KR101594034B1 - Composition Comprising Extracts of Artemisia rubripes, Tubocapsicum anomalum and Camellia japonica (leaves) Improving Skin Allergy, Inflammation and Geriatric Odor - Google Patents

Abstract

Compositions for removing anti-skin allergies, anti-inflammatory and senile body odor containing a combination of almonds, almonds and camellia leaves. The present invention inhibits the expression of iNOS and COX-2, which are mediators of inflammation, and inhibits the secretion of -hexosaminidase, which is an index of allergic response, in water or mellitus, do. In addition, it provides senile body odor removal effect. The effect of this complex function can be maximized by the combination of three effective plants and proper extraction method.

Description

(Composition Comprising Extracts of Artemisia rubripes, Tubocapsicum anomalum and Camellia japonica (Leaves) Improving Skin Allergy, Inflammation and Geriatric Odor, which is a composition for removing anti-skin allergies, anti-

The present invention relates to a composition for the removal of anti-skin allergies, anti-inflammatory and senile body odor containing an ethyl acetate fraction of a combined extract of almonds, allium and camellia leaves.

Skin diseases such as atopic dermatitis are chronic allergic diseases such as atopic asthma and allergic rhinitis, which are common in infants and children and are associated with itching accompanied by chronic itching. The main symptom of atopic dermatitis is itch and incidentally there are various symptoms such as dry skin, pore keratosis, eczema, white skin necrosis and delayed blanch reaction. Patients with atopic dermatitis have elevated serum IgE and are often positive when tested for allergic skin reactions and their symptoms are aggravated by environmental or emotional factors.

More than 50% of patients develop within 3 months to 1 year after birth and 30% develop between 1 and 5 years. That is, most cases are those that develop before 5 years of age. 80% of patients may develop asthma or rhinitis during boyhood, and skin symptoms often improve when respiratory allergies occur. Atopic dermatitis is generally more severe and persistent in infants and tends to improve with age. In other words, between two and three years, about 80% of the symptoms improve but sometimes it continues to be adults. The cause of atopic dermatitis has not yet been clarified yet, but about 30% of pediatric patients are known as food allergies.

To treat atopic dermatitis, moisturizers are used on dry skin to keep the skin from drying, and corticosteroids to treat dermatitis, and appropriate antihistamines to treat pruritus or sleep disorders caused by it. The use of adrenocortical hormones is limited due to the high risk of adverse effects due to endocrine disruption in the human body, and natural products that inhibit histamine secretion can be used to improve atopic skin diseases. Atopic skin diseases are accompanied by bacterial infections and inflammation of scars caused by scratching along with itching. The cause of the inflammation reaction is physiological, chemical action or bacterial infection. It is a defense mechanism of the body. It removes foreign substances and restores the damaged area. Once the stimulus is applied, histamine, serotonine , bradykinin, prostaglandins, hydroxyeicosatetraenoic acid (HETE), and leukotriene are released, resulting in increased vascular permeability and inflammation.

Monocytes are activated by bacterial infections and transformed into macrophages with excellent phagocytosis. Macrophages induce inflammation by secretion of NO, prostaglandin E2, and proinflammatory cytokine, and are important regulatory cells in both inflammation and immune responses. In order to function like this, it must be activated. LPS (lipopolysaccharide), one of the cell wall components of Gram-negative bacteria and well known as endotoxin, is the most well-known external factor involved in macrophage activation. Pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β (interleukin-1 beta) have been observed in monocytes and macrophages such as RAW 264.7 cells. inflammatory cytokines secreted by the inflamed area is increased.

When LPS stimulates macrophages, nitric oxide (NO) is produced in the process of converting L-arginine into L-citrulline by an enzyme called iNOS (inducible nitric oxide synthase), resulting in the generation of NO from macrophages. In mammals, NO is synthesized by three kinds of nitric oxide synthase (NOS): nNOS (neuronal NOS), eNOS (endothelial NOS) and iNOS (inducible NOS). Among these, NO produced by nNOS and eNOS is produced for normal biological function, and concentration in tissues is kept low to a certain level.

However, NO produced by iNOS is overexpressed and exhibits deleterious effects on living organisms such as pathological vasodilation, cytotoxicity, and tissue damage. Prostaglandin E2 (PGE2), an inflammatory factor, is produced by the action of an enzyme called COXs (cyclooxygenaes), which is released by an enzyme called phospholipase A2, And induce an inflammatory reaction. COX is classified as COX-1 and COX-2. COX-1 acts on normal biological functions such as platelet formation, gastric wall protection and maintenance of renal function in the body, and COX-2 synthesizes PGE2 as an inflammatory mediator . PGE2 is known to be deeply involved in the development of cancer, such as promoting inflammation (pain, fever, etc.), immune response, and angiogenesis. All nonsteroidal antiinflammatory drugs (NSAIDs), including selective COX-2 inhibitors, exhibit anti-inflammatory, antipyretic and analgesic effects. However, long-term use of these drugs has serious side effects. For example, secondary anemia due to gastrointestinal peptic ulcer bleeding, platelet function suppression, inhibition of induction of labor, side effects to the kidney, liver damage, hypersensitivity. In order to overcome the side effects of these drugs, natural products such as plants are more preferable as a safer material. In this regard, researches are being actively carried out to find and use the anti-inflammatory materials in natural products.

1. Korean Registered Patent No. 10-0556524 (Feb. 23, 2006) 2. Korean Patent No. 10-1219520 (2013.01.02)

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above-mentioned problems, and it is an object of the present invention to provide a composition for removing allergic skin, anti-inflammatory and senile body odor containing ethyl acetate fraction .

In order to accomplish the above object, an embodiment of the present invention is characterized by a composition for removing allergic skin, anti-inflammatory and senile body odor containing an extract of Allium tuberosum, Alkalineum and Camellia sinensis. The complex extract may be an alcohol extract of dried crumbs of almond, alum and camellia leaves.

Another embodiment of the present invention is a composition for removing anti-skin allergies, anti-inflammatory and senile body odor containing fractions obtained by fractionation of ethyl acetate from an extract of Allium, Alkaline and Camellia leaves.

A method for preparing an anti-allergy, anti-inflammatory and senile body odor removing composition according to the present invention comprises the steps of: mixing a dried plant with shrubs, alum balsam and camellia leaves, pulverizing and pulverizing the mixture to prepare a complex; A complex extraction step of extracting the mixed composite with water or an alcohol solvent; And extracting fractions by fractionating the extracted complex extract with an ethyl acetate layer.

The complex extraction step is carried out at 20 to 100 ° C with an alcohol solvent such as water or alcohol at a volume of 3 to 20 times the dry weight of the composite, filtered under reduced pressure, and the filtrate is concentrated under reduced pressure at 20 to 100 ° C in a vacuum rotary condenser The method comprising the steps of:
The composite of the complex extraction step may comprise a weight ratio of 3: 1: 1 to the top part of the alum and the camellia leaves.

The step of extracting the fraction includes dissolving the extracted complex extract using water, and further fractionating the extract into an ethyl acetate layer and concentrating the extract under reduced pressure.

The alcohol, alcohol or water (H ₂ O) extracts of the herbal, altruistic and camellia leaf complexes of the present invention not only inhibit iNOS and COX1 / 2 production induced by LPS in RAW 264.7 cells, but also inhibit histamine secretion inhibition beta-hexosaminidase, which is an indicator of beta-hexosaminidase, can be effectively used as a composition for the prevention and treatment of skin allergies and inflammatory diseases. In addition, the compound extract of the present invention is excellent in anti-skin allergic and anti-inflammatory effects as well as in the sensory evaluation to remove the senile body odor.

Fig. 1 is a diagram showing a large-scale extraction process for making a combination extract of almonds, alums and camellias.
FIG. 2 is a graph showing the effect of the extracts of hyaluronic acid, alginate and camphor tree ethyl acetate complex (EP) on inhibition of iNOS expression and NO formation in RAW 264.7 cells.
Figure 3 shows the inhibitory effect of COX (cyclooxygenase 1/2), an important factor in the inflammation mediator, of the extracts of the ethyl acetate complex (EP) from the hayworm, alginate and camellia leaves in RAW 264.7 cells.
FIG. 4 shows the effect of suppressing the secretion of β-hexosaminidase, which is an index factor of the allergic reaction of the extracts of ethyl acetate complex (EP) on the rumen, alginate and camellia leaves in RBL-2H3 cells.
FIG. 5 shows the results of the cytotoxicity test (MTT assay) of the extract of ethyl acetate complex (EP) of hayworm, alginate and camellia leaves in RAW 264.7 cells.

Thistle, Alkaline and Camellia are native plants in Korea.

Alcorrhiza (Scientific name: Tubocapsicum anomalum) is a perennial plant belonging to the family Solanaceae. It grows in South Korea, Japan, the middle of Taiwan and also in Africa. Flowers bloom in July-August, light yellow color, 1 ~ 5 pcs per leaf, pedicel is thickened when the fruit is ripe, 1.5-2.5cm long, bend down. Calyx leaf is cup shape, upper edge is horizontal, hairs-free and low, corolla is bell-shaped, is shallowly divided into 5 pieces, lobe is triangular with fingertip shape and tip is pointed and tilted.

Artemisia rubripes is a perennial plant that grows about 1.5 cm high and is a plant of Asteraceae. The stem with a slight purple light stood straight and struck a few branches. Leaves are alternate phyllotaxis and are divided into two feathers deeply. The split pieces are lanceolate, the edges are flat and have no saw teeth. The surface is dark green and the back side is covered with white fluff. There are many rounded flowers with no petals at the ends of the cracked branches, forming conical flower ears. The diameter of a flower is around 2mm and it is surrounded by blue calyx covered with fluffy. The color of the flower is pale brown. It is found only in Gangwon Province and North Gyeonggi Province, and is often found in sunny grasses such as mountain slopes and streams. I tear off the freshly growing young shoots and tie them in cold water, then eat them with herbs or put them into rice cakes and eat them.

Camellia japonica (camellia japonica) is an evergreen broad-leaved tree belonging to the genus Camellia. Most of them grow to about 7m. Large ones sometimes grow up to 20 meters. The thick foliage surface is glazed, and the back of the pale green is glazed. There are sawtooths on the edge of the leaf. The flower runs without peduncle at the end of branch or leaf axil. Most of them bloom with five petals, but occasionally they bloom with seven sheets of flowers. The fruit is opened in the shape of a tall green droplet at night and then ripened into brown and spread into three branches. There is a seed in it that looks like a little big pine nut.

In these three plants, the top part of the honeycomb and alum and the camellia leaves are mixed with the dried plant in a weight ratio of 3: 1: 1, extracted with ethanol or water (H ₂ O) and concentrated under reduced pressure.

This is further fractionated into ethyl acetate and concentrated under reduced pressure to obtain a composition for anti-skin allergy, anti-inflammation and senile body odor removal.

The present invention encompasses extraction from water, or an organic solvent, especially alcohol and alcohol, from a mixture of almonds, allium and camellia leaves and fractionation with ethyl acetate.

The method for preparing the complex extract according to the present invention is as follows.

1. Thistle, Alkaline and Camellia Leaf Composite Extracts are shredded, the top part of Alkaline and camellia leaves are shaded, and dried plants are mixed in a weight ratio of 3: 1: 1 and then crushed and powdered.

2. Extract the mixed composite with an alcohol solvent such as water or alcohol.

At this time, a volume of about 3 to 20 times, preferably about 3 to 10 times, the volume of the dry weight of the composite is dissolved in an alcohol solvent such as water, alcohol, preferably water or alcohol at room temperature or 20 to 100 占 폚, Is subjected to the extraction method such as hot water extraction, cold extraction, warming or reflux cooling extraction or ultrasonic extraction for 1 to 5 times at an extraction temperature of 20 to 70 캜 for about 0.5 to 2 days, preferably 1 to 1 day, Preferably three consecutive times, is obtained by filtration under reduced pressure, and the filtrate is concentrated in a vacuum rotary condenser at a temperature of 20 to 100 ° C, preferably 40 to 70 ° C under reduced pressure to give a water, an alcohol or a mixed solvent thereof An extract can be obtained.

3. Extract the combined extracts with ethyl acetate to extract fractions.

The extracted complex extract is dissolved with water and then further fractionated into an ethyl acetate layer and concentrated under reduced pressure to prepare an ethyl acetate fraction.

The complex extract or the ethyl acetate fraction of the present invention is useful as an anti-skin allergic or anti-inflammatory agent for active ingredients, The body odor removal effect is excellent.

In addition to the anti-skin allergic and anti-inflammatory effects, pharmacologically acceptable emulsifiers, diluents and excipients may be added to the plant extract composition for removing the senile body odor, or a base material for cosmetic production may be used.

The plant extract composition for removing allergic and anti-inflammatory senile body odor of the present invention may be in the form of solutions, suspensions, emulsions, gels, creams, lotions, powders, soaps, cleansing, bodywashes, cleansing and sprays ≪ / RTI >

The plant extract composition for the improvement of skin allergy and inflammation and the removal of the senile body odor of the present invention has an advantage that it is excellent in the removal of nonane which is a causative substance of skin aged body odor and satisfies the user with a plant extract composition. This is for the purpose of illustrating the present invention, and the present invention is not limited by these examples.

EXAMPLES Preparation of a Combined Extract of Thymbalsus, Alkalineum and Camelliaceae

The ethyl acetate complex extract (EP) of the almonds, allium and camellia leaves used in the present invention was prepared by shredding the above-ground portions of almonds and algae and leaves of camellia trees to dry the plants and mixing 1 kg in a weight ratio of 3: 1: After 3 L of methane (ethane) was added, the mixture was cooled, repeatedly extracted three times at regular intervals, and then filtered under reduced pressure through a filter paper (Wattsman, USA). The filtrate was extracted with a vacuum rotary evaporator at 45 ° C 120 to 200 g of SD were obtained. After dissolving in water (H ₂ O), the residue was fractionated into an ethyl acetate layer and concentrated under reduced pressure to give an ethyl acetate fraction (FIG. 1).

Experimental Example 1 Effect of Ethyl Acetate Complex Extract (EP) on iNOS Production as an Inflammatory Mediator

RAW 264.7 cells (10 × 10 ⁴ cells / well) were inoculated into a 24-well plate using DMEM medium, and after 12 hours, fresh medium containing the sample and LPS (1 μg / ml) were simultaneously treated and cultured for 24 hours. The amount of NO produced was measured using the Griess reagent in the form of NO ^{2 -} present in the cell culture. 100 μl of the cell culture supernatant and 100 μl of Griess reagent (1% sulfanilamide, 0.1% naphtylethylenediamine in 2.5% phosphoric acid) were mixed and reacted on 24-well plates for 10 minutes. Absorbance was measured at 540 nm using an ELISA reader. The results are shown in FIG. As can be seen in FIG. 2, it was confirmed that EP effectively decreased NO production in RAW 264.7 cells induced by LPS in a concentration-dependent manner. NO is synthesized from L-arginine by nitric oxide synthase (NOS). Significant increases in NO under pathologic conditions are synthesized by iNOS, which stimulate inflammatory processes and exhibit synergistic effects with other inflammatory mediators. In addition, iNOS is strongly stimulated when exposed to inflammatory cytokines and bacterial endotoxin. Compounds may reduce NO production by iNOS and may be attractive as anti-inflammatory agents. For this reason, the effects of polyphenols on iNOS activity have been extensively studied for development as anti-inflammatory agents. Figure 2 shows the effect of EP on NOS enzyme in LPS-induced cells. This extract reduced the amount of iNOS produced by LPS in a concentration-dependent manner. Therefore, it can be seen that EP has inhibitory effect on iNOS expression or inhibition of iNOS expression in LPS-induced RAW 264.7 cells.

Experimental Example 2 Evaluation of inhibition of cyclo-oxygenase-1 and -2 by EP

The screening process for the screening of COX-1/2 inhibitors against EP was performed according to the method of Kaiman in the following manner. Briefly, COX-1/2 (Cayman, Cayman) was incubated in an incubate with the diluted extract at a ratio of 1: 500 for 15 minutes. After incubation, Quantablu (Pierce) substrate was added and allowed to mature for 45 minutes at 25 ° C. Maturation was followed by reading the luminescence with a Wallac Victor plate reader, The results are shown in Fig. 3 and Table 1.

Experimental Example 3 Anti-skin allergic effect of ethyl acetate complex extract (EP)

The secretion of β-hexosaminidase was measured to investigate the inhibitory effect on degranulation, which is an index of skin allergic response. RBL-2H3 cells were suspended in DMEM containing 10% FBS, and the cells were distributed to a 48-well plate (Corning, NY, USA) at a density of 5 × 10 ⁵ cells / ml. Subsequently, the cells were sensitized with anti-DNP IgE (30 ng / ml) and incubated for 24 hours at 37 ° C in a 5% CO ₂ incubator. Cells of each well were washed twice with buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH, pH 7.2), and then 5.6 mM glucose, 1 mM CaCl ₂ and 0.1 % BSA was added and EP was added at concentrations of 0, 2, 10 and 50 μ / ml for 1 hour at 37 ° C in 5% CO ₂ incubated with DNP-HSA (10 μg / ml) for 1 hour. 40 μl of the supernatant was transferred to a 96-well plate, and 80 μl of substrate buffer (1 mM p-nitrophenyl-N-acetyl-bD-glucosaminide, 0.05 mM sodium citrate, pH 4.5) was added and incubated at 37 ° C for 30 minutes. 200 μl of stop solution was added to each well to terminate the reaction. Absorbance was measured at 405 nm using a microplate reader (Molecular Devices Co. Ltd., USA). The inhibition (%) was calculated from the absorbance values of EP treatment group and control group according to the following equation, and it is shown in FIG. 4 and Table 1.

Inhibition (%) = (1-T / C) x 100

C: Cell (+), DNP-HSA (+), test sample (0)

T: Cell (+), DNP-HSA (+), test sample (+)

sample ^* IC ₅₀ (μg / mL) iNOS COX1 / 2 β-hexosaminidase MeOH extract 24.9 28.4 29.6 The EtOAc fractions (EP) 10.8 11.2 14.1

* IC ₅₀ : 50% inhibitory concentration

<Experimental Example 4>

The test for confirming the removal of the senile body odor was conducted by the following method. A total of 36 men and 18 women and 18 women aged 60 or older were selected as test subjects and divided into 3 groups of 3 men and 3 women per group. For each group, comparative formulations and BP body cleanser preparations were used as samples. Each of the subjects was buried in 7ml of the sample using a sample, and after bubbling, the whole body was cleaned with a shower towel for about 5 minutes and then washed. After the use of the first day sample, a cotton patch (10 cm x 10 cm) was attached to the back side of the new undergarment, and after 24 hours, the body odor of the patch was evaluated, and the second day sample After the use, it was evaluated by the same method, and after 24 hours, it was evaluated by the same method until the third day. The standards for body odor were as follows: a shower was used in the same manner as before the raw material was used, and a face patch (10 cm x 10 cm) was attached to the center of a new underwear backlight. The odor of the patch was set to 10 after 24 hours, And the concentration of body odor from day 1 to day 3 was indicated. That is, the closer to 10, the stronger the smell, the lower the weaker.

Sensory test

18 subjects in the 60s and over used the sample with the best comparison of the subject's self-evaluation, and each sample was used daily by 18 subjects. 10 cm x 10 cm) were evaluated once per week by the partner panel and averaged to be shown in Table 2 below. (The closer to 10, the stronger the smell.)

Sensory test results (average) sample 1 parking 2 parking 3 parking Comparative formulation 1 5.2 5.8 5.0 The EtOAc fractions (EP) 3.3 2.9 1.8

<Experimental Example 5> Cytotoxic effect of ethyl acetate complex extract (EP) on alfalfa, alginate and camellia leaves

The cytotoxicity of RA-264.7 cells, which were AD-macrophages obtained in the above-mentioned Examples, was measured in order to confirm anti-inflammatory activity in an appropriate concentration by treating LPS (1 / / ml). RAW 264.7 cells (10 x 10 ⁴ cells / well) were treated with test drug (EP) in DMEM medium and cultured for 24 hours. 500 μl of the medium and 5 μl of the MTT solution were added to a 24-well plate with the new medium, followed by further reaction for 4 hours. After removing the medium, formazan was dissolved in 200 μl DMSO and the absorbance was measured at 540 nm using a microplate reader. The measurement value is an average value of three repeated experiments, and the results are shown in Fig. 5 below. As shown in FIG. 5, the cytotoxicity of EP to RAW 264.7 cells did not significantly show up to a concentration of 100 μg / ml.

Claims (5)

A composition for the removal of anti-skin allergies, anti-inflammatory and senile body odor containing ethyl acetate fraction of almond extract, allium and camellia leaf complex extract. The method according to claim 1,
Wherein said complex extract is an alcohol extract of dried crumbs of alfalfa, alum and camellia leaves, for the removal of anti-allergic, anti-inflammatory and senile body odor. Shrubs, alums, and camellia leaves, followed by pulverization and pulverization to prepare a complex;
A complex extraction step of extracting the mixed composite with water or an alcohol solvent; And
And extracting fractions by fractionating the extracted complex extract with an ethyl acetate layer. The method for preparing anti-allergy, anti-inflammatory and senile body odor removing composition according to claim 1, The method of claim 3,
The composite extraction step comprises:
Extracting the complex with an alcohol solvent at 20 to 100 DEG C, filtering under reduced pressure, and concentrating the filtrate under reduced pressure at 20 to 100 DEG C in a vacuum rotary condenser, to remove anti-skin allergies, anti-inflammatory and senile body odor &Lt; / RTI &gt; The method of claim 3,
The composite of the complex extraction step comprises a weight ratio of 3: 1: 1, the top part of the alum and the camellia leaves,
Wherein the fraction extraction step comprises:
Dissolving the extracted complex extract with water, and then fractionating the extract into an ethyl acetate layer and concentrating the extract under reduced pressure. The method for preparing an anti-allergic, anti-inflammatory and senile body odor removing composition according to claim 1,

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