KR102297607B1 - Composition for improved atopy skin and skin moisturizing comprising asiatic tearthumb - Google Patents

Abstract

The present invention relates to a composition for improving atopic skin and moisturizing the skin, including a daughter-in-law's navel, which can improve atopic skin by showing an anti-inflammatory effect by using a daughter-in-law extract, strengthening the skin barrier and increasing the moisture content It is possible to provide a composition for improving atopic skin and moisturizing the skin.

Description

Composition for improving atopic skin and moisturizing skin including daughter-in-law's navel

The present invention relates to a cosmetic composition for improving atopic skin including a daughter-in-law's belly button and moisturizing the skin. More specifically, it relates to a composition for improving atopic skin and moisturizing the skin that can improve atopic skin by exhibiting an anti-inflammatory effect by using the daughter-in-law's navel extract, and strengthens the skin barrier and increases the moisture content.

The skin of the human body is an organ that functions as a protective film to protect the living body from the external environment, and prevents the loss of biological components such as water and electrolytes in the human body, and at the same time prevents the intrusion of harmful substances from the outside. The structure of the skin is largely divided into an epidermal layer, a dermis layer, and subcutaneous fat. The epidermal layer is composed of keratinocytes and melanocytes, and the keratinocyte layer, which is the outermost layer of the skin, is in direct contact with the external environment and acts as a primary barrier against penetration of physical, chemical, or substances. In addition, it is also required to maintain flexibility by preventing moisture loss to the outside of the skin and retaining moisture by forming intercellular lipids into a lamellar layer on their own. As the importance of the stratum corneum is emphasized, research on it is being actively conducted. In particular, studies on the structure and function of lipids between keratinocytes existing in the stratum corneum are being actively conducted. The keratinocyte intercellular lipid exhibits a multi-layered lipid film form called a lamellar structure, and in particular, as ceramide is known to play an important role in the function of keratinocyte intercellular lipid, various moisturizers using this have been developed.

On the other hand, in order to understand the mechanism of atopic dermatitis, the opinion that it is caused by an immunological abnormality, particularly inflammation involving a Th2 immune response, has prevailed. It is suggested that it is caused by an abnormality in the bast skin barrier. That is, in the skin of patients with atopic dermatitis, a decrease in the moisture content of the epidermis and a malfunction of the skin barrier are caused by genetic abnormalities and lipid abnormalities such as ceramides constituting the skin barrier. Due to this, the skin penetration of antigens increases, the immune response increases, and various aggravating factors such as changes in skin pH caused by the use of detergents aggravate the deterioration of the skin barrier.

Atopic dermatitis is an inflammatory disease accompanied by pruritus, dry skin, eczema, etc. In climate, it tends to be more severe. Such atopic dermatitis is caused by a complex entanglement of various causes, and remission and recurrence are repeated. Allergic diseases caused by atopic predisposition include allergic dermatitis, allergic rhinitis, asthma, allergic conjunctivitis, and atopic urticaria, and these diseases may appear alone or simultaneously with other diseases. Atopic dermatitis affects a very large number of people, with 0.5 to 1% of the total population and 5 to 10% of children suffering from atopic dermatitis. 50% of patients heal within two stones, but 25% continue into adolescence, and the remaining 25% continue without atopic dermatitis even into adulthood.

The cause of such atopic dermatitis is not clearly known, but it has been found that there are mainly many genetic factors and it is related to a deficiency of the immune system. In addition, it seems to be caused by a complex action of dry skin, the characteristic of feeling itchy skin more easily than normal people, infection by bacteria, viruses, fungi, emotional factors, and environmental factors.

The main symptoms of atopic dermatitis are severe itching, dry skin, rash, oozing, scabs, and scaly skin (scaly). In particular, the severe itching of atopic dermatitis causes psychological damage and makes it difficult to live a normal life.

Since atopic constitution is fundamentally very difficult to treat, atopic dermatitis is being controlled through appropriate treatment while avoiding triggers rather than aiming for a cure. Common atopic dermatitis synergist agents currently used include moisturizing agents, antihistamines that reduce pruritus (itchiness), topical steroids that have therapeutic effects through anti-inflammatory, vasoconstriction, and immunosuppressive actions. Prescription for atopic dermatitis Drug therapy such as steroids, antihistamines, and antibiotics is mainly used. Steroid drugs such as glucocorticoids and cyclospoline can cause diabetes and high blood pressure, and cause serious side effects such as Cushing's syndrome, ophthalmic diseases (cataracts, glaucoma), kidney and liver toxicity. In particular, steroids (adrenal corticosteroids) have anti-inflammatory and immunosuppressive effects and have excellent effects, but side effects such as skin weakness, systemic hormonal symptoms, and addiction may appear when applied for a long period of time. Antihistamines relieve itching symptoms by preventing histamine from being released from mast cells, but are used as a temporary measure and may have side effects such as insomnia, anxiety, loss of appetite, etc.

Accordingly, there is a demand for a new concept atopic dermatitis treatment that is effective for atopic dermatitis and has no side effects.

KR 10-2036826 B1

It is an object of the present invention to provide a composition for improving atopic skin including a daughter-in-law's belly button and moisturizing the skin.

Another object of the present invention is to provide a composition for improving atopic skin and moisturizing the skin that can improve atopic skin by exhibiting an anti-inflammatory effect by using the daughter-in-law's navel extract, and strengthens the skin barrier and increases moisture content.

In order to achieve the above object, a composition for improving atopic skin and moisturizing skin according to an embodiment of the present invention includes a daughter-in-law (Persicaria perfoliata).

The atopic skin is a symptom selected from the group consisting of dry skin, pruritus, dermatophytosis, and eczematous lesions.

The composition may be selected from the group consisting of Pittosporum tobira Ait. extract, Cinnamomum japonicum Sieb. extract, Camellia sinensis O. Ktze. extract, Ilex crenata extract, and mixtures thereof. includes one more.

The extract is extracted using an extraction solvent selected from the group consisting of water, C ₁ to C _{6 lower alcohols, and mixtures thereof.}

The cosmetic composition according to another embodiment of the present invention is prepared by using the composition for improving atopic skin and moisturizing the skin.

A moisturizing cream according to another embodiment of the present invention is prepared using the composition.

A skin regeneration cream according to another embodiment of the present invention is prepared using the composition.

A lotion according to another embodiment of the present invention is prepared using the composition.

Hereinafter, the present invention will be described in more detail.

As used herein, the term 'extract' has the meaning commonly used as a crude extract in the art as described above, but in a broad sense also includes a fraction obtained by additionally fractionating the extract. That is, the plant extract includes not only those obtained using the above-described extraction solvent, but also those obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity or affinity), etc. Fractions obtained through various purification methods are also included in the plant extract of the present invention.

The extract used in the present invention may be prepared in a powder state by an additional process such as distillation under reduced pressure and freeze-drying or spray-drying.

In addition, as used herein, "anti-inflammatory" is meant to include alleviation (relief of symptoms) of inflammatory diseases as defined below, and suppression or delay of the onset of such diseases.

As used herein, the term "inflammatory disease" refers to an inflammatory reaction specified as a local or systemic biological defense reaction against an external physical or chemical stimulus or infection or autoimmunity from external infectious agents such as bacteria, fungi, viruses, and various allergens. It can be defined as a pathological condition. This inflammatory response involves activation of various inflammatory mediators and immune cell-related enzymes (iNOS, COX-2), secretion of inflammatory mediators (secretion of NO, TNF-α, IL-6, etc.), fluid infiltration, cell migration, and tissue It is accompanied by a series of complex physiological reactions such as destruction, and appears externally by symptoms such as erythema, pain, edema, fever, and deterioration or loss of specific body functions.

In order to achieve the above object, a composition for improving atopic skin and moisturizing skin according to an embodiment of the present invention includes a daughter-in-law.

Preferably, the daughter-in-law includes the group consisting of outpost, leaves, stems, above-ground parts, roots, rhizomes, underground parts, seeds, and mixtures thereof.

Daughter-in-law (Persicaria perfoliata) is a kind of the family Knobaceae, which is native to Asia. Daughter-in-law's belly button has traditionally been used as an antidote, antipyretic, diuretic, etc., and has also been used for respiratory diseases such as cough and whooping cough. It is known that quercetin, caffeic acid, etc. are contained as major components of the daughter-in-law's navel extract. The quercetin (quercetin) is a plant antioxidant and belongs to the flavonoid family among phytochemicals. Quercetin plays a role in regulating the immune system's response to external stressors through cellular signaling pathways called kinases and phosphatases, two types of enzymes and membrane proteins required for proper cellular function.

When the daughter-in-law navel extract is used, the anti-inflammatory effect can be exhibited by the active ingredient contained in the daughter-in-law navel extract to improve atopic skin, and the skin barrier is strengthened and moisture content is increased, and the human body stability is excellent.

In general, in the inflammatory response, lipopolysaccharide (LPS) is an endotoxin derived from the cell wall of Gram-negative bacteria and activates the transcription factor NF-κB by binding to toll-like receptor 4 (TLR4), iNOS and COX-2 It induces the expression of NO, inflammatory cytokines, and various inflammatory regulators such as PGE₂.

In this process, the daughter-in-law extract can be used as an anti-inflammatory agent by inhibiting the production of NO, inflammatory (IL-1β, IL-6, TNF-α), PGE₂, COX-2 and iNOS in a concentration-dependent manner.

The PGE₂ is one of the inflammatory mediators and is synthesized by the enzymatic action of phospholipase A2. It starts with the production of arachidonic acid from membrane phospholipids.

The arachidonic acid becomes prostaglandin G2 by enzymatic action and again becomes prostaglandin H2, an unstable metabolite.

These two processes are catalyzed by cyclooxygenase (COX), and there are two or more isoenzymes of COX. Among them, COX-1 is continuously expressed to regulate platelet aggregation, gastric mucosa, and renal function It is responsible for the physiological functions of the back, and COX-2 is expressed by stimuli such as inflammation.

PGH₂ produced by COX is an unstable intermediate metabolite and is metabolized into PGE₂, PGD₂, PGI₂, PGF₂, thromboxane A2 (TXA₂), etc. by various prostanoid synthases depending on the type of cell or stimulation.

In addition, three types of isoforms of PGE synthase (PGES) involved in the metabolism of PGH₂ to PGE₂, cytosolic PGES (cPGES), microsomal PGES-1 (mPGES-1), and mPGES-2, are known. Among mPGES-1, expression is increased by inflammatory stimuli such as lipopolysaccharid (LPS), inflammatory cytokines such as IL-1β and TNF-α, and nitric oxide (NO), and is functionally closely related to COX-2. It is closely linked to the production of PGE₂.

That is, the production of PGE₂ is functionally closely linked with COX-2 and plays an important role in the inflammatory response.

Accordingly, the present invention suppresses the production of COX-2 and iNOS, and inhibits the expression of inflammatory cytokines (Cytokine), nitric oxide (NO) and COX-2 by using the daughter-in-law extract as an active ingredient, It can exhibit excellent anti-inflammatory effect.

The atopic skin is a symptom selected from the group consisting of dry skin, pruritus, dermatophytosis, and eczematous lesions.

Since atopic skin is chronically dry, eczema is easily induced by various stimuli, it has severe itching, and has the characteristics of repeated recurrence. These pruritus and eczema are repeatedly weakened by dry skin and, if not properly managed, eventually progress to subacute and chronic lesions such as lichenification, pigment change, and erythroderma. With immunological abnormalities, superficial folliculitis, phytilosporum folliculitis, epilepsy, superficial dermatophytosis, warts, and recurrent herpes simplex are common. Bacterial infection is usually caused by coagulase-positive Staphylococcus aureus, and it mainly appears as folliculitis lesions with superficial pruritus on the arms and legs. In some cases, it may cause recurrent cellulitis with systemic symptoms.

In addition, atopic dermatitis is an inflammatory disease accompanied by pruritus, dry skin, eczema, etc. or a disease caused by abnormalities in the stratum corneum, the outermost skin barrier, due to genetic, environmental, and immunological causes. tends to be more severe in dry climates. Therefore, the use of a moisturizer to improve the dryness of the skin of atopic dermatitis and the increase in the recovery of the skin barrier function occupies an important position in the treatment of atopic dermatitis.

The dry skin refers to a skin condition in which discomfort can be felt due to dryness, and refers to a condition in which moisture in the skin is insufficient to 10% or less of normal. Clinically, it refers to a skin condition with small red spots and fissures, with scales and a rough surface. If you have a skin disease or systemic disease that causes dry skin, it is important to treat the underlying disease. The basic principle of general dry skin treatment is to supply and maintain moisture in the stratum corneum while minimizing the loss of moisture in the stratum corneum. It is helpful to supply moisture directly to the skin through a shower or bath, but if there is no ability to maintain the supplied moisture, showering or bathing is not helpful. may become drier. Therefore, in order to maintain the moisture and lipid components of the skin, it is better not to forcefully remove the stratum corneum. In addition, it is important to reduce excessive washing and soap use, protect the skin from the external harmful environment, and use an appropriate moisturizer. If dry skin is not controlled, it can progress to dermatitis.

Skin pruritus, also called skin itch, refers to a chronic skin disease in which skin itch is the main symptom without primary dermatitis, and is accompanied by secondary scratches and scabs. Wind and heat loss or damp heat loss is concentrated in the base, or wind and wind stays in the body for a long time and turns into fire, drying the essence and blood. It is caused by not being able to nourish the donation. It appears as paroxysmal, and worsens when exposed to heat or consuming stimulant foods or alcohol. It can lead to secondary pigmentation, lichen-like changes, and eczema-like changes as it ages. Systemic symptoms may include sleep disturbances, nervous breakdowns, and loss of appetite. Local symptoms include pruritus of the anus, pruritus of the scrotum or genitalia, and pruritus of the head, legs, and palms. It is often seen in adults, especially the elderly.

Dermatomycosis is a phenomenon in which erythematous redness and wheal (edema) occur when a physical stimulus is applied to the skin. When the skin is scratched or pressed with more than a certain amount of pressure, it is localized to the area and becomes itchy, red, and swollen similar to hives. As a result, it looks like writing on the skin. Even mild irritation can occur anywhere on the skin of the body, and if it is scratched because it is itchy, hives may appear even more severe. It occurs in about 5% of the Korean population and usually shows a chronic course.

The composition may be selected from the group consisting of Pittosporum tobira Ait. extract, Cinnamomum japonicum Sieb. extract, Camellia sinensis O. Ktze. extract, Ilex crenata extract, and mixtures thereof. includes one more.

The money tree (Pittosporum tobira Ait.) is an evergreen shrub of the Pittosporaceae family, and grows in Sanyana Beach in the southern archipelago region of Korea. It grows to 2~3m in height and has no hair on its branches. The leaves are 4~10cm long and 2~3cm wide, and the leaves are shiny dark green and grow alternately in a long obovate shape. Flowers that bloom in May-June as white chrysanthemums are bisexual and have a strong fragrance. The fruits are round or oval capsules and mature around October. It is widely used as an ornamental plant in the southern region because of its delicate scent and beautiful leaves and flowers from the root bark. The leaves, stems, and bark of the money tree are used for high blood pressure, arteriosclerosis, and joint pain because they are called chilihyang (七里香).

Cinnamomum japonicum Sieb. is an evergreen tree of the Lauraceae family and mainly grows in the archipelago region in the southern part of Korea. It grows up to 15m in height, and the bark of the mature stem is black, and the young branches are green and hairless. The leaves are alternate, 6~15cm long, 2~5cm wide, narrow ovate or long oval, and the back side is white. It is a bisexual flower that blooms in June with a pale yellow brittle flower. The oval-shaped fruit is about 12mm in diameter and matures in purple-black color around December.

The tea tree (Camellia sinensis O. Ktze.) is an evergreen shrub of the Theaceae family and is cultivated as an economic crop in the southern regions of Korea. The leaves are alternate, long oval, 2~15cm long and 2~5cm wide. The upper side is dark green, but the back side is light green, and there is an reticular vein on the upper side. Flowers are white, 1-3 axillary in October-November. The young leaves of the tea tree are called tea leaves and are often consumed as tea and have diuretic and detoxifying properties. The root of the tea tree is called many root (茶樹根) and the fruit is called Daja (茶子), and it is mainly used as a herbal medicine.

Ilex crenata grows wild in the south of the Byeonsan Peninsula in Jeollabuk-do, Korea, and can be planted in Gangneung, Wonju, Taean, Yesan, and south of Daejeon. The currently known varieties of Quercus are 40 taxa, with evergreen small leaves and dense, delicate twigs. The bark is gray, the branches and leaves are thick, and the height is about 3m. The leaves are alternate, in the shape of an upside down egg or a long oval shape, pointed on both sides, and have fine sawtooths on the edge. The length of the leaf is 1.5~3cm, the width is 16~20mm, and the front side is shiny and dark green.

The composition for improving atopic skin and skin moisturizing is used by further including any one selected from the group consisting of money tree extract, saengdal tree extract, tea tree extract, hollywood extract and mixtures thereof, in addition to the daughter-in-law's navel extract, as a complex extract When used, atopic skin improvement and skin moisturizing effect can be more excellently exhibited.

Preferably, the composition of the present invention contains 40 to 60 parts by weight of a money tree extract, 40 to 60 parts by weight of a saengdal tree extract, 40 to 60 parts by weight of a tea tree extract, and 40 to 60 parts by weight of an extract from a sagebrush, based on 100 parts by weight of the daughter-in-law extract. can do. When used as a complex extract within the above range, the atopic skin improvement and skin moisturizing effect are further increased due to the complex action between each component.

The extract is extracted using an extraction solvent selected from the group consisting of water, C ₁ to C _{6 lower alcohols, and mixtures thereof.}

Specifically, washing the natural product to prepare a daughter-in-law navel extract; drying after washing; pulverizing the natural product after drying; leaching the pulverized product using an organic solvent; drying the sample after leaching; leaching with water; And including the step of leaching, it is possible to obtain a natural extract.

The natural extract extracted using the organic solvent may further include the step of performing fractionation using an organic solvent.

The method for preparing the extract may be a conventional extraction method in the art, such as an ultrasonic extraction method, a leaching method, and a reflux extraction method. Specifically, it may be an extract obtained by extracting a natural product from which foreign substances have been removed by washing and drying with water, an alcohol having 1 to 6 carbon atoms, or a mixed solvent thereof, and may be an extract extracted by sequentially applying the solvents to the sample.

The reflux extraction method is water, based on 100 mL of an alcohol having 1 to 6 carbon atoms, 10 to 30 g of a pulverized product of a natural product, a reflux time of 1 to 3 hours, and 50 to 100% alcohol or water having 1 to 6 carbon atoms. More specifically, based on 100 mL of an alcohol having 1 to 6 carbon atoms or 100 mL of water, 10 to 20 g of a pulverized product of a natural product, a reflux time of 1 to 2 hours, and 70 to 90% of an alcohol or water having 1 to 4 carbon atoms.

The leaching method is performed at 15 to 30° C. for 24 to 72 hours, and water or 50 to 100% alcohol having 1 to 6 carbon atoms is used as an extraction solvent. More specifically, the process is carried out at 20 to 25° C. for 30 to 54 hours, and the extraction solvent is water or 70 to 80% alcohol having 1 to 6 carbon atoms.

In the ultrasonic extraction method, the reaction is performed at 30 to 50° C. for 0.5 to 2.5 hours, and the extraction solvent is water or 50 to 100% alcohol having 1 to 6 carbon atoms. Specifically, extraction is performed at 40 to 50° C. for 1 to 2.5 hours, and the extraction solvent is water or 70 to 80% alcohol having 1 to 6 carbon atoms.

The extraction solvent may be used in an amount of 2 to 50 times, more specifically, 2 to 20 times, based on the weight of the sample. For extraction, the sample may be left for 1 to 72 hours for leaching in the extraction solvent, and more specifically, for 24 to 48 hours.

After extraction, the extract may be fractionated by sequentially applying a fresh fractionation solvent. The fractionation solvent used for fractionation is any one or more selected from the group consisting of water, hexane, butanol, ethyl acetic acid, ethyl acetate, methylene chloride and mixtures thereof, preferably ethyl acetate or methylene chloride.

After obtaining the extract or fraction, a method such as concentration or freeze-drying may be additionally used.

The cosmetic composition according to another embodiment of the present invention is prepared by using the composition for improving atopic skin and moisturizing the skin.

The cosmetic composition of the present invention is in a form including the composition for improving atopic skin and moisturizing the skin. The cosmetic composition may be prepared in various forms according to a conventional cosmetic preparation method. For example, the cosmetic composition may be prepared in the form of cosmetic products, lotions, creams, lotions, etc. containing the composition for improving atopic skin and moisturizing the skin, which are diluted with a conventional cleansing solution, an astringent solution, and a moisturizing solution. can be used In addition, the cosmetic composition may include conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments and fragrances commonly used in the field of cosmetic compositions.

A moisturizing cream according to another embodiment of the present invention is prepared using the composition.

A skin regeneration cream according to another embodiment of the present invention is prepared using the composition.

A lotion according to another embodiment of the present invention is prepared using the composition.

According to the composition for improving atopic skin and moisturizing the skin of the present invention, atopic skin can be improved by showing an anti-inflammatory effect by using a daughter-in-law extract, and atopic skin improvement and skin moisturizing that strengthens the skin barrier and increases moisture content composition can be provided.

1 is a graph of experimental results for cell toxicity of a daughter-in-law navel extract according to an embodiment of the present invention.
Figure 2 is a graph of experimental results for the inhibition of nitric oxide (NO) production of the daughter-in-law navel extract according to an embodiment of the present invention.
Figure 3 is a graph of the experimental results for the inhibition _{of PGE 2} production of the daughter-in-law navel extract according to an embodiment of the present invention.
Figure 4 is a graph of the experimental results for the inhibition of TNF-α production of the daughter-in-law navel extract according to an embodiment of the present invention.
5 is a graph showing the results of IL-1β production of the daughter-in-law umbilical cord extract according to an embodiment of the present invention.
6 is a Western blot analysis result for the decrease in the expression of cytokines according to the treatment of the daughter-in-law navel extract according to an embodiment of the present invention.
7 is a result of evaluating the effect of the daughter-in-law navel extract according to an embodiment of the present invention on skin itching symptoms.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

[Preparation Example 1: Preparation of extract]

1. Preparation of daughter-in-law's belly button extract

The daughter-in-law, including the leaves and stems, was thoroughly washed under running water and then completely air-dried. The dried daughter-in-law was pulverized with a blender and leached for 48 hours at room temperature using 70% ethanol, respectively, and then the sample was filtered to prepare a daughter-in-law extract (PE).

2. Preparation of other natural extracts

Using the same method as the method for preparing the daughter-in-law's belly button extract (PE), a money tree extract (J2), a saengdal tree extract (J3), a tea tree extract (J4), and a Quercus tree extract (J5) were prepared.

3. Preparation of Complex Extracts

The daughter-in-law's belly button extract (PE), money tree extract (TE), raw moon tree extract (CE), tea tree extract (SE) and Quercus tree extract (IE) are mixed within the weight range as shown in Table 1 below to improve atopic skin and moisturize skin It was prepared as a composition for

MX1 MX2 MX3 MX4 MX5 PE 100 100 100 100 100 TE - 30 40 60 80 CE - 30 40 60 80 SE - 30 40 60 80 IE - 30 40 60 80

(Unit: parts by weight)

[Experimental Example 1: Anti-inflammatory effect]

Cell culture and samples

For the cell line, RBL-2H3 cells (rat basophilic leukemia mast cells) were distributed from the Korea Cell Line Bank, and 10% feta bovine serum (FBS, Hyclone, South Logan, UT, USA) and 1% antibiotics (100 U/ml penicillin G, 100 μg/ml streptomycin, Hyclone) was added with DMEM medium and cultured at 37° C. and humidified with 5% CO2.

Cytotoxicity assessment

MTT analysis was performed using the MTT assay, which is an alternative animal test method suggested by the Ministry of Food and Drug Safety and the test standard presented by the Ministry of Food and Drug Safety. RBL-2H3 cells cultured in a cell culture dish were separated with Trypsin, mixed with a new medium, counted using a counting chamber and a microscope, and then dispensed at a concentration of 1 to 105 cells/ml in 96 well plates, respectively. Samples were prepared by concentration and cultured for 24 hours.

Cell line RBL-2H3 cells were dispensed and cultured for 24 hours, and then the daughter-in-law extract (PE) was added by concentration (un, con 50 μg/ml, 100 μg/ml, 150 μg/ml, 200 μg/ml, 250 μg/ ml), and after removing the medium, the MTT solution was added at a concentration of 1 μg/ml, and after reacting at 37° C. for 4 hours, the MTT solution was removed and the reaction product formed by dispensing 100 μl of DMSO was dissolved, and for 30 minutes After the reaction, absorbance at 540 nm was measured with an ELISA Reader.

The viability for each cell was compared and analyzed by expressing the difference in absorbance with respect to the control as a percentage.

Cell viability (%) = [(Absorbance of the sample added group) / Absorbance of the non-added group] X 100

1 is a result of confirming the cytotoxicity of the daughter-in-law umbilical cord extract (PE) in RBL-2H3 cells. As a result of measuring the cell viability, the concentration was increased, but there was little difference in the viability.

NO production inhibition activity experiment

As a first step to measure the anti-inflammatory effect of the daughter-in-law (PE) extract at each concentration on the inflammatory response, NO in the cell culture was quantified and compared. To measure NO scavenging activity, RBL-2H3 cells were aliquoted in a 96 well plate at 2x104 cells/ml and _{cultured for 24 hours at 37°C, 5% CO 2} conditions, and then the daughter-in-law extract (PE) was added by concentration (un, con 50 μg/ml, 100 μg/ml, 150 μg/ml, 200 μg/ml, 250 μg/ml).

To measure the amount of NO generated in the cell culture medium using Griess reagent, Griess reagent A was mixed with 35 ml of distilled water (DW), 15 ml of 100% acetic acid, and 0.5 g of sulfanilamide. Grease reagent B was prepared by adding 20 ml of DW, 30 ml of 100% acetic acid and 0.05 g of N-(1-Naphthyl)ethylenediamine.

After mixing 100 μl of cell culture supernatant and 100 μl of Griess reagent and reacting at room temperature for 10 minutes, absorbance was measured at 540 nm, which is the form present in the cell culture solution, and the amount of NO generated is sodium nitrite (NaNO ₂ ) compared with the standard.

As can be seen from the results of Figure 2, it was confirmed that the higher the concentration of the daughter-in-law's belly button extract (PE), the higher the NO production inhibitory activity.

Inflammatory cytokines and PGE ₂ production inhibitory activity experiment

In order to verify the effect of daughter-in-law extract (PE) on the production of inflammatory cytokines such as Prostaglandin E2 (PGE ₂ ), Interleukin (IL)-1β, and Tumor Necrosis Factor-α (TNF-α), cells were subjected to the above conditions. After incubation, the supernatant was collected and _{the contents of PGE 2} , IL-1β, and TNF-α (Enzo LifeScience) were quantified using an ELISA kit.

As can be seen from the results of Figures 3, 4 and 5, it was confirmed that the daughter-in-law extract (PE) significantly _{inhibited PGE 2} , TNF-α and IL-1β.

Confirmation of expression changes of iNOS, COX-2, TNF-α, and IL-1β through Western blot analysis. After incubation, Western blot analysis was performed.

Prepared cells were treated with a lysis buffer containing 250 mM NaCl, 25 mM Tris-HCL (pH 7.5), 10 mM ethylenediaminetetraacetic acid, 1% Nonidet P-40, 0.1 mM phenyl-methysulfonylfuoride, and protease inhibitor. was dissolved. The concentration of the isolated protein was measured using a protein quantification reagent (Bio-Rad, Herculs, CA, USA). Equal amounts of proteins were separated using 10% sodium dodecy sulfate (SDS)-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Schleicher % Schuell, Keene, NH, USA). Each membrane (membrane) was treated with 5% skim milk for 1 hour to block non-specific proteins.

Corresponding antibodies and enhanced chemiluminescence (ECL, Amersham Corp. Arlington Heights, IL, USA) solution was treated and then photosensitized with an X-ray film to confirm the protein expression level. The primary antibody used in this experiment was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) and Cell signaling (Beverly, MA, USA), and the secondary antibody was purchased from Amersham Life Science Co. and used.

As a result, as can be seen in FIG. 6 , it was confirmed that NO production in RBL-2H3 cells was reduced by treatment with the daughter-in-law umbilical cord extract (PE). It was confirmed that this decrease was due to a decrease in the expression of imducible NOS (iNOS).

Therefore, it will be said that daughter-in-law extract (PE) in RBL-2H3 cells exhibits anti-inflammatory effects due to reduced production of NO, PGE2, IL-1β, and TNF-α.

[Experimental Example 2: Anti-inflammatory effect of complex extract]

As described above, the anti-inflammatory activity test results for the daughter-in-law navel extract (PE, MX1) were confirmed. Therefore, the anti-inflammatory activity test was conducted for the complex extracts MX2 to MX5, and for the relative evaluation, the anti-inflammatory degree of the daughter-in-law extract (PE, MX1) was set to the index 5, and the degree of the anti-inflammatory effect to the other complex extracts was indexed. was described as

Anti-inflammatory activity test results for the complex extract are shown in Table 2 below.

PE, MX1 MX2 MX3 MX4 MX5 Inhibition of NO production 5 6 9 7 5 Inhibition of inflammatory cytokine production 5 6 8 6 4 Inhibition of production of PGE ₂ 5 5 8 7 5

(Unit: Index)

As shown in Table 2 above, for the inhibition of NO production, inhibition of the production of inflammatory cytokines and the _{inhibition of the production of PGE 2} for confirming the anti-inflammatory effect, the relative comparison results for the daughter-in-law extract (PE, MX1), MX2 to 4, it was confirmed that it exhibited an excellent anti-inflammatory effect equal to or greater than that.

[Experimental Example 3: Skin moisturizing effect]

For 10 women aged 20 to 25, the compositions MX1 to MX5 for improving atopic skin and moisturizing the skin were prepared as moisturizing creams, and the skin moisturizing effect was evaluated.

The test subjects used the moisturizing cream after washing their face twice every morning (between 7:00 and 9:00) and in the evening (between 7:00 and 9:00) every 12 hours for 4 weeks. At the 4th week, the moisturizing effect of the skin was evaluated by measuring the skin moisture evaporation rate.

The skin moisture evaporation rate was measured with Tewamter TM300 and was shown as an average value of 10 subjects, and the results are shown in Table 3.

PE, MX1 MX2 MX3 MX4 MX5 Water evaporation rate (g/h/m ² ) 17.23 18.24 19.77 20.22 18.34 Decrease rate of water evaporation compared to before first use (%) 39.16 35.59 30.19 28.50 35.24

As shown in Table 3, when the compositions MX1 to MX5 for improving atopic skin and moisturizing the skin prepared in the Preparation Example of the present invention are prepared as a moisturizing cream and continuously used, it can be confirmed that the skin moisture evaporation rate is gradually reduced. there was.

In particular, in the case of the compositions MX3 to MX4 for improving atopic skin and moisturizing the skin, even after 12 hours have elapsed after application, it was confirmed that the effect was significantly reduced compared to the moisture evaporation rate of the skin before use.

Formulation Example 1. Moisturizing cream

A moisturizing cream containing the MX1 to MX5 was prepared in a conventional manner according to the following composition ingredients and composition ratios.

MX1 3.0 wt%

8.0 wt% glycerin

Butylene glycol 5.0 wt%

Glyceryl Stearate / PEG-100 Stearate 1.0 wt%

Polysorbate 60 0.5 wt%

0.5 wt% sorbitan stearate

Phytosqualane 5.0 wt%

jojoba oil 3.0 wt%

15.0 wt% caprylic/capric triglycerides

Aloe Butter 2.0 wt%

beeswax 1.0 wt%

0.5% by weight of stearyl alcohol

0.5% by weight of beheryl alcohol

0.5% by weight of stearic acid

0.2 wt% of carboxyl vinyl polymer

0.1% by weight of xanthan gum

Arginine 0.5 wt%

Dimethicone 0.5 wt%

Beta glucan 0.2 wt%

0.5% by weight of preservative

Purified water remaining

Formulation Example 2. Moisturizing Cream

[Experimental Example 4: Atopic Dermatitis Animal Model Test]

Atopic dermatitis induction and sample administration

In the atopic dermatitis animal model experiment, MX3 to MX4, which were shown to be the best in the anti-inflammatory and skin moisturizing effect test, were used as samples, and the sample was orally administered to an animal model induced by atopic dermatitis, and the degree of skin itchiness was evaluated. did.

Specifically, atopic dermatitis was induced primarily by applying 1% DNCB (Dinitrochlorobenzene) to Nc/Mga mice whose back was shaved, and after a week, 1% DNCB and 100 mg of AD ointment were applied to the depilated area and secondary induced atopic dermatitis.

Effect on skin itching symptom change

Changes in skin itch symptoms, a typical clinical symptom of atopic dermatitis, were evaluated. In order to compare and observe the itch symptoms before and after inducing atopic dermatitis and after administration of the samples of MX3 to MX4, the animal's behavior was photographed once every 2 weeks under dark conditions for 1 hour, and the number of scratching was measured by observing it. did.

The results are shown in FIG. 7, and it was confirmed that all of the samples of MX3 to MX4 clearly relieved skin itching symptoms at 2nd and 4th weeks. It can be seen that samples MX3 to MX4 relieved atopic dermatitis-related clinical symptoms significantly and significantly compared to the atopic dermatitis-inducing group (Vehicle) at 3 weeks and 4 weeks, and the effect was positive control group (CJLP) when considering the dosage. -133) (Novarex Co., Ltd., Korea).

Although the preferred embodiment of the present invention has been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements by those skilled in the art using the basic concept of the present invention as defined in the following claims are also provided. is within the scope of the

Claims (8)

40 to 60 parts by weight of Pittosporum tobira Ait. extract, 40 to 60 parts by weight of Cinnamomum japonicum Sieb. extract, and 40 to 60 parts by weight of extract, Camellia sinensis O. Ktze. 40 to 60 parts by weight of the extract and 40 to 60 parts by weight of the extract of Ilex crenata
A composition for improving atopic skin and moisturizing the skin. The method of claim 1,
The atopic skin is a symptom selected from the group consisting of dry skin, pruritus, dermatosis, and eczematous lesions.
A composition for improving atopic skin and moisturizing the skin. delete The method of claim 1,
The extract is extracted using an extraction solvent selected from the group consisting of water, C ₁ to C _{6 lower alcohols and mixtures thereof.}
A composition for improving atopic skin and moisturizing the skin. A cosmetic composition comprising the composition for improving atopic skin according to any one of claims 1, 2 and 4 and moisturizing the skin. A moisturizing cream comprising the composition according to any one of claims 1, 2 and 4. A skin regeneration cream comprising the composition according to any one of claims 1, 2 and 4. 5. A lotion comprising the composition according to any one of claims 1, 2 and 4.

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