preparation Example 1 and in serum-free DMEM medium containing no yeast extract for 48 hours as a control.

② Sebaceous cell : SZ95

SZ95 cells, the human sebaceous cell line, were obtained from Dr. Benjamin Franklin at the Free University of Berlin University Medical Center in Berlin, Germany. Human immortalized sebocytes (SZ95) from Zouboulis were cultured in a CO ₂ incubator (37 ° C, 5% CO ₂ ) using Dulbecco's modified eagle medium (DMEM) / F12 (1: 1) supplemented with 10% FBS. Then, the culture medium was changed at a cycle of 48 hours.

Experimental Example  2: PPARs  Measurement of expression level

The expression levels of PPARs (PPAR-α, PPAR-β, and PPAR-γ) were measured in sebum cells cultured in Experimental Example 1 as follows. Expression level was measured by Western blotting.

The cultured sebum cells were treated with alfalfa extract (20 μg / ml and 50 μg / ml) and linolenic acid (200 μg / ml), respectively, and cultured for 48 hours. Subsequently, the medium was removed and washed, and then dissolved in 80 μl of lysis buffer. The supernatant was collected by centrifugation at 4 ° C. and 12,000 rpm for 20 minutes. The amount of protein contained in the cell lysate was measured by the Bradford method. 30 [mu] l of protein was separated by SDS-PAGE containing 10% polyacrylamide and electroblotted with PVDF membrane for 1 hour at 120V. The PVDF membrane was then left in 5% skim milk for 1 hour and then placed in a primary antibody overnight at 4 ° C. After washing three times with TBST, the secondary antibody was reacted for 1 hour at room temperature, and then reacted with ECL kit (Amersham-Pharmacia) three times with TBST and quantitated using BioRad GelDoc.

The band intensities of the respective test values were read, and the increase in the content was converted into the percentage of the control. The results are shown in Table 1 by RT-PCR in sebaceous cells.

density
(μg / ml) PPAR-α (%) PPAR-β (%) PPAR-γ (%) Almond extract 20 101.9 212.8 158.7 Almond extract 50 110.7 250.1 207.4 Linolenic acid 200 162.5 175.1 157.3

The expression of PPAR-α, PPAR-β, and PPAR-γ proteins were examined after treatment with alfalfa extract at concentrations of 20 and 50 μg / ml for 48 hours. And increased concentration-dependent protein expression. Linolenic acid, a positive control, also increased the expression of PPAR-β and PPAR-γ.

Experimental Example  3: SREBP -1c, FAS Of expression level

The expression levels of SREBP-1c and FAS (fatty acid synthase) were measured in sebum cells cultured in Experimental Example 1 as follows. Expression level was measured by Western blotting.

The cultured sebum cells were treated with alfalfa extract (20 μg / ml and 50 μg / ml) and linolenic acid (200 μg / ml), respectively, and cultured for 48 hours. Subsequently, the medium was removed and washed, and then dissolved in 80 μl of lysis buffer. The supernatant was collected by centrifugation at 4 ° C. and 12,000 rpm for 20 minutes. The amount of protein contained in the cell lysate was measured by the Bradford method. 30 [mu] l of protein was separated by SDS-PAGE containing 10% polyacrylamide and electroblotted with PVDF membrane for 1 hour at 120V. The PVDF membrane was then left in 5% skim milk for 1 hour and then placed in a primary antibody overnight at 4 ° C. After washing three times with TBST, the secondary antibody was reacted for 1 hour at room temperature, and then reacted with ECL kit (Amersham-Pharmacia) three times with TBST and quantitated using BioRad GelDoc.

The band intensity of each test value was read, and the increase in the content was compared with that of the control. The results are shown in Table 2.

density
(μg / ml) SREBP-1
(%) FAS (%) Almond extract 20 131.0 209.1 Almond extract 50 185.4 222.9 Linolenic acid 200 126.2 144.3

The expression of SREBP-1 and FAS (fatty acid synthase) protein was increased after 48 hours of treatment with alfalfa extract (20 and 50 μg / ml). Linolenic acid (200 μg / ml) used as a positive control also increased protein expression.

Experimental Example  4: Measurement of lipid

Lipid production was measured by nile red staining and flow cytometry.

The keratinocyte cultured in Experimental Example 1 was treated with alfalfa extract (20 μg / ml and 50 μg / ml) and linolenic acid (200 μg / ml), respectively, and cultured for 48 hours. The keratinocytes were harvested from each month, and then stained with 1 uM Nile red solution for 10 minutes. The fluorescence intensity was measured at 552/636 (Ex / Em) nm. The measurement results are shown in Fig. In FIG. 1, fluorescence intensity was measured by FACS after Nile red staining. As a result, lipid production was increased in the group treated with alfalfa extract, and cells treated with linolenic acid 200 μg / ml (87.30) (50 μg / ml, 95.22) was found to be larger than that of the extracts.

Experimental Example  5: Cholesterol measurement

The SZ95 cells cultured in Experimental Example 1 were dispensed into a 10-cm culture dish at a ratio of 1 × 10 ^5, and the resulting extracts were treated at 25, 50, 100 and 200 μg / ml, respectively, for 5 days and cholesterol was quantified . For the cholesterol determination, the cells cultured for 5 days were treated with lysis buffer (10 μL of EDTA, 100 mM SPB solution, 1 μL of PMSF, and 10 μl of Triton X-100) and sonicated. A chloroform: methanol (2: 1) solution was added to the cell lysate, mixed and centrifuged at 2000 rpm for 15 minutes to collect the lower layer liquid and concentrate it with nitrogen gas (N ₂ ).

The concentrated sample was dissolved in 300 μl of ethanol and measured with a total cholesterol kit (AM 202-K). 100 μl of the sample was added to 1 ml of the enzyme solution, and the mixture was incubated at 37 ° C. for 5 minutes, and the absorbance was measured with an ELISA reader at a wavelength of 500 nm. Cholesterol in the standard solution was adjusted to 3 mg / mL by adding 20 μl of the standard solution to 3 ml of the enzyme solution.

The amount of cholesterol measured is shown in Table 3. As shown in Table 3, the control group was 0.77 ml / ml, but the cholesterol increased about twice as much as the control group.

Experimental Example  6: Triglyceride  Measure

SZ95 cells cultured in Experimental Example 1 were dispensed into a 10 cm culture dish at a dose of 1 × 10 ^5, and the extracts of the alum extracts were treated at 25, 50, 100 and 200 μg / ml, respectively, for 5 days and cholesterol was determined. For the method of quantifying triglyceride, the cells cultured for 5 days were treated with lysis buffer (10 μL of EDTA, 1 mM of SPB solution, 1 μL of PMSF, and 10 μL of Triton X-100) and then disrupted by ultrasonication. A chloroform: methanol (2: 1) solution was added to the cell lysate, mixed and centrifuged at 2000 rpm for 15 minutes to collect the lower layer liquid and concentrate it with nitrogen gas (N ₂ ).

To the concentrated sample, 300 μL of ethanol was added and dissolved, and triglyceride was measured using a triglyceride kit (GPO-PAP). 100 μL of the sample was added to 1 mL of the enzyme solution, the mixture was incubated at 37 ° C. for 5 minutes, and the absorbance was measured with an ELISA reader at a wavelength of 546 nm. For the standard solution of triglyceride, 20 μl of the standard solution was added to 3 ml of the enzyme solution to give a value of 3 mg / ml.

Formulation Example  One: Yeast extract  Preparation of compositions containing

Formulation Examples 1, 2 and 3 (Preparation Example 1) containing an almond extract were prepared, and a composition containing glycerin and 1,3-butylene glycol as moisturizing agents (Comparative Formulation Example 1 ), A composition containing glycerin and sorbitol as a moisturizing agent (Comparative Formulation Example 2), and a composition containing no extract or moisturizer (Comparative Formulation Example 3) are shown in Table 5 below.

division ingredient
(Unit: wt%) Formulation Example 1 Formulation Example 2 Formulation Example 3 compare
Formulation Example 1 compare
Formulation Example 2 compare
Formulation Example 3 A Almond extract
(Production Example 1) 0.1 - - - - - Almond extract
(Production Example 1) - 1.0 - - - - Almond extract
(Production Example 1) - - 3.0 - glycerin - - - 5.0 5.0 1,3-butylene glycol - - - 5.0 - - Sorbitol - - - - 5.0 - EDTA-2Na 0.02 0.02 0.02 0.02 0.02 0.02 Purified water to 100 to 100 to 100 to 100 to 100 to 100 B Cetostearyl alcohol 2.0 2.0 2.0 2.0 2.0 2.0 Glyceryl stearate 1.5 1.5 1.5 1.5 1.5 1.5 Microcrystalline 0.7 0.7 0.7 0.7 0.7 0.7 Squalene 5.0 5.0 5.0 5.0 5.0 5.0 Liquid paraffin 3.0 3.0 3.0 3.0 3.0 3.0 Triethanolhexanone 5.0 5.0 5.0 5.0 5.0 5.0 Polysorbate 1.2 1.2 1.2 1.2 1.2 1.2 Sorbitan stearate 0.5 0.5 0.5 0.5 0.5 0.5 Tocopheryl acetate 0.2 0.2 0.2 0.2 0.2 0.2 Cyclomethicone 3.0 3.0 3.0 3.0 3.0 3.0 BHT 0.05 0.05 0.05 0.05 0.05 0.05 C Incense, preservative Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount Suitable amount

Experimental Example  7: Almond extract  Moisturizing effect

As intercellular lipids are produced and increased, the homeostasis, immunity and moisturizing power of the skin also increases. In order to confirm the moisturizing effect of the extract of Ankyo, the clinical moisturizing effect of the compositions of Formulation Examples 1, 2 and 3 was measured as follows. A, B, and C groups were divided into five groups (A, B, C, D, and E) by a randomly selected group of 25 healthy adult men and women who felt skin dry through the questionnaire. (Comparative Formulation Example 1) containing glycerin and 1,3-butylene glycol as moisturizing agents, a composition comprising glycerin and sorbitol as a moisturizing agent (Comparative Formulation Example 2 ) Was applied to the supernumerary skin for 8 weeks twice a day, respectively. The moisturizing effect was evaluated by the Corneometer CM 820 (Corage + Khazaka, Germany) after every two weeks and after the test. The experimental results are shown in Table 6 below.

division Before use After 2 weeks of use After 4 weeks of use Formulation Example 1 22.8 35.7 44.1 Formulation Example 2 22.9 37.5 48.2 Formulation Example 3 23.1 40.3 54.8 Comparative Formulation Example 1 23.8 32.5 36.1 Comparative Formulation Example 2 23.0 32.0 36.5 Comparative Formulation Example 3 22.9 33.4 34.3

As shown in Table 6, it was found that the moisturizing effect of Formulations 1, 2 and 3 containing the extract of Yeast extract of the present invention (Formulations 1, 2 and 3) I could confirm.

Experimental Example  8: Ceramide  Measure

The samples of the skin keratin samples were collected using the tape method and the ceramide contents were measured under the following conditions. The content of ceramides per unit protein of the sample extracted by the tape method was determined, and the content of the protein was determined by a BSA protein assay kit.

For the ceramide analysis, UPLC-MS used Quattro Premier XE MS model connected with Waters Aquity UPLC system.

<Analysis condition>

Column: Acquity BEH C * (2.1 mm X 50 mm, 1.7 um)

Mobile phase 5 mM ammonium formate / methanol (4: 6, 0.1% formic acid)

Flow rate: 0.3 ml / min

Column temperature: 40 ° C

<MS analysis condition>

Ionization conditions-ESI cation mode

Source temperature -130 ℃

Desolvation temperature -350 ℃

Congas station 800 L / h

Capillary voltage -3.2 Kv

Ion Monitor MRM

Ceramide Formulation Example 3 (ng / mg) Comparative Formulation Example 3 (ng / mg) C16 ceramide 130.9 86.5 C18 ceramide 124.2 76.7 C24 ceramide 198.3 135.6

As a result of the test, it was confirmed that the content of the three ceramides per unit protein was increased when Formulation Example 3 and Comparative Formulation 3 were compared.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (6)

A composition for external application for skin for maintaining skin homeostasis by increasing skin lipid biosynthesis, which comprises a combination extract as an active ingredient. The composition according to claim 1, wherein the skin lipid biosynthesis is enhanced by promoting transcription factors of sterol regulatory factor binding protein (SREBP-1c), peroxisome proliferation-activating receptors (PPARs) or fatty acid synthetase (FAS) . The composition for external application for skin according to claim 1, wherein the increase in the lipid biosynthesis of the skin is achieved by increasing the expression amount of cholesterol, ceramide or triglyceride. delete The composition for external application for skin according to any one of claims 1 to 3, wherein the tangle extract is contained in an amount of 0.00001 to 40.0% by weight based on the total weight of the composition. The composition according to any one of claims 1 to 3, wherein the composition is in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, cleansing oil, powder foundation, emulsion foundation, wax foundation, Massage cream, and spray. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;

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