KR20210150831A - Cosmetics using aloe extract effective for preventing atopic dermatitis - Google Patents

Abstract

The present invention relates to cosmetics using an aloe extract effective for preventing atopic dermatitis, and more particularly, to a method for preparing a fermented and hydrolyzed aloe extract with an effect of preventing and ameliorating atopic dermatitis and cosmetics prepared by using the same, with an aim at preparing a fermented extract capable of increasing skin penetration of active ingredients and antioxidant activity and enhancing anti-inflammatory and anti-allergic effects by extracting and fermenting aloe. To this end, the method for preparing a fermented aloe extract with an effect of preventing and ameliorating atopic dermatitis according to the present invention comprises the steps of: taking 1.0 kg of aloe shell, adding 2 liters (L) of ethanol (EtOH), and leaving at 50 to 60℃ for 8 hours while occasionally shaking; filtering the ethanol extracted solution under reduced pressure by using filter paper while shaking, and taking the filtrate; putting the filtrated liquid in a vacuum concentrator and concentrating under reduced pressure at 50 to 60℃ under constant conditions so as to make a soft extract; and dissolving 50,000 units of β-glucosidase in 10 milliliters of water, adding to the soft extract, and reacting at 40 to 50℃ for 8 hours while shaking so as to ferment and hydrolyze.

Description

Cosmetics using aloe extract effective for preventing atopic dermatitis

The present invention relates to a cosmetic using an aloe extract effective for preventing atopic dermatitis, and more particularly, by extracting and fermenting aloe to increase skin penetration and antioxidant activity of active ingredients, and to enhance anti-inflammatory and anti-allergic effects. , and relates to a method for producing a fermented and hydrolyzed aloe extract showing atopic dermatitis prevention and improvement effects for the purpose of developing cosmetics containing the same, and to cosmetics manufactured using the same.

Aloe is a perennial tropical plant of the genus Aloe belonging to the Liliaceae family. Aloe vera (A. barbadensis Mill.), Aloe arborescens Mill., Aloe saponaria Haw., etc. are mainly cultivated in Korea. Aloe has been used as a folk medicine for a long time and has been mainly used as a bitter taste and laxative. All have different degrees of action, but have anti-inflammatory action, anti-ulcer and cell regeneration action, antibacterial and antifungal action, antiviral action, blood sugar improvement action, gastric secretion inhibitory action, hepatocellular protection action, sacrificial action, immunomodulatory action, anticancer action, etc. Have.

The field where aloe is most widely used is as a cosmetic product. It has excellent skin moisturizing properties, and has excellent anti-inflammatory, anti-allergic, antioxidant and skin softening properties, so it is used in various layers from children to the elderly.

In particular, atopic dermatitis is a chronic disease, and since it shows a sensitive reaction depending on the type of cosmetics, research to use aloe extract for atopic dermatitis has been continuously conducted.

Atopic dermatitis (AD) is a chronic recurrent skin disease accompanied by pruritus, and it is known that about 50% of patients begin before 1 year of age, and about 30% develop between 1 and 5 years of age. Although the mechanism of atopic dermatitis has not been fully elucidated, susceptibility to viral infections such as herpes simplex or papillomatosis, decreased delayed immune response to specific microbial antigens, increased IgE production, decreased susceptibility to contact allergens, and fragmentation Various immunological abnormalities have been reported, such as decreased lymphocyte response to promoters and antigens, and decreased chemotaxis of granulocytes and monocytes. In addition, the prevalence of this disease and the progression to intractable adult atopic dermatitis are gradually increasing in the industrialized society, which is the basis for the importance of environmental factors such as industrialization as well as genetic factors in the occurrence of atopic dermatitis. It is known that symptoms such as pruritus, itchiness, lichenification, rash, scale, and body dryness are characteristic of this disease.

For the treatment of atopic dermatitis, basic methods for dermatitis and methods to identify and remove triggers are applied. 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 relieve symptoms quickly and effectively when the lesion is acutely aggravated, but when used continuously, it can cause adrenal suppression, skin atrophy, capillary dilatation, reduced pigmentation, steroid acne, hirsutism, and growth disorders. There are concerns about such as short-term use and limited use for sensitive skin such as areas with thin epidermis or a lot of blood vessels.

In the case of calcineurin inhibitors, they can be used as an adjunct to existing treatments and minimize the problems that may arise from the use of topical steroids. It can be applied, but it is approved and used only for short-term use in children over 2 years of age as a second-line treatment.

It can be seen that it is necessary to develop cosmetics that help prevent and improve atopy from a long-term perspective while using drugs for atopy treatment.

The present invention has been devised to solve these problems, and an object of the present invention relates to the development of cosmetics that help prevent and improve atopic dermatitis when used for a long period of time without showing side effects on atopic skin, skin allergies, pruritus, In order to protect, prevent and improve damaged skin from contact dermatitis, skin inflammation caused by microorganisms, skin rash and atopic diseases, enzymes are added to aloe extract to ferment and hydrolyze, and golden fermented extract, fermented eosin-chief extract, etc. The purpose is to develop various types of cosmetics such as body lotion, body cleanser, facial cream and body cream by combining them.

In the method for producing a fermented aloe extract exhibiting an effect of preventing and improving atopic dermatitis according to the present invention for achieving the above object, 1.0 kg of aloe peel is taken and 2 liters (L) of ethanol (EtOH) is added, followed by 8 at 50 to 60 ° C. A step of leaving it to stand while shaking occasionally for a period of time;

Filtering under reduced pressure using filter paper while shaking the ethanol extraction solution, and taking the filtrate;

Putting the filtrated liquid in a vacuum concentrator and concentrating under a reduced pressure at 50 ~ 60 ℃ under constant conditions to make a soft joe extract;

Dissolving 50,000 units of beta-glucosidase in 10 milliliters of water, adding it to soft extract, and shaking it for 8 hours at 40-50° C.

In addition, cosmetics produced using the fermented aloe extract according to the present invention take 100 g of the aloe fermented and hydrolyzed extract, and uniformly mix 200 g of hydrolyzed golden extract, 100 g of fermented eoseongcho ferment extract and 20 kg of aloe vera to ferment aloe and After obtaining the hydrolysis-extraction complex, purified water 40 kg, glyceryl stearate 6 kg, PEG 100 stearate 4 kg, sorbitan stearate 3 kg, polysorbate 20 3 kg, dimethicone 0.5 kg, triethanolamine 1 kg, caprylic caprylic triglyceride ceride 4kg, carbomer 941 1kg, butylene glycol 1kg, isododecane 0.5kg, glycerin 5kg, diethylhexyl carbonate 3kg, dicapryl carbonate 3kg, stearyl alcohol 5kg, glyceryl behenate 1kg, phenoxyethanol 0.5kg , Disteadimonium hectorite 1kg, sorbitan sesquioleate 3kg, methylparaben 0.3kg, propylparaben 0.2kg, disodiumEDTA 0.5kg, mixed and heated at 70 ~ 75℃ for 4 hours After preparing the lotion base, it is introduced into a homogenizer together with the aloe hydrolysis extraction mixture and homogenized at 3,000 to 3,500 RPM for 30 minutes to prepare a body lotion.

In addition, 100 g of the aloe fermented and hydrolyzed extract was taken, and 200 g of golden hydrolyzed extract, 100 g of fermented fish extract and 20 kg of aloe vera were uniformly mixed to obtain an aloe fermented and hydrolyzed extract complex, followed by purified water 50 kg, lauric acid 6 kg , myristic acid 4kg, monoalkylphosphate 1kg, oxybenzone 0.5kg, glycol distearate 4kg, triethanolamine 1kg, polyquatenium 1kg, laurylsulfonic acid triethanolamine 1kg, lauramide DEA 1kg, cyclomethicone 0.5kg , cocamide propylbetaine 2kg, K-Quyl hydrolyzed collagen 1kg, butylene glycol 1kg, PEG-7-glyceryl cocoate 5kg, glycerin 5kg, glyceryl behenate 1kg, phenoxyethanol 1kg, dimethicone 0.5 kg, sorbitan sesquioleate 3kg, propylene carbonate 1kg, methylparaben 0.3kg, propylparaben 0.2kg, disodium EDTA 0.5kg, mixed while heating at 70 ~ 75℃ for 4 hours to form a body cleanser base After preparing the aloe fermentation and hydrolysis extraction complex, it is put into a homogenizer and homogenized at 3,000 to 3,500 RPM for 30 minutes to prepare a body cleanser.

In addition, 100 g of the aloe fermented and hydrolyzed extract was taken, and 200 g of golden hydrolyzed extract, 100 g of fermented eoseongcho ferment extract and 20 kg of aloe vera were uniformly mixed to obtain an aloe fermented and hydrolyzed extract, followed by purified water 35 kg, glyceryl stearate 5kg, PEG 100 Stearate 3kg, Sorbitan Stearate 4kg, Polysorbate 20 3kg, Dimethicone 1kg, Triethanolamine 1kg, White Beeswax 2kg, Carbomer 941 1kg, Butylene Glycol 0.5kg, Isododecane 1kg , glycerin 5kg, diethylhexyl carbonate 1kg, dicapryl carbonate 1kg, stearyl alcohol 4kg, glyceryl behenate 1kg, phenoxyethanol 1kg, disteadimonium hectorite 1kg, sorbitan sesquioleate 2kg, methylparaben 0.3 kg, 0.2 kg of propylparaben, and 0.5 kg of disodium EDTA were mixed and mixed while heating at 70 to 75° C. for 4 hours to prepare a facial cream base, and then to the homogenizer together with the aloe fermentation and hydrolysis extraction complex It is characterized in that it is added and homogenized for 30 minutes at 3,000 to 3,500 RPM to prepare 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 fish extract and 20 kg of aloe vera were uniformly mixed to obtain an aloe fermentation and hydrolysis extraction complex, and then purified water 40 kg, squalane 1 kg, ceti Aryl alcohol 1kg, macadamia seed oil 1kg, grape seed oil 0.5kg, butylene glycol 1kg, polyglutamic acid 1kg, glycerin 5kg, diethylhexyl carbonate 1kg, dicapryl carbonate 0.5kg, stearic acid 3kg, caprylic/ Capric triglyceride 1kg, stearyl alcohol 1kg, phenoxyethanol 0.5kg, green tea seed oil 0.5kg, aracadyl alcohol 0.4kg, behenyl alcohol 0.3kg, aracadyl glucoside 0.3kg, cetyl palmitate 0.2kg, sorbitanol Bate 0.5kg, Sorbitan Palmitate 1kg, Cetearyl Oliveate 0.5kg, PEG-32 0.2kg, Ceramide-3 0.3kg, Dimethicone 0.5kg, Allantoin 0.2kg, Carbomer 940 0.5kg and Triethanolamine 0.1kg After preparing the body cream base by mixing while warming at 70 to 75° C. for 4 hours, it is put into a homogenizer together with the aloe fermentation and hydrolysis extraction complex, and homogenized with 3,000 to 3,500 RPM for 30 minutes to create a body cream characterized in that it is manufactured.

Other objects and effects of the present invention will become apparent from the following detailed description, and the detailed description and examples indicating preferred embodiments of the present invention do not limit the scope of the present invention.

According to the cosmetic using the aloe extract effective for preventing atopic dermatitis according to the present invention, in order to protect, prevent and improve damaged skin from contact dermatitis, skin inflammation caused by microorganisms, skin allergy, pruritus, skin rash, and atopic diseases, aloe As a result of developing various types of cosmetics such as body lotion, body cleanser, facial cream, and body cream by combining fermented and hydrolyzed extract with golden hydrolyzed extract, eoseongcho bee extract, and aloe vera, it has strong penetration into the skin and high antioxidant activity. It has high antibacterial activity against Gram-positive and negative bacteria and shows good prevention and improvement effects on atopy in mice 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 applied as a main ingredient in the present invention is a perennial tropical plant of the genus Aloe belonging to the Liliaceae family.

Aloe vera (A. barbadensis Mill.), Aloe arborescens Mill., Aloe saponaria Haw., etc. are mainly cultivated in Korea.

Aloe has been used as a folk medicine for a long time and has been mainly used as a bitter taste and laxative. All have different degrees of action, but have anti-inflammatory action, anti-ulcer and cell regeneration action, antibacterial and antifungal action, antiviral action, blood sugar improvement action, gastric secretion inhibitory action, hepatocellular protection action, sacrificial action, immunomodulatory action, anticancer action, etc. It is known to have

As active ingredients contained in aloe, barbaloin, aloesin, aloenin, isobarbaloin, isoaloeresin, and aloeemodin have been reported.

A study on the pharmacological activity of aloe vera extract showed that aloe extract showed a distinct inhibitory effect on carrageenan-induced foot edema in rats and inhibited the production of _{PGE 2 in vitro.} As a study on the anti-inflammatory effect of aloe vera gel on the human rectal mucosa, it _{is reported that when aloe is administered, it suppresses the production of PGE 2} and thus exhibits an anti-inflammatory effect.

As a study on the immunomodulatory effect of Aloe vera gel on mice, it has been reported that when Aloe vera extract is administered, it promotes antibody production and increases the number of plaque forming cells. Immunomodulation of polysaccharides contained in Aloe vera In a study on the chemical properties of aloe, the structural characteristics of polysaccharide components contained in aloe were identified through enzymatic and acid hydrolysis.

Barbaloin, aloesin, aloenin, aloe-emodin, which are known as active ingredients of aloe, are mainly contained in the bark of the aloe plant. Aloe vera was mixed here.

Golden (Scutellaria Radix), which is contained as a complex ingredient together with aloe, is the root of Scutellaria baicalensis GEORGI, a perennial herbaceous plant belonging to the Lamiaceae family. It has been used for antibacterial, diuretic, laxative, atherosclerotic prevention, gastric juice suppression, sedation, and blood pressure lowering effects.

The active ingredients of gold are baicalin, baicalein, and wogonine, etc., and the study on the pharmacological action shows that by inhibiting lipid oxidation in the liver of rats, It is known that the content is reduced and is effective in hyperlipidemia induced by ethanol (EtOH) ingestion. In addition, the antioxidant action of golden ingredient protects the skin from cellular damage caused by oxidation and protects the skin from free radicals and free radicals. It has been reported to prevent cell oxidation and damage.

Baicalin, the main component of gold, has a flavonoid structure and has various biological activities such as anti-allergy, antibacterial and antiviral, and in addition, inhibition of lipoxygenase, inhibition of microsome lipid peroxidation, Antioxidant activity such as inhibition of oxygen radical production, and inhibition of fibroblast damage induced by _{reactive oxygen species (H 2} O ₂ , —OH and O ₂ ^{—) has recently been reported.}

Baicalin, which has the highest content in gold, is hydrolyzed by beta-glucuronidase secreted by E. coli in humans and animals to produce baicalin, with antioxidant activity, anti-inflammatory effect, and anti-inflammatory properties. It has been reported that the allergy effect and the skin damage suppression effect are remarkably enhanced.

In addition, Houttuynia cordata is a perennial herbaceous plant belonging to the Saururaceae family. The stem is thin and reddish-purple, the leaves are heart-shaped and pointed, and the leaves are similar to sweet potatoes or buckwheat leaves, and the juice of the leaves and stems. It has a smell very similar to the “fishy smell” of fish, so it was called Eoseongcho (魚腥草). Eoseongcho has diuretic, analgesic, hemostasis, tissue regeneration, vasodilation and antidiarrheal action, and in the herbal remedies, donguibogam, botanical dictionary, it is said that it is effective in diarrhoea, syphilis, boil, white poison, hemorrhoids, prolapse and eliminates heavy metal poison.

As a result of a study on the separation and utilization technology of functional ingredients to improve medicinal properties and edibility of eoseongcho, quercetin, quercitrin, rutin and myricetin were identified as flavonoids of eoseongcho, and among these ingredients, quercetin is known to be contained in onions.

As a result of a study on the structural characteristics of quercetin and quercetin glycosides contained in Ewoongcho and onion, these phenolic compounds reported pharmacological actions such as reduction of carcinogenic activity, inhibition of growth of mutant cancer cells, lowering of blood pressure, and strengthening of capillaries. , quercetin has known pharmacological actions such as inhibition of lipid peroxide formation, antiviral, antibacterial, and antimutagenic action.

Accordingly, the present invention provides anti-inflammatory, anti-allergic, pruritus suppression, contact dermatitis improvement and anti-atopic dermatitis along with antioxidant action by combining golden hydrolyzed extract, fermented fish extract and aloe vera with aloe fermented and hydrolyzed extract as the main component of cosmetic preparations. It can enhance the effect, does not irritate or damage the skin of children and infants, and by using a safe, non-toxic and less irritating base, it does not show any rejection reaction even for women and sensitive skin, and it does not cause skin aging while maintaining skin moisture and flexibility. We developed formulations such as body lotion, body cleanser, facial cream and body lotion by formulating a prescription that can delay

The features of the present invention for achieving the above object are as follows, and the composition ratio corresponds to the present invention, and the composition amount can be implemented in the range of ± 10%.

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

1. Preparation of aloe hydrolyzate hydrogel

After 1 kg of aloe was incised, the skin and yellow endothelial secretions were taken, air-dried at 50°C for 3 days, then 600 ml of ethanol was added, extracted with shaking in a water bath at 60°C for 6 hours, and filtered. The filtrate was concentrated under reduced pressure and then freeze-dried to obtain a brown powder of aloe (yield: 15.4%), which was used as a sample.

For the preparation of aloe hydrogel, first, aloe extract powder is dissolved in purified water, propylene glycol, labrosol and ethanol are sequentially added and mixed, and then Carbopol 940 is added to swell and triethanolamine ( triethanolamine) to prepare a hydrogel by adjusting the pH to 6.5.

For the preparation of aloe hydrolyzed hydrogel (hereinafter ALH gel), aloe extract powder was dissolved in purified water, 1,000 units and 5,000 units of beta-glucosidase were added, respectively, and reacted at 50°C for 4 hours. ALH) 1% and aloe hydrolyzate (ALH) 5% (see Table 1).

Table 1. Preparation method of aloe hydrolyzate hydrogel

2. Dermatitis induction and sample processing using house dust mite (DPE)

To induce atopic dermatitis, the neck, ears, and back of 8-week-old Nc/Nga mice were cleaned of hair removal and then left for 24 hours to heal micro-wounds on the skin after hair removal was finished, and 4% SDS solution was sprayed onto the hair removal area. Then, make a culture solution with 0.5% Tween 20 and phosphate buffer solution (PBS), and add a 1% solution prepared by adding house dust mite extract (D. Atopic dermatitis was induced by applying 50 μl each for 8 weeks. Physiological saline was applied to the control group, and 10 μg of cyclosporin A was administered intravenously once a day for 8 weeks. It was applied to the skin.

3. Immune Cell Count Measurement

Take a certain amount of skin tissue from the back of a mouse induced by dermatitis by applying weekly house dust mite (DPE), cut it into small pieces, add collagenase 1 mg/ml (in 2% FBS + RPMI1640), and shake at 37°C (180 rpm, 20 min) The method of recovering the supernatant after culturing in an incubator was repeated 4 times. The cultured supernatant was treated with ACK solution (8.3 g NH ₄ Cl, 1 g KHCO ₃ in 1 ℓ of demineralized water + 0.1 mM EDTA) at room temperature for 5 minutes to lyse red blood cells and washed twice with D-PBS again. After staining with 0.04% trypan blue, the number of cells was measured. After adjusting the measured skin tissue-infiltrating cells to 5 × 10 ⁵ cells, immunofluorescence staining was performed at 4°C. PEanti-CD3e, FITC-anti-CD69, FITC-anti-CCR3, PE-anti-Gr-1, FITC-anti-CD11b, PE-anti-Gr-1, PE-anti-B220 and FITC-anti- IgE was added and reacted on ice for 30 minutes. After the reaction, after washing with phosphate buffered saline at least 3 times, CD3 ⁺ /CD69 ⁺ , CCR3 ⁺ , B220 ⁺ /IgE ⁺ , CD11b ⁺ /Gr-1 ⁺ After analyzing the cell number as a percentage (%), the absolute cell number in each tissue was calculated by applying the total cell number.

As a result of observing the number of immune cells, the total number of immune cells in the normal group was 29 ± 2.5 (×10 ⁴ /g), 119.5 ± 23.5 (×10 ⁴ /g) in the control group, and 52.0 ± 5.0 (×10 ⁴ /g) in the CsA group. g), ALH 5% administration group 56.0 ± 7.0 (×10 ⁴ /g), ALH 1% administration group 55 ± 8.0 (×10 ⁴ /g) showed a significant reduction effect 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-administered group, The ALH 5% administration group showed 7.5 ± 2.9 (×10 ⁴ /g) and the ALH 1% administration group 5.9 ± 3.5 (×10 ⁴ /g), indicating a significant reduction effect compared to the control 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), 4.4 ± 1.1 (×10 ⁴ /g) of the 1% ALH group, indicating a significant reduction effect compared to the control group.

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

The number of B220 ⁺ /IgE ⁺ cells was 0.6 ± 0.3 (×10 ⁴ /g) in the normal group, 6.4 ± 0.5 (×10 ⁴ /g) in the control group, 3.3 ± 0.2 (×10 ⁴ /g) in the CsA group, and ALH 5% It was found to be 2.4 ± 0.9 (×10 ⁴ /g) in the administration group and 1.9 ± 1.2 (×10 ⁴ /g) in the ALH 1% administration group, indicating a significant reduction effect compared to the control group.

Table 2. Number of Nc/Nga Mouse Skin Immune Cells 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 ± S.E. (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 the analysis of inflammatory cytokines, Nc/Nga mice that had atopic dermatitis-like skin induced by house dust mite (DPE) were anesthetized with ether after the experiment was completed, blood was collected by cardiac puncture, and serum was separated. IL-6 and TNF-γ concentrations in the isolated serum were measured with an enzyme-linked immuno-sorbent assay (ELISA, Biosource, USA). 100 μl (1/100 dilution) of Nc/Nga mouse serum was dispensed into each well, and after reacting at room temperature for 1 hour, it was washed twice with a washing buffer. 100 μl of HRP-conjugeted Avidin was treated, reacted at room temperature for 1 hour, and washed again. 100 μl of the TMB substrate was dispensed and reacted in the dark for 30 minutes, then 50 μl of stop solution was treated, and absorbance was measured at 450 nm using an ELISA reader.

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

As a result of measuring the amount of cytokine production in the serum, the IL-6 production amount 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 group, and 5% of ALH. The administration group was 146.8 ± 4.23 (pg/ml) and the ALH 1% administration group was 156.25 ± 2.2 (pg/ml), which showed a significant reduction effect compared to the control group.

The amount of TNF-α production in the blood 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/ml) of the ALH 1% administration group showed a significant reduction effect compared to the control group (see Table 3).

Table 3. Cytokine production in Nc/Nga mouse serum and spleen induced by house 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 ± S.E. (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. Measurement of Immunoglobulin in Serum

Immunoglobulin in serum was measured at 8, 12, and 16 weeks of sample administration, about 100 μl of blood was collected from the orbital artery of the mouse using a capillary tube, and then centrifuged at 6,500 rpm in a centrifuge for 20 minutes. After that, 30 μl of serum was isolated. IgE in serum was measured using an ELISA kit. The antibody was diluted with a buffer solution, coated on a 96-well plate, and left at 4°C for 16 hours. After washing each well with a buffer solution 3 times, 100 μl of serum (100-fold dilution) was dispensed. This was reacted at room temperature for 1 hour, washed twice with a buffer solution, treated with 100 μl of HRPconjugated Avidin, reacted at room temperature for 1 hour, and washed again. Here, 100 μl of TMB substrate was dispensed and reacted in the dark for 30 minutes, then 50 μl of stop solution was treated, and absorbance was measured at 450 nm using an ELISA reader.

Serum concentrations 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, left in a refrigerator at 4° C. for 12 hours, washed twice with buffer solution, and then added with biotin-conjugated antibody and reacted for 30 minutes. After washing twice again with buffer solution, 100 μl of HRP-conjugeted Avidin was treated, reacted for 1 hour at room temperature, and washed again. 100 μl of TMB substrate was dispensed and reacted in the dark for 30 minutes, then 100 μl of stop solution was treated, and absorbance was measured at 450 nm using an ELISA reader.

For measurement of serum IgE production, blood was collected from the eyes of Nc/Nga mice at 8 weeks, 12 weeks, and 16 weeks, and serum was separated, and then the IgE production amount was measured. At 8 weeks, due to the change in serum IgE level, in the normal group, 492.5 ± 57.8 (ng/ml), in the control group, 855.5± 68.5 (ng/ml), in the CsA group, 915.5 ± 131.0 (ng/ml), and in the ALH 5% group, 947.5 There was no significant difference as ± 33.8 (ng/ml) and 709.0 ± 83.9 (ng/ml) in the ALH 1% group, 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 administration group, 3,117.9 ± 82.9 (ng/ml) in the ALH 5% administration group, and 3,180.9 ± 5.4 (ng/ml) in the ALH 1% administration group. and the effect lasted up to 16 weeks.

The serum IgG1 production amount was 2,645.0 ± 85.2 (μg/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% administration group did not affect the production amount as 3,706.0 ± 51.1 (㎍ / ㎖).

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

The IgG2a blood concentration was 1,967.5 ± 55.9 (μg/ml) in the normal group, 2,694.5 ± 33.8 (μg/ml) in the control group, 1,473.0 ± 70.1 (μg/ml) in the CsA group, and 2,283.5 ± 150.6 (μg/ml) in the ALH 5% group. ㎖), the ALH 1% administration group showed a significant reduction effect compared to the control group to 2,153.0 ± 118.7 (㎍ / ㎖).

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

Table 4. Immune Antibody Production Amount in Nc/Nga Mouse Serum Induced by House 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 ± S.E. (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. Measurement of antibacterial activity against gram-positive and gram-negative bacteria of aloe and golden complex hydrogel

First, the turbidity of the nutrient agar (Nutrient agar) and citrate disoxycholine agar (Desoxycholate citrate agar) culture medium was adjusted to be the same as the standard turbidity standard (Tubidity Standard, Transmittance 35%) as the test bacteria solution.

As a sample solution, aloe and golden complex hydrogels were dissolved by heating in a water bath, passed through a membrane filter 0.2 μm, and then diluted two-fold to 200, 100, 50, 25, 12.5 and 6.25 μg/ml in stages. After mixing 2 ml each with 18 ml of Mueller Hinton agar medium and hardening, inoculate 5 μl of each test bacterial solution with an automatic dispenser and incubate at 37° C. for 18 hours. was observed.

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

When comparing the bacterial growth inhibition rate against gram-positive and negative bacteria of aloe and gold complex hydrogel, the golden extract showed relatively high antibacterial activity against gram-positive bacteria such as Staphylococcus aureus and Staphylococcus epidermis, and showed a low level against gram-negative bacteria. On the other hand, the antibacterial activity of the hydrogel showed antibacterial activity against both Gram-positive and negative bacteria, but the growth inhibition rate was low (see Table 5).

Table 5. Antibacterial effect (%) of aloe and gold complex hydrogel against gram-positive and gram-negative bacteria

7. Results of atopic dermatitis induction and sample treatment and observation of inhibitory effect on atopic dermatitis induced in NC/Nga mice

First, the neck, ears, and back of the 8-week-old NC/Nga mice were cleaned of hair removal and then left for 24 hours to heal the micro-wounds on the skin when the hair removal was finished.

And after spraying 4% SDS solution on the hair removal area, 0.5% Tween 20 and PBS were used to make a culture medium, and the house dust mite (D. pteronyssinus) extract (DPE, Yonsei University Department of Microbiology, Seoul) antigen was added to Younggi. 10 mg/ml, 50 μl was applied 3 times a week for 8 weeks, and 5% and 1% of ALH in the experimental group were administered at appropriate concentrations for 8 weeks (8 weeks to 16 weeks).

The mice induced by dermatitis by applying house dust mite (DPE) were divided into three groups, with physiological saline in the control group, cyclosporin A in the positive control group, and 5% and 1% concentration of golden hydrolyzate in the experimental group. was applied to the skin for 8 weeks (8 weeks to 16 weeks). At 4 week intervals of drug treatment, sensory evaluation was performed. For the dermatitis of NC/Nga mice, the Wilcoxon method commonly used in atopic dermatitis was used.

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

Referring to Figure 1, when observing the inhibitory effect of atopic dermatitis induced in NC/Nga mice, Figure 1A is a photograph of the back of an 8-week-old NC/Nga mouse, and Figure 1B is a photo of the back region of NC/Nga mice after hair removal for 8 weeks. In the post photograph, keratinization progressed and skin inflammation was severe. In contrast, CSA (Fig. 1C), a positive control, was injected intraperitoneally and showed significant improvement. As a result of applying the gold hydrolyzate, which is the experimental group, to the skin for 8 weeks (Fig. 1D), it was confirmed that the scratching behavior was relatively reduced compared to the control group, and also keratosis, lichenification, skin exfoliation symptoms, and dermatitis symptoms were significantly improved.

Figure 1. Therapeutic effect of aloe and gold complex on atopic dermatitis in NC/Nga mice induced by dust mites.

In addition, referring to Table 6, as a result of measuring the severity of dermatitis every 4 weeks by a sensory method, the control group was 2.1 ± 0.4 at 8 weeks, 2.5 ± 0.16 at 12 weeks, 2.6 ± 0.2 at 16 weeks, and CsA at 8 weeks 1.8. ± 0.41, 12 weeks 1.7 ± 0.25, 16 weeks 0.9 ± 0.4, 5% golden hydrolyzate is 8 weeks 1.5 ± 0.16, 12 weeks 1.7 ± 0.3, 16 weeks 0.8 ± 0.1, 1% golden hydrolyzate is 8 weeks 1.4 ± 0.24 , showed a significant decrease from week 12 to 2.0 ± 0.35 at 12 weeks and 1.6 ± 0.4 at 16 weeks.

Table 6. Visual evaluation index of aloe and gold complex for atopic dermatitis in NC/Nga mice induced by 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 gruup by t-test (***p<0001)

8. Atopic dermatitis induction and sample processing and microscopic observation of skin and ears of NC/Nga mice

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

The tissue was formatted with paraffin, and blocks were made with a thickness of 5 μm. Tissue sections are epidermis, dermis, keratinocytes, neutrophils, eosinophils, and other cells that cause inflammation and hematoxylin and eosin to identify edema (hematoxyline and eosin, H&E) staining and toluidine staining to stain mast cells were performed.

Immunohistochemical staining (immuno histochemical stain) made a block (block), which included rat antimouse CD4 mAb (RM4-5; PharMingen, San Diego, CA), rat anti-mouse (rat anti-mouse) CCR3 mAb (53-6.7; Becton Dickinson, Mountain View, CA) antibody was used.

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

And after deparaffinization (Deparaffinized), it was pretreated in a microwave oven (microwave oven) for 15 minutes using 0.01M citrate buffer (citrate buffer pH 6.0).

After treatment in 3% hydrogen peroxidase for 10 minutes to inhibit the action of peroxidase in tissues, proteins were blocked with normal serum in order to inhibit non-specific protein binding to antigens in tissues. Then, it was attached to the primary single antibody for 1 hour and then washed with a buffer solution.

After the end of the experiment, part of the skin and ears of the mice were cut, and the general shape of the tissues and the thickness of the ears were observed through hematoxylin and eosin and toluidine blue staining. As a result, the H&E-stained control group In the skin and ears of (Fig. 2B), the epidermis/dermis was markedly enlarged with edema, and infiltration of leukocytes was also observed.

On the other hand, in the group administered with aloe gold complex (Fig. 2D), the thickness of the epidermis was relatively reduced compared to the control group, and the infiltration of leukocytes was significantly reduced, thereby reducing edema.

In the toluidine staining that stains mast cells, it can be seen that the control group (Fig. 2F) had a lot of infiltrated mast cells (arrows) around the dermis.

However, almost no mast cells were observed in the Aloe golden complex administered group (Fig. 2H) compared to the control group, as shown in the photo.

In addition, as a result of measuring the thickness of ear tissue through a fossil decomposition device under a 400x optical microscope, the normal group was 21.1 ± 1.4 (x10/μm), the control group was 129.5 ± 2.5 (x10/μm), and CsA was 57.4 ± 7.3 ( x10/μm), the group administered with aloe gold complex 5% showed 37.1 ± 0.3 (x10/μm), and the group administered with 1% aloe gold complex showed 62.2 ± 1.5 (x10/μm), indicating that the ear thickness was significant in the group administered with aloe gold complex compared to the control group. appeared to decrease negatively. (See Table 7)

Figure 2. Microscopic observation 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. Comparison of ear thickness in 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 ofCsA, and Scutellariae extracts for 8 weeks (Nikon, Japan, orignal magnification, ear x 400)

Claims (5)

Taking 1.0 kg of aloe shell, adding 2 liters (L) of ethanol (EtOH), and leaving it at 50 to 60° C. for 8 hours while shaking occasionally;
Filtering the ethanol extraction solution under reduced pressure using filter paper while shaking, and taking the filtrate;
Putting the filtered solution into a vacuum concentrator and concentrating under a reduced pressure at 50 to 60° C. under constant conditions to make a soft extract;
Dissolving 50,000 units of beta-glucosidase (β-glucosidase) in 10 milliliters of water, adding it to the yeonjo extract, and reacting it with shaking at 40 to 50° C. for 8 hours to fertilize and hydrolyze; A method for producing fermented and hydrolyzed aloe extract having atopic dermatitis prevention and improvement effect, comprising: Take 100 g of the aloe fermentation and hydrolysis extract prepared according to claim 1, and uniformly mix 200 g of golden hydrolyzed extract, 100 g of fermented eoseongcho ferment extract and 20 kg of aloe vera to obtain an aloe fermentation and hydrolysis extraction complex,
Purified water 40kg, glyceryl stearate 6kg, PEG 100 stearate 4kg, sorbitan stearate 3kg, polysorbate 20 3kg, dimethicone 0.5kg, triethanolamine 1kg, caprylic caprylic triglyceride 4kg, carbomer 941 1kg, Butylene glycol 1kg, isododecane 0.5kg, glycerin 5kg, diethylhexyl carbonate 3kg, dicapryl carbonate 3kg, stearyl alcohol 5kg, glyceryl behenate 1kg, phenoxyethanol 0.5kg, disteadimonium hectorite 1kg , 3 kg of sorbitan sesquioleate, 0.3 kg of methylparaben, 0.2 kg of propylparaben, and 0.5 kg of disodium EDTA were mixed and heated at 70 to 75° C. for 4 hours to prepare a body lotion base, and then the aloe water Cosmetics manufactured using aloe fermentation and hydrolyzed extract, characterized in that it is put into a homogenizer together with the decomposed extraction mixture and homogenized at 3,000 to 3,500 RPM for 30 minutes to prepare a body lotion. Take 100 g of the aloe fermentation and hydrolysis extract prepared according to claim 1, and uniformly mix 200 g of golden hydrolyzed extract, 100 g of fermented eoseongcho ferment extract and 20 kg of aloe vera to obtain an aloe fermentation and hydrolysis extraction complex,
Purified water 50kg, lauric acid 6kg, myristic acid 4kg, monoalkyl phosphate 1kg, oxybenzone 0.5kg, glycol distearate 4kg, triethanolamine 1kg, polyquatenium 1kg, laurylsulfonic acid triethanolamine 1kg, lauramide DEA 1 kg, cyclomethicone 0.5 kg, cocamide propyl betaine 2 kg, K-Quyl hydrolyzed collagen 1 kg, butylene glycol 1 kg, PEG-7-glyceryl cocoate 5 kg, glycerin 5 kg, glyceryl behenate 1 kg, phenoxy Siethanol 1kg, dimethicone 0.5kg, sorbitan sesquioleate 3kg, propylene carbonate 1kg, methylparaben 0.3kg, propylparaben 0.2kg, disodium EDTA 0.5kg were mixed, and at 70 ~ 75℃ for 4 hours. Aloe fermentation and hydrolysis, characterized in that after preparing a body cleanser base by mixing while warming, it is put into a homogenizer together with the aloe fermentation and hydrolysis extraction complex and homogenized at 3,000 to 3,500 rpm for 30 minutes to prepare a body cleanser Cosmetics manufactured using extracts. Take 100 g of the aloe fermentation and hydrolysis extract prepared according to claim 1, and uniformly mix 200 g of golden hydrolyzed extract, 100 g of fermented eoseongcho ferment extract and 20 kg of aloe vera to obtain an aloe fermentation and hydrolysis extraction complex,
Purified water 35kg, glyceryl stearate 5kg, PEG 100 stearate 3kg, sorbitan stearate 4kg, polysorbate 20 3kg, dimethicone 1kg, triethanolamine 1kg, white beeswax 2kg, carbomer 941 1kg, butylene glycol 0.5kg, isododecane 1kg, glycerin 5kg, diethylhexyl carbonate 1kg, dicapryl carbonate 1kg, stearyl alcohol 4kg, glyceryl behenate 1kg, phenoxyethanol 1kg, disteadimonium hectorite 1kg, sorbitan sesqui 2 kg of oleate, 0.3 kg of methylparaben, 0.2 kg of propylparaben, and 0.5 kg of disodium EDTA were mixed and heated at 70 to 75° C. for 4 hours to prepare a facial cream base, followed by fermentation and hydrolysis extraction of the aloe Cosmetics manufactured using aloe fermented and hydrolyzed extract, characterized in that it is put into a homogenizer together with the complex and homogenized at 3,000 to 3,500 RPM for 30 minutes to prepare a facial cream. Take 100 g of the aloe fermentation and hydrolysis extract prepared according to claim 1, and uniformly mix 200 g of golden hydrolyzed extract, 100 g of fermented eoseongcho ferment extract and 20 kg of aloe vera to obtain an aloe fermentation and hydrolysis extraction complex,
Purified water 40kg, squalane 1kg, cetiaryl alcohol 1kg, macadamia seed oil 1kg, grape seed oil 0.5kg, butylene glycol 1kg, polyglutamic acid 1kg, glycerin 5kg, diethylhexyl carbonate 1kg, dicapryl carbonate 0.5kg, stearic Acid 3kg, caprylic/capric triglyceride 1kg, stearyl alcohol 1kg, phenoxyethanol 0.5kg, green tea seed oil 0.5kg, aracadyl alcohol 0.4kg, behenyl alcohol 0.3kg, aracadyl glucoside 0.3kg, cetylpalmi Tate 0.2kg, sorbitan olivate 0.5kg, sorbitan palmitate 1kg, cetearyl olivate 0.5kg, PEG-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 heated at 70 to 75° C. for 4 hours to prepare a body cream base, and then added to a homogenizer together with the aloe fermentation and hydrolysis extraction complex to 3,000 to 3,500 RPM Cosmetics manufactured using aloe fermented and hydrolyzed extract, characterized in that the body cream is prepared by homogenizing for minutes.