KR20120101897A - Preparation method of aloe fermentation and hydrolysis extract showing prevention and improvement effect of atopic dermatitis and cosmetics prepared using the same - Google Patents

Abstract

PURPOSE: A method for preparing aloe fermentation extract and a cosmetic product using the extract are provided to enhance skin permeability of an active ingredient and antioxidation. CONSTITUTION: A method for preparing aleo fermentation extract which prevents and treats atopic dermatitis comprises: a step of collecting 1.0 kg of aloe peel and adding the aloe peel to 2 L of ethanol; a step of incubating the mixture at 50-60 Deg. C. for 8 hours with shaking sometimes; a step of filtering ethanol extract at reduced pressure to collect liquid; a step of concentrating the liquid at 50-60 Deg. C. under a predetermined condition to prepare soft extract; a step of dissolving 50,000 unit of beta-glucosidase in the water and adding to the soft extract; and a step of reacting the final mixture at 40-50 Deg. C. for 8 hours.

Description

Preparation method of aloe fermentation and hydrolysis extract showing atopic dermatitis prevention and improvement effect and cosmetics produced using the same

The present invention relates to a method for producing aloe fermentation extracts and a cosmetics prepared using the same, which exhibits atopic dermatitis prevention and improvement effect, and more specifically, extracts and ferments aloe to increase skin penetration and antioxidant activity of the active ingredient, A method of preparing aloe fermentation and hydrolyzed extracts which exhibits the effect of preventing and improving atopic dermatitis for the purpose of preparing a fermentation extract that can enhance anti-inflammatory and anti-allergic efficacy, and to develop a cosmetic containing the same, and cosmetics prepared using the same It is about.

Aloe is a perennial tropical plant of the genus Aloe belonging to the genus Liliaceae, and its origin is mainly Africa, and more than 600 species are distributed or grown all over the world. Aloes grown mainly in Korea are Aloe vera ( A. barbadensis Mill. ) And Aloe arborescens ( A. arborescens ) . Mill . ), Aloe sandpaper and Leah (A. saponaria Haw . ) And so on. Aloe has been used as a folk medicine for a long time, and has been used mainly as bitter and laxative. Although there is a difference in degree, anti-inflammatory action, anti-ulcer and cell regeneration action, antibacterial and antifungal action, antiviral action, blood sugar improvement action, gastric secretion inhibition action, hepatocellular protection action, hypogonadism, immunomodulation action, anticancer action, etc. Have.

The most widely used field of aloe is cosmetics, which has excellent skin moisturizing function, and has excellent anti-inflammatory, anti-allergic, antioxidant and skin softening effects, and is used in various layers from children to the elderly.

In particular, since atopic dermatitis is a chronic disease and has a sensitive reaction depending on the type of cosmetics, studies for using aloe extract for atopic dermatitis have been continuously conducted.

Atopic dermatisis (AD) is a chronic relapsing skin disease with pruritus. It is known that 50% of patients develop before 1 year of age and 30% develop between 1 and 5 years of age. The mechanisms of atopic dermatitis have not been fully understood yet, but susceptibility to viral infections such as herpes simplex or scleroderma, reduced delayed immune response to certain microbial antigens, increased production of IgE, decreased sensitivity to contact allergens, cleavage Several immunological abnormalities have been reported, including decreased lymphocyte response to promoters and antigens, and reduced chemotaxis of granulocytes and monocytes. In addition, the prevalence of this disease and the progression to refractory adult atopic dermatitis are gradually increasing in the industrialized society, which is evidence that genetic factors and environmental factors such as industrialization are important in the occurrence of atopic dermatitis. The disease is characterized by pruritus, itching, lichen dermatitis, rash, tearing, and systemic dryness.

The treatment of atopic dermatitis has been applied to identify the basic methods of dermatitis and to identify and eliminate the triggers. In general, the treatment of atopic dermatitis patients in hospitals includes steroids, calcineurin inhibitors such as tacrolimus or pimecronimus, antihistamines, cyclosporin, methotrexate and azathioprine. Drugs such as immunosuppressants are used. Topical steroids can improve symptoms quickly and effectively when the lesion worsens, but can cause adrenal suppression if used continuously, skin atrophy, capillary dilation, decreased pigmentation, steroidal acne, hirsutism, growth disorders There is a risk of such a short-term use and there is a limit to use for sensitive skin such as thin epidermis or a lot of blood vessels. Calcineurin inhibitors can be used as an adjunct to existing treatments and minimize the problems caused by the use of topical steroids to prevent steroid-resistant patients, such as those around the skin, such as the eyes, face and genital area. Although it is applicable, it is approved and used for short-term use in children over 2 years of age as the second therapeutic agent.

It is necessary to develop cosmetics that can help prevent and improve atopy in the long term while using drugs for treating atopy.

The present invention has been made to solve these problems, the object of the present invention relates to the development of cosmetics to help prevent and improve atopic dermatitis during long-term use without showing side effects on atopic skin, skin allergy, pruritus, In order to protect, prevent, and improve damaged skin from contact dermatitis, dermatitis caused by microorganisms, skin rashes and atopic diseases, fermented and hydrolyzed by adding enzyme to aloe extract, and fermented extract of fermented extract of Echo and Echinacea. Its purpose is to develop various kinds of cosmetics such as body lotion, body cleanser, facial cream and body cream.

Aloe fermentation extract manufacturing method showing the effect of preventing and improving atopic dermatitis according to the present invention for achieving the above object, take 1.0 kg of aloe shell and put 2 liters (L) of ethanol (EtOH) 8 at 50 ~ 60 ℃ Left to shake occasionally for time;

Shaking the ethanol extract solution and filtering under reduced pressure using filter paper and taking the filtrate;

Putting the filtrate into a vacuum condenser and concentrating under reduced pressure at 50 to 60 ° C. in a predetermined condition to produce a soft extract state;

It is characterized in that the beta glucosidase (β-glucosidase) 50,000unit dissolved in 10 milliliters of water and added to the soft-core extract, reacting while shaking for 8 hours at 40 ~ 50 ℃ characterized in that the step consisting of bilhyo and hydrolysis.

In addition, the cosmetics prepared using the aloe fermentation extract according to the present invention, take the aloe fermentation and hydrolysis extract 100g, and evenly mix 200g golden hydrolyzed extract, 100g Eochocho fermentation extract and 20kg aloe vera fermentation and aloe After obtaining the hydrolysis extract complex, purified water 40kg, glyceryl stearate 6kg, Fiji 100 stearate 4kg, sorbitan stearate 3kg, polysorbate20 3kg, dimethicone 0.5kg, triethanolamine 1kg, caprylliccapryltriglycol 4 kg of ceride, 1 kg of carbomer 941, 1 kg of butylene glycol, 0.5 kg of isododecane, 5 kg of glycerin, 5 kg of dimethylhexyl carbonate, 3 kg of dicaprylcarbonate, 5 kg of stearyl alcohol, 1 kg of glyceryl behenate, 0.5 kg of phenoxyethanol , Disteadimonium hectorite 1kg, sorbitan sesquioleate 3kg, methylparaben 0.3kg, propylparaben 0.2kg, disodium ID TA 0.5g was mixed and warmed at 70-75 ° C. for 4 hours to prepare a body lotion base, and then added to the homogenizer together with the aloe hydrolysis extraction mixture and homogenized for 30 minutes at 3,000 to 3,500 ALPM for body lotion. It characterized in that the manufacturing.

In addition, 100 g of the aloe fermented and hydrolyzed extracts were taken, and 200 g of the golden hydrolyzed extract, 100 g of fermented green tea extract and 20 kg of aloe vera were uniformly mixed to obtain an aloe fermented and hydrolyzed extract complex, followed by 50 kg of purified water and 6 kg of lauric acid. , 4 kg of myristic acid, 1 kg of monoalkyl phosphate, 0.5 kg of oxybenzone, 4 kg of glycol distearate, 1 kg of triethanolamine, 1 kg of polyquathenium, 1 kg of laurylsulfonic acid triethanolamine, 1 kg of laramide DEA, 0.5 kg of cyclomethicone , Cocamidpropylbetaine 2kg, K-kuyl hydrolyzed collagen 1kg, Butylene glycol 1kg, Fiji-7-glyceryl cocoate 5kg, Glycerin 5kg, Glyceryl behenate 1kg, Phenoxyethanol 1kg, Dimethicone 0.5 kg, sorbitan sesquioleate 3 kg, propylene carbonate 1 kg, methyl paraben 0.3 kg, propyl paraben 0.2 kg, disodium ID 0.5 kg, and mixed at 70 ~ 75 ℃ 4 By homogenisation after the mixing and heating the thus prepared base body cleanser to Homo to put into a homogenizer with the Aloe fermentation and hydrolysis extracted complex for 30 min with 3000-3500 rpm for between characterized in that the body made of a cleanser.

In addition, 100 g of the aloe fermentation and hydrolysis extracts were taken, and 200 g of the golden hydrolysis extract, 100 g of Echo fermented extract and 20 kg of aloe vera were uniformly mixed to obtain an aloe fermentation and hydrolysis extract complex, followed by purified water 35 kg, glyceryl stearate. 5kg, Fiji 100stearate 3kg, sorbitan stearate 4kg, polysorbate20 3kg, dimethicone 1kg, triethanolamine 1kg, white biswax 2kg, carbomer941 1kg, butylene glycol 0.5kg, isododecane 1kg , 5 kg of glycerin, 1 kg of dimethylhexyl carbonate, 1 kg of dicapryl carbonate, 4 kg of stearyl alcohol, 1 kg of glyceryl behenate, 1 kg of phenoxyethanol, 1 kg of disteadimonium hectorite, 2 kg of sorbitan sesquioleate, methylparaben 0.3 kg, propylparaben 0.2kg, disodium IDT 0.5kg mixed, and warmed at 70 ~ 75 ℃ for 4 hours to mix the facial cream base Prepared after the Aloe fermentation and put into the homogenizer with the hydrolysis extract complex was homogenized for 30 minutes at 3000-3500 rpm characterized by made of a facial cream.

In addition, 100 g of the aloe fermented and hydrolyzed extract was taken, and 200 g of golden hydrolyzed extract, 100 g of fermented green tea extract and 20 kg of aloe vera were uniformly mixed to obtain an aloe fermented and hydrolyzed extract complex, followed by purified water 40 kg, squalane 1 kg, ceti 1 kg aryl alcohol, 1 kg macadamia seed oil, 0.5 kg grape seed oil, 1 kg butylene glycol, 1 kg polygtamic acid, 5 kg glycerin, 1 kg diethyl carbonate, 0.5 kg dicaprylcarbonate, 3 kg stearic acid, caprylic / Capric triglyceride 1kg, stearyl alcohol 1kg, phenoxyethanol 0.5kg, green tea seed oil 0.5kg, arachadyl alcohol 0.4kg, behenyl alcohol 0.3kg, arachadyl glucoside 0.3kg, cetyl palmitate 0.2kg, sorbitanol 0.5 kg of bait, 1 kg of sorbitan palmitate, 0.5 kg of cetearyl olivate, 0.2 kg of Fiji-32, 0.3 kg of ceramide-3, 0.3 kg of dimethicone, 0.2 kg of allantoin, 0.5 kg of Carbomer 940 and 0.1 kg of Triethanolamine were mixed and mixed with warming at 70-75 ° C. for 4 hours to prepare a body cream base, and then added to the homogenizer together with the aloe fermentation and hydrolysis extraction complexes, then 3,000- It is characterized by preparing a body cream by homogenizing for 30 minutes at 3,500 ALPM.

Further objects and effects of the present invention will become apparent from the following detailed description, and the detailed description and examples showing the preferred embodiments of the present invention are not intended to limit the scope of the present invention.

According to the present invention, a method for preparing aloe fermentation and hydrolyzed extracts having an atopic dermatitis prevention and improvement effect and cosmetics prepared using the same, according to the present invention, contact dermatitis, dermatitis caused by microorganisms, skin allergy, pruritus, skin rash and atopic dermatitis In order to protect, prevent and improve the damaged skin from diseases, various kinds of body lotions, body cleansers, facial creams and body creams are combined with aloe fermented and hydrolyzed extracts with golden hydrolysis extracts, honey vinegar extracts and aloe vera. As a result of the development of cosmetics, the skin has a strong penetrating ability, high antioxidant activity, high antimicrobial activity against gram-positive and negative bacteria, and good prevention and improvement of atopic dermatitis induced by house dust mites.

Hereinafter, one preferred embodiment according to the present invention will be described in detail.

In order to achieve the above object, aloe, which is applied as a main ingredient in the present invention, is a perennial tropical plant of the genus Aloe belonging to the genus Liliaceae, and its origin is mainly Africa, and about 600 species are distributed or grown all over the world. Aloes grown mainly in Korea are Aloe vera ( A. barbadensis Mill.) And Aloe arborescens ( A. arborescens ). Mill., A. saponaria Haw.) And the like.

Aloe has been used as a folk medicine for a long time, and has been used mainly as bitter and laxative. Although there is a difference in degree, anti-inflammatory action, anti-ulcer and cell regeneration action, antibacterial and antifungal action, antiviral action, blood sugar improvement action, gastric secretion inhibition action, hepatocellular protection action, hypogonadism, immunomodulation action, anticancer action, etc. It is known to have.

Barbaloin, aloesin, aloenin, isobarbaloin, isoaloeresin and aloe-emodin have been reported as active ingredients in aloe.

Studies on the pharmacological activity of Aloe vera extract have been shown to have a clear inhibitory effect on carrageenan-induced foot edema in rats and to inhibit the production of PGE ₂ in vitro. The anti-inflammatory effect of aloe vera gel on human rectal mucosa has been reported to exhibit anti-inflammatory effect by inhibiting the production of PGE ₂ when aloe is administered.

Studies on the immunomodulatory activity of mice with aloe vera gel have been reported to promote antibody production and increase the number of plaque forming cells upon administration of aloe vera extract, and immunomodulation of polysaccharides contained in aloe vera In the study of chemical properties, the structural properties of polysaccharides contained in aloe were identified through enzyme and acid hydrolysis.

Barbaloin, aloesin, aloenin, aloe-emodin, etc., which are known as active ingredients of aloe, are mainly contained in the bark of aloe plants, so the use part of the aloe plant used in the present invention mainly ferments and hydrolyzes the extract of the bark. Here aloe vera was mixed.

Scutellaria Radix, a complex with aloe, is a skin-derived root of Scutellaria baicalensis GEORGI, a perennial herbaceous plant belonging to Lamitae and Labiatae. It has been used for the purpose of antibacterial, diuretic, laxative, atherosclerosis prevention, gastric juice secretion, sedation, blood pressure lowering action.

The active ingredients of gold are Baicalin, Baicalein and Wogonine. In pharmacological studies, gold triglycerides and free fatty acids in serum were inhibited by inhibiting lipid oxidation in the liver of rats. It is known to reduce the content and have an effect on hyperlipidemia caused by ethanol (EtOH) ingestion, and also to protect the cellular damage caused by oxidation by the antioxidant activity of the golden component and to skin caused by free radicals and free radicals It has been reported to prevent oxidation and damage of cells.

Baicalin, the main component of gold, has a flavonoid structure and various biological activities such as anti-allergic, antibacterial and antiviral, and also inhibits lipoxygenase, inhibits lipid peroxidation of microsomes, the inhibition of oxygen radical generation, and more recently active oxygen (H ₂ O _2, -OH, and O ₂ ^-) is the antioxidant activity, such as inhibition of fibroblast injury induced by it is reported.

Baikalin, the highest content of gold, is hydrolyzed by β-glucuronidase secreted by E. coli from humans and animals. It has been reported that allergic effect and skin damage inhibitory effect are remarkably enhanced.

In addition, Houttuynia cordata is a perennial herbaceous plant belonging to the Saururaceae, whose stems are thin and reddish purple, and the leaves are heart-shaped and pointed, and the leaves are similar to sweet potatoes or buckwheat leaves. The smell is very similar to the "fishy fishy" in Eoseongcho (魚腥草) was called. Eoseongcho acts as a diuretic, analgesic, hemostatic, tissue regeneration, vasodilatation, and haejujeok, the herbaceous tree, Dongbobom, botanical metabolism is effective for killing, syphilis, boil, white poison, hemorrhoids, detoxification and elimination of heavy metal poison.

As a result of research on the separation and use of functional ingredients for improving the medicinal and edible solubility of eosungcho, the flavonoid constituents of eochochocho were identified as quercetin, quercitrin, rutin and myricetin. Among these ingredients, quercetin is known to be contained in onion.

Studies on the structural properties of quercetin and quercetin glycosides in fish and onions have reported pharmacological effects of these phenolic compounds such as decreased activity of carcinogenic substances, decreased growth of mutant cancer cells, lowered blood pressure and strengthened capillaries. Pharmacological action of quercetin is known to inhibit lipid peroxidation, antiviral, antibacterial and antimutagenic.

Therefore, the present invention is a combination of aloe fermentation and hydrolysis extracts of golden hydrolyzed extracts, fish vinegar fermentation extracts and aloe vera as the main components of cosmetic preparations with anti-oxidation, anti-inflammatory, anti-allergic, antipruritic, contact dermatitis with anti-atopic dermatitis It can enhance the effect, does not irritate and damage the skin of children and infants, and by using a safe and non-toxic and low irritation safe mechanism, it does not reject at all to women and sensitive skin, maintains skin hydration and flexibility while maintaining skin moisture and flexibility Formulations such as body lotion, body cleanser, facial cream and body lotion have been developed by formulating a prescription that can delay.

Features of the present invention for achieving the above object is as follows, the constituent ratio corresponds to the present invention, it can be carried out in the range ± 10% of the constituent amount.

Hereinafter, the operation and experimental results of the mixture or composition according to the present invention will be described.

1. Aloe Hydrolyzate Hydrogel  Produce

After dissecting 1 kg of aloe, the skin and the yellow endothelial secretion were taken, dried at 50 ° C. for 3 days, and 600 ml of ethanol was added thereto, followed by filtration with shaking at 60 ° C. for 6 hours. The filtrate was concentrated under reduced pressure and lyophilized to obtain a brown powder (yield 15.4%) of aloe, which was used as a sample.

To prepare aloe hydrogel, first dissolve the Aloe X powder in purified water, add propylene glycol, labrasol and ethanol sequentially, mix, and swell with carbopol 940 (carbopol940). triethanolamine) was adjusted to pH 6.5 to prepare a hydrogel.

Preparation of aloe hydrolyzed hydrogel (hereinafter referred to as ALH gel) is performed by dissolving aloe extract powder in purified water, adding 1,000 units of beta-glucosidase and 5,000 units, respectively, and reacting at 50 ° C. for 4 hours. ALH) 1%, Aloe Hydrolyzate (ALH) 5% was prepared (see Table 1).

Table 1. Preparation of Aloe Hydrolyzate Hydrogel

2. House dust  mite( DPE Induction of dermatitis and sample processing

To induce atopic dermatitis, clean the area around the neck, ears, and back of 8-week-old Nc / Nga mice, leave it for 24 hours to heal fine wounds after epilation, and then spray 4% SDS solution on the epilation site. After that, make a culture solution with 0.5% Tween 20 and phosphate buffer solution (PBS) , and add 1% solution made with house dust mite extract ( D. pteronyssinus, DPE, Yonsei University Microbiology Department) to 3 times a week. 50 μl each time was applied for 8 weeks to induce atopic dermatitis. Physiological saline was applied to the control group. Cyclosporin A was administered once daily for 10 weeks by intravenous injection of 10 ㎍, 1% of aloe hydrolyzate (ALH) and 5% of aloe hydrolyzate (ALH) for 8 weeks. Applied to the skin.

3. Immunity Cell number  Measure

Apply weekly house dust mite (DPE) to take a certain amount of skin tissue from the back area of the dermatitis-induced mouse, and finely cut 1 mg / ml of collagenase (in 2% FBS + RPMI1640) for shaking at 37 ° C (180 rpm, 20 min) was repeated four times to recover the supernatant after incubation in the incubator. ACK solution (8.3 g NH ₄ Cl, 1 g KHCO _{3) in} cultured supernatant in 1 L of demineralized water + 0.1 mM EDTA) for 5 minutes at room temperature to dissolve erythrocytes, washed twice with D-PBS, stained with 0.04% trypan blue, and then counted. It was. The measured skin tissue infiltrating cells were adjusted to 5 × 10 ⁵ cells and subjected to immunofluorescence staining at 4 ° C. PE-anti-CD3e, FITC-anti-CD69, FITC-anti-CCR3, PE-anti-Gr-1, FITC-anti-CD11b, PE-anti-Gr-1, PE-anti-B220 and FITC- respectively Anti-IgE was added and reacted on ice for 30 minutes. After the reaction, the cells were washed with phosphate-buffered saline at least three times, and then CD3 ⁺ / CD69 ⁺ , CCR3 ⁺ , B220 ⁺ / IgE ⁺ , CD11b ⁺ / Gr-1 ⁺ using a flow cytometer Cell Quest program. After analyzing the cell number as a percentage (%), the absolute cell number in each tissue was calculated by applying the total cell number.

The number of immune cells in the normal group was 29 ± 2.5 (× 10 ⁴ / g), control group 119.5 ± 23.5 (× 10 ⁴ / g), CsA-administered group 52.0 ± 5.0 (× 10 ⁴ / g), 56.0 ± 7.0 (× 10 ⁴ / g) in the ALH 5% group, 55 ± 8.0 (× 10 ⁴ / g) in the ALH 1% group showed significant reduction effects compared to the control group (see Table 2).

The number of CD3 ⁺ / CD69 ⁺ cells in the skin was 2.2 ± 1.4 (× 10 ⁴ / g) in the normal group, 23.0 ± 1.9 (× 10 ⁴ / g) in the control group, 9.5 ± 0.5 (× 10 ⁴ / g) in the CsA-treated group, It was shown as 7.5 ± 2.9 (× 10 ⁴ / g) in the ALH 5% group and 5.9 ± 3.5 (× 10 ⁴ / g) in the ALH 1% group.

The number of CCR3 ⁺ cells was 0.7 ± 0.3 (× 10 ⁴ / g) in the normal group, 9.4 ± 0.7 (× 10 ⁴ / g) in the control group, 5.3 ± 1.8 (× 10 ⁴ / g) in the CsA group, and 5.4 ± in the ALH 5% group. 1.6 (× 10 ⁴ / g), ALH 1% administration group 4.4 ± 1.1 (× 10 ⁴ / g) showed a significant reduction compared to the control group.

CD11b ⁺ / Gr-1 ⁺ cell number was 1.0 ± 0.8 (× 10 ⁴ / g) in control group, 9.2 ± 0.8 (× 10 ⁴ / g) in control group, 2.2 ± 0.8 (× 10 ⁴ / g) in CsA group, ALH The 5% group showed 4.1 ± 1.3 (× 10 ⁴ / g) and the ALH 1% group showed 3.4 ± 1.2 (× 10 ⁴ / g), which showed a significant reduction effect compared to the control group.

The number of B220 ⁺ / IgE ⁺ cells was 0.6 ± 0.3 (× 10 ⁴ / g) in control group, 6.4 ± 0.5 (× 10 ⁴ / g) in control group, 3.3 ± 0.2 (× 10 ⁴ / g) in CsA group, ALH 5% 2.4 ± 0.9 (× 10 ⁴ / g) administration group, 1.9 ± 1.2 (× 10 ⁴ / g) administration group ALH showed significant reduction effect compared to the control group.

Table 2. Nc / Nga Mouse Skin Immune Cell Number Induced by House Dust Mite (DPE)

Nc / Nga mice model followed by the administration of ALH (5%, 1% /) for 8 weeks. The mice dorsal skin were removed and skin cell absolute number were measured by analyzed by flow cytometer. Values are represented as mean ± SE (n = 4). Statistically significant value compared with Nc / Nga-normal mice group by student's t- test (* p <0.05, ** p <0.01, *** p <0.001).

4. Cytokine Measurement

For inflammatory cytokine analysis, blood was collected by cardiac puncture after anesthesia with anesthetized Nc / Nga mice, which resulted in atopic dermatitis-like skin caused by house dust mite (DPE). IL-6 and TNF-γ concentrations in serum were measured using an enzyme-linked immuno-sorbent assay (ELISA, Biosource, USA). Each well was dispensed with 100 μl (1/100 dilution) of Nc / Nga mouse serum and reacted at room temperature for 1 hour, followed by washing with two wash buffers. 100 μl of HRP-conjugeted Avidin was treated and reacted at room temperature for 1 hour, followed by washing again. 100 μl of TMB substrate was dispensed and reacted in the dark for 30 minutes, and then treated with 50 μl of stop solution to measure absorbance at 450 nm using an ELISA reader.

To measure cytokine production in splenocytes, spleen cells (1 × 10 ⁴ / well) of mice infected with atopic dermatitis were coated with anti-CD28 (1 μg / ml) and anti-CD3 (1 μg) by applying house dust mite (DPE). / Ml) was incubated for 48 hours in a 96-well plate coated. In order to measure IL-4 and IFN-γ by ELISA kit, 100 μl of splenocyte culture supernatant was dispensed into each well, reacted at room temperature for 1 hour, washed twice, and then biotin-conjugated antigen. Was added and reacted for 30 minutes. Again washed with buffer twice, treated with 100 μl of HRP-conjugeted Avidin, and reacted at room temperature for 1 hour and then washed again. 100 μl of TMB substrate was dispensed and reacted in the dark for 30 minutes, and then treated with 100 μl of stop solution to measure absorbance at 450 nm using an ELISA reader.

As a result of measuring cytokine production in serum, IL-6 production was 137.9 ± 0.1 (pg / ml) in the normal group, 200.6 ± 16.9 (pg / ml) in the control group, 153.0 ± 3.72 (pg / ml) in the CsA-administered group, and 5% in ALH. The administration group 146.8 ± 4.23 (pg / ㎖), ALH 1% administration group 156.25 ± 2.2 (pg / ㎖), showed a significant reduction compared to the control group.

Serum TNF-α production was 8.2 ± 1.5 (pg / ml) in the normal group, 36.3 ± 5.4 (pg / ml) in the control group, 25.6 ± 3.0 (pg / ml) in the CsA group, and 23.2 ± 1.1 (pg / ml in the ALH 5% group. ), 19.9 ± 0.3 (pg / ㎖) ALH 1% group showed a significant reduction compared to the control group (see Table 3).

Table 3. Cytokine Production in Nc / Nga Mouse Serum and Spleen Induced by Dust Mite (DPE)

To determine amount of IL-6 and TNF-α by ELISA, blood was collected from the retro-orbital plexus under ether anesthesia and serum was obtained by 10,000 rpm centrifugation and stored at -20 ° C until use. Splenocytes from Nc / Nga mice, were stimulated with anti-CD3 antibodies for 48 h. The culture supernatants were assayed for IL-4 and IFN-γ by ELISA. Amount of cytokines are shown in picogram per milliliter and values are represented as mean ± SE (n = 4). Statistically significant value compared with Nc / Nga-normal mice group by student's t- test (* p <0.05, * p <0.01, *** p <0.001).

5. In serum Immunoglobulins  Measure

Serum immunoglobulin was measured by capillary tube from the orbital artery of mice at 8, 12, and 16 weeks of sample administration, and then centrifuged for 20 minutes at 6,500 rpm using a capillary tube. 30 μl of serum was isolated. Serum IgE was measured using an ELISA kit. The antibody was diluted with a buffer solution, coated on a 96-well plate, and left for 16 hours at 4 ° C. Each well was washed three times with buffer solution and then 100 μl of serum (100-fold dilution) was dispensed. It was reacted at room temperature for 1 hour, washed twice with buffer solution, and then treated with 100 μl of HRP-conjugated Avidin, reacted at room temperature for 1 hour, and washed again. 100 μl of TMB substrate was dispensed therein and reacted in the dark for 30 minutes, and then treated with 50 μl of stop solution, and the absorbance was measured at 450 nm using an ELISA reader.

Serum concentration measurements of total IgG1, IgG2a, IgG2b, and IgM were measured using ELISA after the end of the experiment. 100 μl (1/200 dilution) of serum was dispensed into each well, and the mixture was left in a 4 ° C. cold room for 12 hours, washed twice with buffer, and then reacted for 30 minutes with a biotin-conjugated antibody. After washing twice with buffer solution, 100 μl of HRP-conjugeted Avidin was treated and reacted at room temperature for 1 hour, and then washed again. 100 μl of TMB substrate was dispensed and reacted in the dark for 30 minutes, and then treated with 100 μl of stop solution to measure absorbance at 450 nm using an ELISA reader.

Serum IgE production was measured at 8 weeks, 12 weeks, and 16 weeks in the eyes of Nc / Nga mice, and serum was isolated. Changes in serum IgE levels resulted in 492.5 ± 57.8 (ng / ml) in the normal group at 8 weeks, 855.5 ± 68.5 (ng / ml) in the control group, 915.5 ± 131.0 (ng / ml) in the CsA group, and 947.5 in the 5% ALH group. ± 33.8 (ng / mL), 709.0 ± 83.9 (ng / mL) in the ALH 1% group, but there was no significant difference, but at 12 weeks, 734.4 ± 138.8 (ng / mL) in the normal group and 3,347.1 ± 30.5 ng / ml in the control group. ), 3,041.5 ± 47.8 (ng / ml) in the CsA group, 3,117.9 ± 82.9 (ng / ml) in the ALH 5% group, and 3,180.9 ± 5.4 (ng / ml) in the ALH 1% group. The effect lasted for up to 16 weeks.

Serum IgG1 production level was 2,645.0 ± 85.2 (㎍ / ml) in the normal group, 3,774.0 ± 76.4 (µg / ml) in the control group, 3,238.5 ± 111.4 (µg / ml) in the CsA group, and 3,691.0 ± 130.1 (µg in the ALH 5% group / Ml), ALH 1% group was 3,706.0 ± 51.1 (㎍ / ㎖) did not affect the production amount.

IgM blood levels were 647.0 ± 30.9 (µg / ml) in the normal group, 929.5 ± 37.6 (µg / ml) in the control group, 458.5 ± 56.5 (µg / ml) in the CsA group, and 714.0 ± 23.4 (µ / ml in the ALH 5% group. ㎖), ALH 1% administration group was 680.5 ± 59.7 (㎍ / ㎖) showed a significant decrease compared to the control group.

IgG2a blood levels were 1,967.5 ± 55.9 (µg / ml) in normal group, 2,694.5 ± 33.8 (µg / ml) in control group, 1,473.0 ± 70.1 (µg / ml) in CsA group, and 2,283.5 ± 150.6 (µg / ml) in ALH group ㎖), ALH 1% administration group was 2,153.0 ± 118.7 (㎍ / ㎖) showed a significant reduction compared to the control group.

IgG2b blood levels were 6,968.0 ± 46.1 (㎍ / ml) in the normal group, 9,284.5 ± 355.8 (㎍ / ml) in the control group, 6,191.5 ± 141.1 (㎍ / ml) in the CsA-administered group, and 7,907.0 ± 94.1 (µg / ml) in the ALH 5% -administered group. ㎖), ALH 1% administration group was 7,324.0 ± 287.6 (㎍ / ㎖) showed a significant reduction compared to the control group (see Table 4).

Table 4. Immune Antibody Production in Nc / Nga Mouse Serum Induced by Dust Mite (DPE)

Blood was collected from the retro-orbital plexus under ether anesthesia and heparinized immediately thereafter. The plasma samples were assayed for amount of immunoglobulines by a sandwich ELISA. Values are represented as mean ± SE (n = 4). Statistically significant value compared with Nc / Nga-control mice group by student's t- test (* p <0.05, ** p <0.01, *** p <0.001).

6. Aloe & Golden Compound Hydrogel  Determination of antimicrobial activity against gram positive and gram negative bacteria

First, the test bacteria were prepared by adjusting the turbidity of nutrient agar and citric acid desoxycholate citrate agar to the same level as the standard turbidity standard (Tubidity Standard, Transmittance 35%).

Aloe and golden complex hydrogels were dissolved by heating in a water bath with a sample solution, and passed through 0.2 μm of a membrane filter, followed by diluting stepwise twice to 200, 100, 50, 25, 12.5 and 6.25 μg / ml. 2 ml of each was mixed and hardened with 18 ml of Mueller Hinton agar medium, and 5 µl of test solution was inoculated using an automatic dispenser and incubated for 18 hours at 37 ° C. Observed.

Separately, 8 ml of Mueller Hinton broth, 1 ml of test solution, and 1 ml of sample solution (1000 µg / ml) were added to a sterilized test tube, incubated at 37 ° C for 18 hours, and the respective permeability was measured at 600 nm. The growth inhibition rate of bacteria was calculated by food. A culture in which only the bacterial solution was added without adding a sample was used as a control.

Comparing the bacterial growth inhibition rate against gram positive and negative bacteria of aloe and golden complex hydrogels, the gold extract showed relatively high antibacterial activity against gram positive bacteria such as Staphylococcus aureus and epidermal staphylococcus, and low values for gram negative bacteria. On the other hand, the antimicrobial activity of hydrogel showed antimicrobial activity to both Gram-positive and negative bacteria, but its growth inhibition rate was low (see Table 5).

Table 5. Antimicrobial Effects of Aloe and Golden Complex Hydrogels on Gram-positive and Gram-negative Bacteria (%)

7. Atopic dermatitis induction and sample processing NC Of Nga  Observational Effect of Atopic Dermatitis in Mice

First, the neighboring areas of the neck, ears, and back of 8-week-old NC / Nga mice were depilated cleanly and left for 24 hours to heal fine wounds after skin removal.

After spraying to 4% SDS solution hair removal site, the culture medium was made with 0.5% Tween 20 and PBS to put the house dust mite (D. pteronyssinus) extract (DPE, Yonsei University Microbiology, Seoul) antigen. 10 mg / ml, 50 μl was applied three times a week for 8 weeks, and 5% and 1% of the experimental group ALH were administered at an appropriate concentration for 8 weeks (8 to 16 weeks).

DPE-induced dermatitis-induced mice were divided into three groups, physiological saline in the control group, cyclosporin A in the positive control group and golden hydrolyzate in the experimental group 5% and 1%. It was applied to the skin for 8 weeks (from 8 to 16 weeks). Sensory evaluation was performed at 4 week intervals of drug treatment. The dermatitis of NC / Nga mice was the Wilcoxon test, which is commonly used in atopic dermatitis.

The visual evaluation index was expressed as the sum of scores of each of the following five items. The assessment items were divided into Erythema, Pruritus & Dry skin, Edema & escoriation, Erosion, and Lichenification. Each of these items was scored as none (0), weak (1), severity (2), and severe (3).

Referring to Figure 1, the effect of inhibiting atopic dermatitis induced in NC / Nga mice is shown in Figure 1A is a picture of the back of the 8-week-old NC / Nga mice, Figure 1B is applied to DPE for 8 weeks after depilation Afterwards, keratinization progressed and skin inflammation was severely shown. In contrast, the positive control CSA (Figure 1C) was injected intraperitoneally and showed significant improvement. When the hydrolyzate of the experimental group was applied to the skin for 8 weeks (Fig. 1D), the scratching behavior was relatively reduced compared to the control group, and it was confirmed that the keratinization, thyroiditis, skin dropout symptoms and dermatitis symptoms were remarkably improved.

Figure 1.Therapeutic Effect of Aloe and Golden Complexes on Atopic Dermatitis in House Dust Mites-induced NC / Nga Mice

In addition, referring to Table 6, as a result of measuring the severity of dermatitis every four weeks by the sensory method, the control group is 8 weeks 2.1 ± 0.4, 12 weeks 2.5 ± 0.16, 16 weeks 2.6 ± 0.2, CsA 8 weeks 1.8 ± 0.41, 12 weeks 1.7 ± 0.25, 16 weeks 0.9 ± 0.4, gold hydrolyzate 5% for 8 weeks 1.5 ± 0.16, 12 weeks 1.7 ± 0.3, 16 weeks 0.8 ± 0.1, 1% for gold hydrolyzate 8 weeks 1.4 ± 0.24 12 weeks 2.0 ± 0.35, 16 weeks 1.6 ± 0.4 showed a significant reduction from 12 weeks.

Table 6. Visual Evaluation Index of Aloe and Golden Complexes for Atopic Dermatitis in NC / Nga Mice Induced by House Dust Mites

Effects of ALH on clinical skin features and severity in DPE-induced dermatitis model of NC / Nga mice Clinical skin index of dermatitis was defined as the sum of the individual scores graded as 0 (none), 1 (mild), 2 (moderate) and 3 (severe) for each of five signs and symptoms (itch, erythema / hemorrhage, edema, excoriation / erosion and scaling / dryness): Symptoms were evaluated by skin dryness, eruption and wound on the three parts of the body: ear, face and head, and back Statistically significant value compared with NC / Nga-CT mice grouup by t-test (*** p <0001)

8. Atopic dermatitis induction and sample processing NC Of Nga  Microscopic Observation of Skin and Ear of Mice

After the end of the experiment, the skin of the left ear tip and dorsal neck was removed and fixed in 10% paraformaldehyde for 2 hours.

The tissue was formatted in paraffin and made a block with a thickness of 5 μm. The tissue part is hematoxylin and eosin that identifies the inflamed epidermis, dermis, keratinocytes, neutrophils, eosinophil and other cells and edema. (hematoxyline and eosin, H & E) staining and toluidine staining were performed.

Immuno histochemical stains create blocks, which include rat anti-mouse CD4 mAb (RM4-5; PharMingen, San Diego, Calif.), Rat anti-mouse mouse) CCR3 mAb (53-6.7; Becton Dickinson, Mountain View, CA) antibody was used.

Tissue sections were sliced to 4 μm thickness and then attached to a probe-on plus slide (Fisher Scientific, U.S.A) and dried.

Deparaffinized and dehydrated, and pretreated with a microwave oven (microwave oven) for 15 minutes using 0.01M citric acid buffer (citrate buffer pH 6.0).

In order to inhibit the action of peroxidase in the tissues, 10% treatment with 3% hydrogenated supernatant was followed by blocking the protein with normal serum to inhibit nonspecific protein binding with antigens in the tissues. The cells were then attached to the primary single antibody for 1 hour and washed with buffer.

After the experiment, the skin and ears of the mouse were excised, and then the normal morphological changes and the thickness of the ear were examined by hematoxylin and eosin staining and toluidine blue staining. In the skin and ears of (Fig. 2B), the epidermis (epidermis / dermis) significantly expanded due to edema and white blood cell infiltration was also observed.

Compared with the control group (Fig. 2D), the thickness of the epidermis was relatively decreased, and the infiltration of leukocytes was significantly reduced compared to the control group.

In toluidine staining of mast cells, the control group (Fig. 2F) shows that a lot of mast cells (arrows) infiltrate around the dermis.

However, in the group administered with the Aloe vera complex (Fig. 2H), mast cells were hardly observed as shown in the photograph.

In addition, the thickness of the ear tissue was measured using a fossil decomposer in a 400 times optical microscope. The normal group was 21.1 ± 1.4 (x10 / μm), the control group was 129.5 ± 2.5 (x10 / μm), and the CsA was 57.4 ± 7.3 ( x10 / μm), 37.1 ± 0.3 (x10 / μm) in the 5% Aloe Golden Complex group, and 62.2 ± 1.5 (x10 / μm) in the 1% Aloe Golden Complex group were significantly higher than the control group. It has been shown to decrease. (See Table 7)

Figure 2. Microscopic observations of skin and ears of NC / Nga mice induced by atopic dermatitis

Ear

H & E toluidine blue

Skin

H & E toluidine blue

Histologic examination of ear and skin lesion in DPE-induced dermatitis model of NC / Nga mice The animals were administrated with saline (control) or SBE for 8 weeks Ear, skin biopsy was stained with hematoxylin and eosin (H & E) and toluidine blue (A , E; normal B, F: cotrol, C, G: CsA, D, H; SBE) for examining inflammatory cells and mast cell, respectively Bright microscopy (Nikon, Japan, orignal magnification, ear x 200, skin x 400)

Table 7. Ear Thickness Comparison of NC / Nga Mice Induced by Atopic Dermatitis

Ear thickness of NC / Nga mice by DPE stimulation After atopy dermatitis ear in NC / Nga mice model followed by the administration of CSA, and Scutellariae extracts for 8 weeks (Nikon, Japan, orignal magnification, ear x 400)

Claims (5)

Take 1.0 kg of aloe shell and add 2 liters (L) of ethanol (EtOH) and leave to shake occasionally for 8 hours at 50 ~ 60 ℃;
Shaking the ethanol extract solution and filtering under reduced pressure using filter paper and taking the filtrate;
Putting the filtrate into a vacuum condenser and concentrating under reduced pressure at 50 to 60 ° C. in a predetermined condition to produce a soft extract state;
Dissolving 50,000 units of beta-glucosidase in β-glucosidase in 10 milliliters of water and adding to the soft-core extract, reacting with shaking for 8 hours at 40 to 50 ° C. for bilitation and hydrolysis; Method for producing aloe fermentation and hydrolysis extract showing atopic dermatitis prevention and improvement effect, characterized in that consisting of.
100 g of aloe fermented and hydrolyzed extract prepared by claim 1 were taken, and 200 g of golden hydrolyzed extract, 100 g of fermented green tea extract and 20 kg of aloe vera were uniformly mixed to obtain an aloe fermented and hydrolyzed extract complex.
Purified water 40kg, Glyceryl stearate 6kg, Fiji 100 stearate 4kg, Sorbitan stearate 3kg, Polysorbate20 3kg, Dimethicone 0.5kg, Triethanolamine 1kg, Caprylic capryl triglyceride 4kg, Carbomer 941 1kg, 1 kg of butylene glycol, 0.5 kg of isododecane, 5 kg of glycerin, 3 kg of dimethylhexyl carbonate, 3 kg of dicapryl carbonate, 5 kg of stearyl alcohol, 1 kg of glyceryl behenate, 0.5 kg of phenoxyethanol, 1 kg of disteadimonium hectorite , 3 kg sorbitan sesquioleate, 0.3 kg methyl paraben, 0.2 kg propyl paraben, 0.5 kg disodium IDT and mixed with warming for 4 hours at 70 ~ 75 ℃ to prepare a body lotion base after the aloe water Aloe fermentation, characterized in that the body lotion is prepared by injecting the homogenizer with the decomposition extraction mixture and homogenized for 30 minutes at 3,000 ~ 3,500 ALPM. Cosmetics prepared using hydrolyzed extracts.
100 g of aloe fermented and hydrolyzed extract prepared by claim 1 were taken, and 200 g of golden hydrolyzed extract, 100 g of fermented green tea extract and 20 kg of aloe vera were uniformly mixed to obtain an aloe fermented and hydrolyzed extract complex.
50 kg of purified water, 6 kg of lauric acid, 4 kg of myristic acid, 1 kg of monoalkyl phosphate, 0.5 kg of oxybenzone, 4 kg of glycol distearate, 1 kg of triethanolamine, 1 kg of polyquathenium, 1 kg of laurylsulfonic acid triethanolamine, laramide DEA 1 kg, cyclomethicone 0.5 kg, cocamidpropyl betaine 2 kg, K-kuyl hydrolyzed collagen 1 kg, butylene glycol 1 kg, Fiji-7-glyceryl cocoate 5 kg, glycerin 5 kg, glyceryl behenate 1 kg, phenoxy 1 kg of ethanol, 0.5 kg of dimethicone, 3 kg of sorbitan sesquioleate, 1 kg of propylene carbonate, 0.3 kg of methylparaben, 0.2 kg of propylparaben, 0.5 kg of disodium ID, and mixed at 70-75 ° C. for 4 hours After mixing with warming to prepare a body cleanser base, it is added to the homogenizer together with the aloe fermentation and hydrolyzed extract complexes and homogenized for 30 minutes at 3,000 ~ 3,500 arpm to prepare a body cleanser. Aloe fermentation and cosmetics prepared by using the hydrolysis extract characterized in that.
100 g of aloe fermented and hydrolyzed extract prepared by claim 1 were taken, and 200 g of golden hydrolyzed extract, 100 g of fermented green tea extract and 20 kg of aloe vera were uniformly mixed to obtain an aloe fermented and hydrolyzed extract complex.
Purified water 35kg, Glyceryl stearate 5kg, Fiji 100 stearate 3kg, Sorbitan stearate 4kg, Polysorbate20 3kg, Dimethicone 1kg, Triethanolamine 1kg, Whitebiswax 2kg, Carbomer941 1kg, Butylene glycol 0.5 kg, 1 kg of isododecane, 5 kg of glycerin, 1 kg of dimethylhexyl carbonate, 1 kg of dicapryl carbonate, 4 kg of stearyl alcohol, 1 kg of glyceryl behenate, 1 kg of phenoxyethanol, 1 kg of disteadimonium hectorite, sorbitan sesqui Oleate 2kg, methylparaben 0.3kg, propylparaben 0.2kg, disodium IDT 0.5kg and mixed with warming for 4 hours at 70 ~ 75 ℃ to prepare a facial cream base, the aloe fermentation and hydrolysis extraction Aloe fermented and hydrolyzed, characterized in that the homogenizer was added to the homogenizer together with the complex and homogenized at 3,000 to 3,500 ALPM for 30 minutes to prepare a facial cream. Cosmetics prepared using decomposed extract.
100 g of aloe fermented and hydrolyzed extract prepared by claim 1 were taken, and 200 g of golden hydrolyzed extract, 100 g of fermented green tea extract and 20 kg of aloe vera were uniformly mixed to obtain an aloe fermented and hydrolyzed extract complex.
Purified Water 40kg, Squalan 1kg, Cetiaryl Alcohol 1kg, Macadamia Seed Oil 1kg, Grape Seed Oil 0.5kg, Butylene Glycol 1kg, Polygtamic Acid 1kg, Glycerin 5kg, Diethylhexylcarbonate 1kg, Dicaprylcarbonate 0.5kg, Stearic 3 kg of acid, 1 kg of caprylic / capric triglycerides, 1 kg of stearyl alcohol, 0.5 kg of phenoxyethanol, 0.5 kg of green tea seed oil, 0.5 kg of arachadyl alcohol, 0.3 kg of behenyl alcohol, 0.3 kg of arachadyl glucoside, cetyl palmi Tate 0.2kg, Sorbitan olivate 0.5kg, Sorbitan palmitate 1kg, Cetearyl olivate 0.5kg, Fiji-32 0.2kg, Ceramide-3 0.3kg, Dimethicone 0.5kg, Allantoin 0.2kg, Carbomer 940 0.5 kg and 0.1 kg of triethanolamine are mixed and mixed with warming at 70-75 ° C. for 4 hours to prepare a body cream base, and then added to the homogenizer together with the aloe fermentation and hydrolysis extraction complexes. It was homogenized for 30 minutes, 3,000 to 3,500 rpm the cosmetics prepared by using the aloe extract fermentation and hydrolysis, characterized in that for producing the body cream.