KR20100119729A - Method of extracting alginic acid and therapeutic composition for atopy using thereof - Google Patents

Abstract

PURPOSE: A composition containing alginic acid oligomer for treating atopic dermatitis is provided to improve atopic dermatitis treatment without toxicity. CONSTITUTION: A composition for treating atopic dermatitis contains 10-20 weight parts of alginic acid oligomer, 15-25 weight parts of tangerine peel extract, 3-7 weight parts of noni extract and 5-15 weight parts of Orostachys Herba extract as an active ingredient. The composition has emulsion form containing aqueous layer and oil layer. The oil layer contains jojoba oil, German Rampion oil, or shea butter. The composition further contains emulsifier or tackifier.

Description

Alginate extraction method and atopic therapeutic composition using the alginic acid oligomer TECHNICAL FIELD

The present invention relates to atopic treatment compositions comprising alginic acid extraction methods and alginic acid oligomers.

In general, atopy is a biological response that occurs as a result of the production of IgE antibodies and refers to an allergic reaction in which the body becomes extremely sensitive without direct contact with allergens. Symptoms of atopic dermatitis include atopic dermatitis, allergic conjunctivitis, allergic rhinitis, and asthma, and the onset of these symptoms has a large genetic impact.

Most current treatments for atopic dermatitis treat the allergic reaction in a manner that inhibits the production of IgE antibodies using artificially made chemicals.

However, modern people prefer so-called eco-friendly products using natural plants rather than products using artificially made chemicals. In addition, as the side effects of artificial chemicals are highlighted, many studies are being conducted in the industry to develop products using natural ingredients extracted from natural plants.

One technical problem to be achieved by the embodiments of the present invention is to provide an atopic therapeutic composition.

Another technical problem of the present invention is to provide a method for extracting alginic acid from seaweed.

Another technical problem to be achieved by the embodiments of the present invention is to provide a method for preparing an atopic therapeutic composition comprising the alginic acid oligomer derived from the alginic acid.

Atopic therapeutic compositions for achieving the above object include alginic acid oligomers, tangerine peel extracts, noni extracts, vortex extracts as active ingredients.

When the total weight of the atopy treatment composition is 100 parts by weight, the alginic acid oligomer may be 10 to 20 parts by weight, 15 to 25 parts by weight of the tangerine extract, 5 to 15 parts by weight of the Wasong extract, the noni extract It may be 3 to 7 parts by weight. The atopic therapeutic composition may further comprise aloe extract, fish extract, glycerin, silk amino acid, menthol in addition to the above components.

The atopy treatment composition is in an emulsion form including an aqueous layer and an oily layer, and the tangerine peel extract, the noni extract, and the turmeric extract are dissolved in the aqueous layer. The oily layer may include at least one of jojoba oil, evening primrose oil, camellia oil, and shea butter.

The atopic therapeutic composition may comprise an emulsifier or thickener. The emulsifier or thickener may be olive emulsifying wax or cetyl alcohol.

The present invention includes a method for extracting alginic acid from seaweed, wherein the alginic acid extraction method comprises the steps of preparing a fine seaweed or kelp, adding an acid to the seaweed or kelp, adding a base to the mixture and heat treatment And adding ethanol to the mixture, and filtering the mixture and then drying the filtered solution to obtain alginic acid.

The method for preparing the atopic therapeutic composition includes extracting alginic acid from seaweed, making the alginic acid an alginic acid oligomer by protein recombination, and mixing a tangerine peel extract, noni extract and wasong extract with the alginic acid oligomer. It includes.

The atopy treatment composition according to the present invention has little toxicity to eyes or skin as a result of eye irritation test and skin irritation test, and has an ability to improve atopic dermatitis. The composition according to the present invention is effective in the treatment of atopic dermatitis as a natural skin improver without addition of artificial preservatives or phosphorus oxidizing agents which can cause skin irritation.

According to the embodiments of the present invention, natural active ingredients can be extracted from citrus dermis, noni and wasong, and alginic acid can be extracted from readily available materials such as seaweed and shimashima. In addition, the method of extracting the citrus dermis, noni, vortex extract active ingredient and alginic acid low molecular weight may be provided with a high alginate oligomerization method anti-inflammatory, antibacterial effect. Natural active ingredient extract formed by this method and oligomers of alginic acid and alginic acid show an excellent effect in improving the pathology of atopic skin diseases.

1 is a graph showing the yield according to each method in the case of extracting alginic acid from seaweed and kelp.
Figure 2 is a graph showing the viscosity of alginic acid extracted from seaweed and alginic acid extracted from seaweed and control.
3 is a flow chart schematically showing a natural material extraction method according to steam distillation.
4 is a flowchart schematically showing a natural material extraction method according to the solvent extraction method.
5 is a graph showing the concentration of the extraction component according to the extraction time, when extracting the natural herbal extract.
Figure 6 is a graph showing the change in the content of the active ingredient according to the citrus dermis extraction time.
8A and 8B are eye pictures of the non-washed group 24 hours after applying the atopy treatment composition according to the present invention.
9A and 9B are eye photographs of the non-washed group at 72 hours after applying the atopy treatment composition according to the present invention.
10A and 10B are eye pictures of the washing group 24 hours after applying the atopy treatment composition according to the present invention.
11A and 11B are eye pictures of the washing group at 72 hours after applying the atopy treatment composition according to the present invention.
12 shows treatment compartments and control compartments arranged on rabbit skin.
13 and 14 are photographs showing the skin at 24 hours and 72 hours after application of the atopy treatment composition according to the present invention.
15 is a schematic diagram conceptually illustrating an atopy induction model.
Figure 16 is a graph of toxicity results showing the relative value of toxicity when viewed as 100 treated with nothing in each cell.
17 is a graph showing the ratio of IgE secretion according to the concentration of herbal extracts.
18 is a schematic diagram conceptually illustrating an atopy induction model.
Figure 19 is a photograph showing the ear thickness of normal rats and atopic dermatitis induced rats.
20 is a graph showing the concentration of IgE in fluorine.
21 is a graph showing the IgE concentration according to the concentration of the herbal extract.
22 is a graph showing the effect of the herbal extracts on the cytokine expression level of CD4 + T cells.
Figure 23a to 23k is a photograph of each part of the human body before application of the herbal composition of the present invention.
24A to 24K are photographs of respective parts of the human body after application of the herbal composition of the present invention, corresponding to each of FIGS. 23A to 23K.

Hereinafter, an alginic acid extraction method, an alginic acid oligomer forming method, and an atopic therapeutic composition using the alginic acid oligomer according to an embodiment of the present invention will be described in detail.

In the present embodiment, first, a method of extracting alginic acid from seaweed will first be described, and a method of forming alginic acid oligomers by lowering the alginic acid will be described, and an atopy treatment composition and manufacturing method including the alginic acid oligomer will be described.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The objects, features and effects of the present invention described above will be readily understood through embodiments related to the accompanying drawings. However, the present invention is not limited to the embodiments described herein and may be applied and modified in various forms. Rather, the following embodiments are provided to clarify the technical spirit disclosed by the present invention, and furthermore, to fully convey the technical spirit of the present invention to those skilled in the art having an average knowledge in the field to which the present invention belongs. Therefore, the scope of the present invention should not be construed as limited by the embodiments described below.

Example  1. Extracting Alginate from Kelp / Seaweed

First Aquaculture  Lightly wash seaweed and kelp with fresh water, In the shade Fragmentation Powdered  A certain amount of the alginic acid was extracted under the following three conditions.

A) acid alkali Hydrothermal extraction

1000 g of 0.2 N H₂SO₄ was added to 20 g of crushed seaweed and stirred at 70 ° C. for 7 hours, followed by filtration using whatman No. 4 filter paper and a nylon cloth. After filtration, the filtrate is discarded and the residue is used. After 1000 ml of 1% Na₂CO₃ was added to the residue and stirred for 10 hours, 1,000 ml of purified water was added to the solution.

After storing the solution for one day, Whatman No. 4 After filtering using filter paper and nylon cloth, discard the residue and take the filtrate. The acidity of the filtrate is adjusted to pH 12.

1N H₂SO₄ was added to 3,000 ml of the filtrate and neutralized to pH 7. In this example, the pH was 7 when 1N mL of 1N H 2 SO 2 was added. When the same amount of ethanol is added to the filtrate, a supernatant layer is formed. 1,000 ml of a mixture of ethanol (500 ml) and acetone (500 ml) were prepared in the residue, and the residue was washed with a mixture. 4 Filter using filter paper and nylon cloth.

The filtrate is dried at 30 ° C. for 24 hours and then weighed.

The final sample obtained by the method described above was 6.5 g in weight, with a final yield of about 32.5%.

B) alkali Hydrothermal  Extraction

Into 20 g of ground wakame 600ml purified water to make a mixture and the mixture was stirred for 30 minutes while raising the temperature from 22 ℃ to 75 ℃. The mixture is filtered with a nylon cloth, the filtrate is discarded and a residue is obtained. 400 ml of 1% Na₂CO₃ was added to the residue, followed by stirring at 75 ° C. for 4 hours. The seaweed melts and sticks, so pay attention to the temperature and stir. After the stirring is completed, 1,600 ml of purified water is added. Filter using Whatman No. 4 filter paper and a nylon cloth, and adjust the pH to pH 2 while adding 10% HCl to the filtrate. The filtrate was centrifuged at 3000 rpm for 10 minutes at 20 ° C. to obtain a dark green-like precipitate. The precipitate is placed in a beaker, and 400 ml of 50% ethanol is added to the precipitate, and neutralized to pH 7 with 10% NaOH while stirring. The neutralized mixture is filtered and further filtered by addition of 99% ethanol. After filtration, the filtrate is discarded and the residue is washed with 1: 1 ethanol and acetone.

The residue is dried at 30 ° C. for 24 hours and weighed.

The final sample obtained by the above-mentioned method weighed 4.5 g, and the final yield of alginic acid amount was 22.5%.

All) Mountain singer  Heat treatment

Dried at room temperature, dry 20 g of seaweed, lightly wash with fresh water, and then dry in the shade. The seaweed was pulverized with a blender and 600 ml of sulfuric acid (H₂SO₄, Duksan, EP) solution was added to the ground seaweed to precipitate for 1 hour, and then washed 5 times by filtration using a filter.

600 ml of a 5% sodium carbonate (Na₂CO₃, Duksan, EP) solution was added to the remaining seaweed residue after the filtration and heat-treated for 3 hours, and then 1,800 ml of purified water was added thereto and stirred at 60 ° C. for 12 hours. After the stirring, the solution is centrifuged (4,000 rpm, 4 ° C., 30 min), and then the same amount of ethanol is added to the supernatant. The solution was filtered again and dried to obtain 14.4 g of a dark brown solid alginic acid extract.

The extracted alginic acid was pulverized with a grinder, 200 ml of purified water was added to the pulverized alginic acid, stirred for 1 hour at room temperature, dissolved, and 20 ml of 0.05% hypochlorous acid (NaOCl, Duksan, EP) was added to measure the degree of decolorization and pH. At this time, the acid value treatment to pH 2-3 using hydrochloric acid and then separated by precipitation at 60 ℃. Here, the precipitated solution is gelled, and the precipitated gel is separated and 30 ml of 50% ethanol is added.

The solution is precipitated under acidic conditions (pH 2 ~ 3) to form a gel. Here, the solution is gradually formed into a gel at a temperature of 60 ° C., and neutralized to 30% by adding 30 ml of 50% ethanol to the precipitated gel and adding 30 ml of sodium hydroxide (NaOH, Duksan, EP) while stirring for 12 hours. Titrate.

The neutralized titrated alginic acid solution was used as Whatman No. Filter with 4 and nylon cloth and store the filtered residue in the freezer for 1 day. The residue is put into a dryer at 40 ℃ dried and pulverized with a grinder. The powdered alginic acid is added to a 1M hydrochloric acid (Hcl, Duksan, EP) solution at a concentration of 0.5w / v%. The alginic acid solution is heat-treated at 121 ° C. for 1 hour 30 minutes to 3 hours, then neutralized (pH 7.0) and 1 g of activated carbon is added per 100 ml of the alginic acid solution. The alginate solution is then stirred for 12 hours and filtered 2-3 times using a 0.2 μm pore size membrane filter. Ethanol twice the volume is added to the filtered alginic acid and placed in a centrifuge to recover the precipitate after centrifugation. The precipitate is freeze-dried at minus 4 ℃.

The yield of alginic acid extracted by the above method is 72.2%, the acid number and the heat treatment process of the pure alginic acid produced is 59.2%.

D) high temperature Pressurized pretreatment  Alginic Acid Extraction I

20 g of dry powdered seaweed was added to 1,500 ml of purified water in 75 times distilled water (w / v) and left at room temperature for about 20 minutes, and then placed in an autoclave at 121 ° C. for 20 minutes. It is then taken out of the autoclave and re-added 1500 ml of purified water to the solution, only hot water extraction without stirring. The solution is cooled and filtered with filter paper (whatman No. 2) to extract the filtrate. 2,000 ml of methanol was added to the filtrate, stirred for 20 minutes with a 20 minute analog stirrer, and then precipitated and filtered (whatman No 2). Again dried at 50 ° C. for 12 hours to obtain alginic acid. In the case of extracting alginic acid by the above method, the resultant was small so that it was not separated from the filter paper.

E) high temperature Pressurized pretreatment  Alginic Acid Extraction II

1,500 ml of purified water was added to 75 g of distilled water (w / v) in 20 g of dry aid (powder) seaweed and allowed to stand at room temperature for about 30 minutes, and then placed in an autoclave at 121 ° C. for 20 minutes. It is then taken out of the autoclave and re-added 1500 ml of purified water to the solution, only hot water extraction without stirring.

30 ml of 5% sodium carbonate (Na₂CO₃) solution is added to seaweed, heated at 60 ° C for 3-4 hours, and filtered by hot water extraction. 600 ml of 5% sodium carbonate (Na₂CO₃) solution was added to the remaining seaweed residue after the filtration, followed by heat treatment for 3 hours, followed by addition of purified water, followed by stirring at 60 ° C. for 6 hours. After the stirring, centrifuge at 4,000 rpm, 4 ° C. for 30 minutes. After centrifugation, an equal amount of ethanol is added to the supernatant, followed by filtration and drying. In this way, a dark brown 4.7 g dark brown alginic acid extract can be obtained.

In the present method, since the high temperature pressurization pretreatment and the hot water extraction process are performed instead of the process such as acid alkali treatment, it shows the low yield instead of the advantage of shortening the process time.

The difference in alginic acid content according to each of the above-described extraction methods and seaweed and kelp was small, and the water-soluble alginic acid was contained in the spores.

Example  2. Alginate Extraction Method Comparison

Table 1 shows the yields according to the respective methods when the alginic acid is extracted from seaweed and kelp, Figure 1 is a graph showing each yield of Table 3.

Sample Mountain singer
Heat treatment alkali
Hydrothermal extraction Acid, alkali
Hydrothermal extraction High temperature
conduct Seaweed 59.2% 22.5% 32.5% 12.3% Kelp 63.7% 38.4% 39.6% 26.1%

Here, the yield of alginic acid extracted from kelp according to the alginic acid production method according to the acid hydrolysis method is 63.7%, showing a higher yield than other experimental methods. Hereinafter, the alginic acid used in the Example uses what was obtained by the acid hydrolysis heat treatment method.

Example  3. Identification of physical properties of alginic acid

A) Molecular weight of alginic acid

200 μg of the alginic acid dry sample was dissolved in 0.2 M NaCl solution and measured by gel filtration chromatography (SepHarose CL-6B or SepHarose CL-4B gel chromatography). For molecular weight standard, Showa Pullulan standard P-82 kit was used.

B) Structural characteristics of alginic acid

Alginic acid is a linear polysaccharide mainly present in seaweed. Poly-β-D-mannosyluronic acid (poly-M), poly α-L-glusiluronic acid (poly- α-L-gulosyluronic acid (poly-G) or a mixture of poly-MG has three structural features. The poly-M and poly-G are the same as in Chemical Formula 1 and Chemical Formula 2, respectively. According to the selectivity of these structures, alginate lyase is also classified into three types, A1-I, A1-II, and A1-III, respectively corresponding to the poly-M, poly-G, and poly-MG.

[Formula 1]

[Formula 2]

C) viscosity of alginic acid

Viscosity was measured using a rotary viscometer with a 0.1 M NaCl solution of a sample in which 10 g of alginic acid was dissolved at a constant concentration (0.1% to 1%). At this time, the rotational viscometer was used a Brookfield viscometer, while changing the rpm to 5, 7. 5, 15, 30, 70, 100, 120, 10, 180, 200 rpm respectively spindle NO. Measurements were made three times at 20 second intervals using 20. The temperature of the cup was maintained at 24 ° C when measuring the viscosity. Junsei's sodium alginate was used as a control.

Figure 2 is a graph showing the viscosity of alginic acid extracted from seaweed and alginic acid extracted from seaweed and control.

Table 2 below shows the chemical composition of alginic acid contained in seaweed and kelp.

Alginic acid form Block type Absorbance % M / G ratio Control 1 Seaweed MG 1.206 19 0.75 MM 2.121 34 GG 2.737 48 Control 2 Kelp MG 1.323 25 1.17 MM 2.142 41 GG 1.724 33 Test Zone 1 Seaweed
(Hydrolysis 1h30m) MG 1.542 26 1.02 MM 2.173 37 GG 2.157 36 Test 2 Seaweed
(Hydrolysis 3h) MG 1.104 19 1.04 MM 2.301 41 GG 2.189 39 * M / G ratio = (% MM +% MG / 2) / (% GG +% MG / 2)

Example  4. Alginic acid using alginic acid Oligomer ( Alginate oligosaccharides ) Produce

First, LB (Luria-Bertani media) medium is prepared. To prepare the LB medium, 10 g of tryptone, 5 g of yeast, and 10 g of NaCl were added to 1 L of distilled water and slowly stirred to dissolve. After dissolution, sodium hydroxide is added to measure the pH with a pH meter, and the pH of the solution is adjusted to 7.2-7.4. When the pH is adjusted, divide into 200ml each into 5 Erlenmeyer flasks. The five Erlenmeyer flasks were sterilized by placing them in an autoclave at 121 ° C. for 20 min.

The medium in five sterilized flasks was irradiated with UV for 5 minutes, and 5 ml of each medium was collected from each medium to prepare 5 seed media. The five Erlenmeyer flasks are stored on a clean bench. 5μl of ampicillin and 4μl of strain to be used are also added to the prepared medium. At this time, a usable strain is Stenotrophomonas maltophilia CH12-10. The seed medium is placed in a shaking incubator and stored overnight at 37 ° C. and 200-250 rpm.

Put 200 μl of empicillin into the five 200 ml Erlenmeyer flasks, which are the total medium. Then, 5 ml of the seed medium is placed in a 200 ml Erlenmeyer flask, which is a whole medium. Wrapped with a stopper, the whole medium is added to a stirred incubator, set at 37 ° C. and 200 to 250 rpm, and reacted for 1 to 3 hours. . After the reaction for 1 to 3 hours, 1 ml of sample medium and LB medium for measuring the growth of strains were placed in a UV tube, and the OD (optical density) value was measured by a UV spectrometer. At this time, absorbance is measured based on 520nm. When the OD value is 0.6-0.8, IPTG (isopropyl-1-thio-β-D-galactopyranoside) is added again to 1/1000 in the whole medium. The whole medium containing the IPTG is put in a stirred incubator and stored overnight after setting to 25 ℃, 220rpm.

Take out the five stored medium and centrifuge for 30 minutes at 4400rpm, 4 ℃, and collect only the supernatant. Into 1 L of the supernatant, 100 g of sodium alginate using kelp extract alginic acid of the above example was added and completely dissolved.

99% of alcohol is added to the alginic acid solution in a ratio of 1: 1. At this time, after the oligomer precipitate is formed and stored at -4 ° C for at least one hour, the oligomer is filtered through an aspirator. The filtered oligomer is dried in a dryer at 70 ° C., and the weight of the dried oligomer is pulverized.

As a result of repeating the process four times, the yield of alginate oligomer (alginate oligosaccharides) for 100 g of sodium alginate was 54 g for the first experiment, 49 g for the second experiment, 58 g for the third experiment, The fourth experimental dml reached 52 g.

Example  5. Natural Herb Extraction Method

Natural herbal extracts, together with alginic acid oligomers prepared by the methods described above, form the main component of the atopic therapeutic composition. Hereinafter, the action and extraction method of citrus dermis extract, noni extract, wasong extract which are the main components used in the atopy treatment composition will be described in detail.

The tangerine and the peel is a peel of citrus fruits including citrus dermis. The citrus peels include not only fresh tangerines, but also dried citrus peels.

The citrus peel may have various forms. For example, it may simply be in the form of a shell separated from the tangerine, it may be in the form of a powder obtained by grinding the shell. The citrus peel contains about 60 bioactive substances, including carotenoids, bioflavonoids, pectin and terpenes. The bioactive substances are known to perform antioxidant, anti-inflammatory, anti-allergic, antibacterial, antiviral, immune enhancing effects. The citrus peel also contains citric acid, a type of AHA (Alpha Hydroxy Acid), and the citric acid performs the function of exfoliation.

Among the citrus peels, the aromatic metabolites (Essential Oil), which are secondary metabolites secreted from the endocrine glands of plants, have various biological activities such as aroma effects, antibacterial (sterilization) and antiseptic effects due to volatility. Citrus essential oil is a fragrance ingredient contained in oil glands of citrus peel flavedo layer, and is widely used as an additive in detergents, food flavors, medicines, and cosmetics. The main component of the citrus peel is d-limonene (d-limonene, 65-88%) while being harmless to humans and the environment, it has excellent insecticidal and antibacterial properties.

Noni is a plant native to the South Pacific island nation, a tropical climate of volcanic geology. The fruits of Noni have been used as valuable herbs in tropical regions of Asia and South Pacific, such as India, Indonesia and Malaysia. They are known to use noni juice, flowers, roots and stems for malaria, asthma, fever, headache, constipation, hookworm, eye disease, as well as for hemostatic and traumatic treatments. In our country, Noni's roots were started in the name of Hae-gaeok and Paeokcheon. The effectiveness of the Noni has been shown by many researchers, including more than 100 kinds of products, including cleansing blood, treating damaged areas, strengthening cellular functions, stabilizing blood pressure, lowering cholesterol levels and stabilizing mind and body. It is known to have medical effects. The extract of Noni fruit contains about 50 kinds of physiologically active substances in addition to Xeronine, Proxeronine, and Proxeronase. The physiologically active substances are known to perform actions such as relieving pain, relieving chronic fatigue and lethargy, enhancing immune function, and enhancing body physiological activity.

The fruit of Noni and the citrus peel contains a lot of active ingredients such as pectin, hesperidin, naringin, pigments, essential oils, geronine, progeronine. In particular, the fruit of the noni and the citrus peel contains an aromatic oil component (Essential Oil) having an aroma effect, antibacterial (sterilization) effect and antiseptic effect. The aromatic oil component is a secondary metabolite secreted from the endocrine gland of the plant and has various biological activities. For example, citrus essential oils are aromatic components contained in oil glands of the flavedo layer of citrus peel, and are widely used as additives for detergents, food flavors, pharmaceuticals, and cosmetics.

The Warsong is a perennial herbaceous plant called a rock brush. The hawthorn includes leaves and stems except for the roots, and powder ground by drying them.

The vortex is composed of nine flavonoids, fatty acid esters, seco-A-triterpene, 3-oxo-triterpene, and 3-oxo-triterpene. It contains 31 kinds of chemical components such as hydroxy-triterpene component. They are known to have antimutagenic effects, anticancer and antimetastatic and immune function enhancing effects, antioxidant effects and cancer cell proliferation inhibitory effects.

Considering the above-mentioned narcissus through the literature, it is said that Dangboncho is used for dryness and pain in the mouth. The herbaceous wood is said to be taken by extracting the hawsong with water in the colon subcutaneous blood, and when applied to all wounds, it is said to have a convergent effect. The herbal regeneration is said to heal the white poison and kill the species (,), and is said to have an effect on the intestine (유) and milk (癒). In the class of super weakness, it is said that in order to control the hemorrhagic hemorrhage of hemorrhoids, the medicinal herb is washed with the whole solution. [上海 科學 技術 出版社 小學 館 編;中藥 大事 典, 小學 館, 1985, p.526].

Table 3 below shows the active ingredients contained in the citrus peel extract, Noni extract, Wasong extract and seaweeds and their pharmacological action.

Raw material name INCI and
English name Used part Water quality Drug operation Citrus Peel
extract Citrus Grandis Peel Extract Citrus Essential oils d-limonene Antioxidant effect, anti-allergy, antibacterial effect Flavonoids Hesperidin,
nagrin Sterilization, Insecticide Noni
extract Morinda Citrifolia Extract Fruit Geronine
Progeronine Circulation and stamina of the human body
Inflammation treatment, pathogen suppression Wasong
extract Orostachys japonicus Leaf, stem Triterpene
Aflatoxin B1 '
Flavonoids Blue blood, detoxification, hemostasis, moisturizing effect, eczema, wound healing, skin regeneration,
Antioxidant effect, immune enhancing effect Seaweed marine algae Mouth stem alginic acid Antibacterial effect, anticancer effect

To extract the herbal medicine from natural materials, three extraction methods, such as steam distillation, compression, and solvent extraction, can be used. In this embodiment, the solvent extraction method with the most extraction efficiency was used through the experiment of the extraction method.

The solvent extraction method is a batch extraction method of the solid-liquid extraction experiment method. Solvent extraction from solids is mainly used for the separation of natural organic materials. The basic test method is a triangular flask or round bottom flask, which is usually used as an instrument. In case of a micro sample, a test tube or a centrifuge tube is also used. In the case of a centrifuge tube, the solid-liquid separation can be performed quickly by centrifugation after the extraction operation, which is convenient. The sample can also be extracted by crushing the sample in the extract. The solid sample is ground finely or cut in such a way as to allow good contact with the liquid. Soak in a suitable solvent, place or stir at low temperature or room temperature or reflux at low temperature. Ultrasonication is also effective, but heat is generated, so it is necessary to consider stability with respect to heat as with heat extraction.

A) Selection of extraction method

Steam distillation, compression, and solvent extraction are performed through the following processes, respectively.

 (1) steam distillation

3 is a flow chart schematically showing a natural material extraction method according to steam distillation.

Referring to Figure 3, using the steam generated in the steam generator is a method of distilling the raw material in the flask with water vapor to obtain a condensed raw material extract while passing through the cooling tube. First, the raw material to be distilled is about 1/3 of the flask. The flask is then set up to have a 30 ° -45 ° tilt from vertical. Cooling tubes should be as long as possible and suitable for testing. It is preferable to use the glass tube which is connected to the flask in the steam generator as short as possible. If the glass tube is too long, water vapor condenses on the way.

Accurate yield is calculated by measuring the amount of extract relative to the raw material of the extracted extract. In this manner, an extract of mint leaves (menthol) was obtained.

 (2) compression method

The compression method is to extract the essential oil by compressing the raw material, which is suitable for extracting the extracts of citrus peel, aloe, noni, eosungcho and evening primrose seeds. As an example, essential oils of citrus dermis were extracted by the following method.

First, soak citrus dermis in distilled water and store for 1 week. Next, the citrus dermis contained in distilled water is put into a compressor and compressed to extract the citrus dermis extract.

Aloe is placed in a compressor in a pulp state and compressed and extracted by the above method, and noni, eoseongcho and evening primrose seeds are extracted in the same way as citrus dermis.

 (3) Solvent Extraction

4 is a flowchart schematically showing a natural material extraction method according to the solvent extraction method.

First, the raw materials to be extracted are mixed with alcohol ethanol, respectively, and the solution is aged for a predetermined time. That is, after mixing each raw material (for example, citrus dermis 120g, aloe 100g, menthol 20g, sprout 100g) in 1000ml of 30% alcohol ethanol, each mixed raw material can be put into a stirring aging machine and aged for 3 days.

After aging the solution for 3 days, each raw material is filtered through a standard sieve and the extract is separated.

(4) Extraction method selection

The steam distillation method was a low yield of raw material extraction, the compression method is suitable for extracting the extract of citrus dermis and aloe, noni, eoseongcho, evening primrose seed, but extracting menthol using peppermint using a mixer rather than using a compress It is suitable to grind the mint leaves and use the juice. According to the steam distillation method, the amount of the extract of citrus dermis and aloe was not so much compared with the input of raw materials. The solvent extraction method is an extraction using a solvent, the extraction effect of each raw material is about 70% more than other experimental methods, it is determined that it will be suitable for mass production. In the following examples, the natural material was mainly extracted using a solvent extraction method.

B) Extraction of natural substances by extraction

 (1) Mix 120g of well-dried citrus dermis, eoseongcho and Noni each with 1L of 30% alcohol ethanol. Using a stirring incubator (Shaking incubator; Model # VS-8480SR) for 45 days at 45 ℃, 120rpm in a dark place and aged. Aging is carried out in a sealed state so that air is not introduced in the stirring process. After aging, the citrus dermis essential oil is extracted by filtration twice with a 600mesh filter medium → 300mesh filter medium → 200mesh filter medium to remove shells and residues.

(2) 100 g of aloe flesh cut to 5 cm in width is mixed with 1 L of 30% alcohol ethanol. Aged aging for 4 days at 45 ℃, 120rpm conditions in a dark place using a stirred incubator. At this time, aged in a sealed state so that air is not introduced during the stirring process. After aging, the aloe extract is extracted by filtration twice with 600mesh filter medium → 300mesh filter medium → 200mesh filter medium to remove the aloe pulp.

(3) 100 g of evening primrose seeds washed with distilled water are mixed with 1 L of 30% alcohol ethanol. Aged aging for 4 days at 45 ℃, 120rpm conditions in a dark place using a stirred incubator. At this time, aged in a sealed state so that air is not introduced during the stirring process. After aging, the evening primrose seed extract is extracted by filtration twice with 600mesh filter medium → 300mesh filter medium → 200mesh filter medium to remove the buds.

(4) The ratio of the raw material and ethanol used when separating the natural materials is shown in Table 4 below.

C) Extraction of active ingredient from citrus dermis and content analysis

80 g of dried citrus dermis is mixed with 1 L of 30% alcohol ethanol. Aged and stirred for 4 days at 60 ℃ dark 150rpm conditions in a stirred incubator. After aging, the citrus dermis essential oil is extracted by filtration twice with a 600 mesh filter, twice with a 300 mesh filter, and twice with a 200 mesh filter to remove the shells and residues.

The extracted essential oil is concentrated for 60 minutes under the condition of 50 ℃, 100rpm using a concentrator.

5 is a gas chromatographic analysis of the number of components extracted from the citrus dermis by the solvent extraction method, Table 5 shows the components analyzed by the gas chromatography, the area of each component and the time stayed in the column.

In order to analyze the components of the citrus dermis by the gas chromatography, solvent extraction was performed (0.5% ethanol for alcohol, 30%) using a 0.5 cm thin sample. At this time, the ethanol for alcohol was added to the cutting sample and stirred for 96 hours. The extracted result was centrifuged at 2500 rpm for 15 minutes and analyzed by GC 6890.

6 is a graph showing the change in the content of the active ingredient according to the citrus dermis extraction time. The extraction method was extracted by stirring for 24 hours using hot water extraction and solvent extraction (alcohol ethanol, 30%) using a 0.5 cm thin sample and a powder sample, followed by 0, 0.5, 1, 7, 12, 24 hour intervals. 3 ml each of them was centrifuged at 2500 rpm for 15 minutes and analyzed by GC 6,890 nm.

Among the essential oil components of the citrus dermis extract, 32 were detected, and among them, d-limonene (d-limonene, C ₁₀ H ₁₆ ) was the most contained with an area ratio of 72.3%. In the ethanol extract, di-limonene was detected at retention time (RT) of 9.13 minutes, and the amount of extract was relatively high at 3.0 × 10 ⁶ at 24 hours, and the extract was 3 times higher than that of 7 hours. Abnormality was detected.

Table 6 shows the components detected by the solvent extraction and the content ratio of each component as the area ratio of the peak.

la) From Noni  Active ingredient extraction

Add 30-60 kg of Noni fruit to a 100-L container and add 200-500 mL of 20-50% ethanol. The ethanol uses 100 mL of 35% ethanol per 10 kg of Noni. The noni fruit to which ethanol was added is aged at 20 ° C. for about 13 days. The noni fruit and ethanol are filtered two times with a 600 mesh filter, two times with a 300 mesh filter, and two times with a 200 mesh filter in order to remove the skin and residue after ripening, and extracts the noni fruit essential oil.

The extracted Noni fruit essential oil is concentrated under a condition of 50 ° C. and 100 rpm using a concentrator for 60 minutes.

hemp) From Wasong  Active ingredient extraction

Raw walnuts were dried at -40 ° C. for 8 hours using a lyophilizer and then finely crushed using a grinder and then filtered into a 0.5 mm filter medium. The crushed vortex is stored in the -20 ℃ freezer. After washing the vortex with distilled water, 100 g of the vortex is mixed with 1 L of 30% alcohol ethanol. Aged by stirring for 3 days at 120 rpm in a dark place 45 ℃ in a stirred incubator. At this time, it is aged in a sealed state so that air does not flow in the stirring process. In order to remove the residue of the vortex solution after aging, the vortex extract is extracted by filtration twice with 200 mesh filters twice, 300 mesh filters twice, and 600 mesh filters twice. The extracted filtrate of the vortex solution was concentrated under reduced pressure at 60 ° C., completely dried, and then weighed and dried at a concentration of 1000 μg / ml.

F) composition of extract mixture

 The composition of the natural herbal extracts to be combined with the alginic acid oligomer is pulverized with citrus dermis, fish vinegar and noni powder, and then powder and 30% alcohol are added at a ratio of 1: 8 to 10, for example 1: 8.3, 4 at 45 rpm at 120 rpm. Aging daily. The aloe extract is added to the aloe gel and 30% alcohol in a ratio of 1: 9 to 11, for example, 1: 10, and aged at 45 rpm at 120 rpm for 4 days. Evening primrose seed extract is aged for 30 days ethanol at 120rpm at 45 ℃ in a ratio of 1:10.

G) Pharmacological action of natural extracts used in extract mixtures

Table 7 below shows the pharmacological action of the natural extract used in the extract mixture composition.

Ah) Wasong  Effect of Extracts on Antioxidant Activity

Lipids in cosmetics or lipids present in the body's biofilm are oxidized by a chain reaction with free radicals in the presence of free radicals, which is a source of cosmetic quality changes and biological aging. In order to prevent the oxidation reaction, free radical scavenging is used to donate hydrogen atoms through peroxy radical and dehydrogenation reaction in the propagation stage of the chain reaction to maintain the radicals in a relatively stable state. This free radical scavenging is called an antioxidant, the most representative of which is a phenolic compound.

The phenolic compound is widely distributed in the plant system and has various structures and molecular weights as one of secondary metabolites, and is a main component of flavonoids and tannins. Because they have phenylic hydroxy groups, they bind to proteins and other macromolecules. The flavonoids have various physiological activities such as anticancer, antiviral and anti-inflammatory by donating hydrogen atoms to radicals to inhibit their production. Flavonoids also inhibit the oxidation of lipids in vivo, including LDL, by chelating metal ions that catalyze peroxidation reactions. However, when flavonoids are present in high concentrations, the pH is high and iron is present, they act as donors of free radicals. Accordingly, ingestion of flavonoids above a certain concentration is rather harmful to the human body.

The results of analysis of total phenolic compounds and flavonoid contents according to the extraction time by extracting vortex with ethanol and water are shown in Table 8, and the unit is mg / 100g.

Phenolic content Flavonoid content Methanol June 647.2 ± 1.26 238.3 ± 1.43 In July 782.3 ± 3.22 322.7 ± 1.21 August 2,703.1 ± 5.21 634.2 ± 2.33 September 2,632.1 ± 2.38 1,0123. ± 3.24 October 2,428.0 ± 3.52 1,324.6 ± 1.87 November 1,462.8 ± 1.70 1,211.4 ± 1.36 December 1,232.4 ± 1.23 985.3 ± 1.32 water June 525.3 ± 0.55 217.6 ± 2.55 In July 800.2 ± 1.37 253.3 ± 1.98 August 1,985.4 ± 2.34 511.1 ± 2.14 September 1,865.5 ± 1.80 824.9 ± 3.87 October 2,004.2 ± 2.17 941.2 ± 2.84 November 1,312.1 ± 1.99 442.8 ± 3.62 December 998.6 ± 1.86 401.6 ± 2.40

Referring to Table 8, the content of total phenolic compounds was higher in ethanol extracts than water extracts, especially from 2,428.0 ± 3.52 ~ 2,703.1 ± 5.21 mg / 100g in Wasong collected in August ~ October. Na et al. (Na, GM, Han, Hs, Ye SH and Kim, HK 2004. Physiologica activity of medicinal plant extracts.Korean j. Food Preservation, 11: 388-393.) As a result of examining the total phenolic compounds of the extract according to the results, the content of licorice 1.38 ~ 2.23%, health 0.67 ~ 0.95% and the head worms 0.26 ~ 0.81%, which has a similar tendency to the results of the present invention.

The flavonoid content also has the same tendency as the total phenolic compounds. In other words, the flavonoid content of ethanol extract of Wasong collected in September-November was 1,012.1 ± 3.24 ~ 1,324.6 ± 1.87 mg / 100g.

As a result, it can be seen that the active ingredient having an antioxidant effect of vortex is eluted more easily in ethanol than water. For example, Wasong contained more phenolic compounds compared to 1.3% of the phenolic compounds of mulberry and koji mulberry.

character) Wasong  Of extract Antimicrobial  Effect results

Table 9 shows the minimum growth inhibitory concentrations of the ethanol extracts of Wasong using the agar medium dilution method, and shows the results of measuring the minimum inhibitory concentrations of rock ethanol extracts against various microorganisms.

microbe Growth at each concentration (mg / ml) MIC
(mg / ml) 10 5 2.5 1.25 0.625 G (+) S. aureus - - - - + 1.25 B. subtilis - - - - + 1.25 C. diphteriae - - - - + 1.25 S. mutans - - - + + 2.5 C. freundi - + + + + 10 S. epidermidis - - - + + 2.5 P. vulgaris - + + + + 10 G (-) E. coli + + + + + - S. paratyphi A - + + + + 10 S-typhi - + + + + 10

Referring to Table 9, the Warsong extract showed a wide range of antimicrobial effects in Gram-positive bacteria. All five gram-positive bacteria except C. freundi and P. vulgaris showed minimum growth inhibitory concentrations of 1.25-2.5 mg / ml.

Example  6. Alginic acid Oligomer  Preparation of Atopic Skin Enhancer Using

In this embodiment, the alginate oligomer dissolved in the natural herbal extract and purified water in the above embodiment in the aqueous phase and dispersed using a suitable natural extracting emulsifier using a natural extraction oil in the form of an emulsion (emulsion) Prepared. The alginic acid oligomers used in the following examples are all low molecular weight alginic acid oligomers prepared through the above examples. The emulsion can be used as a body cream and body lotion.

Ingredients of the emulsion, citrus peel, Nosong, aloe, aloe, eosungcho extract, jojoba oil, evening primrose oil, camellia oil, olive oil wax, oligomer, natural vitamin E, glycerin, panthenol, grapefruit seed extract is used.

The composition method of the said emulsion is as follows.

① Mix distilled water and 95% alcohol ethanol to prepare 30% alcohol ethanol.

② Mix 120g of dermis per 1L of 30% alcohol. At this time, do not allow other foreign substances to enter.

③ Wrap the foil in the mixed raw material to block the light and put it in the stirring aging machine for 4 days.

(5) The stirring aging conditions of the mixture were set at 45 ° C. and 120 rpm. Maintain a constant rate and temperature during the stirring.

⑤ extract the raw material extract by removing the sludge of the mixture.

Atopic skin disease treatment cream using the raw material extract was prepared as follows using an emulsification method or a multiphase emulsification method.

Here, the emulsification method includes a W / O (water / oil) method and an O / W method, and the multiphase emulsification method includes a W / O / W method and an O / W / O method. The W / O method is a method of emulsifying the aqueous phase by mixing the oil phase layer. The O / W method is a method of mixing and emulsifying an oil phase layer in an aqueous phase layer. The W / O / W method is a method of emulsifying by mixing half the aqueous layer in the oil phase layer, and then mixing the resultant in the remaining half of the aqueous layer to emulsify. The O / W / O method is a method of emulsifying by mixing half of the oil phase in the aqueous phase, and then mixing the resultant in the remaining half of the oil phase.

A) each On oil painting  Viscosity results

Table 10 below shows the viscosity of the atopy skin improver prepared according to the methods a) to d), and FIG. 7 shows the results of Table 10 graphically. In FIG. 7, the x-axis shows the change of time and the y-axis shows the viscosity.

Referring to Table 10, it can be seen that the viscosity of the atopic skin improver increases over time. Here, it can be confirmed that the viscosity in the case of the W / O method is increased to a suitable degree for skin application.

The composition of the atopic dermatitis therapeutic agent cream 52.6 g according to the present invention prepared by the above method is shown in Table 11 below. Here, the alginic acid oligomer (represented as oligosaccharide in the following table) was used 8.5g per 52.6g total weight, but is not limited thereto, 10 to 20 parts by weight can be used when the total weight is 100 parts by weight. At this time, the tangerine peel extract may be 15 to 25 parts by weight, the Warsong extract is 5 to 15 parts by weight, the noni extract may be used 3 to 7 parts by weight.

Example  7. Safety testing of atopic skin improvers of herbal compositions obtained in various ways according to the present invention

Safety test through skin irritation test: Since the high antioxidant material effective for atopic dermatitis is used as a product directly applied to the skin or as the main ingredient of such product, there should be no side effect such as hypersensitivity reaction or rash on human skin. do. This safety was tested using a finished product or a prototype for improving atopic dermatitis.

end) Eye Stimulation Test

 This test was conducted to investigate eye irritation of rabbits with atopic dermatological improvement functional cosmetics. According to Korean Food and Drug Administration Notification No. 2008-75, "Regulations on Screening of Functional Cosmetics," etc. The regulation was carried out in compliance with the regulations on management (December 1, 2006).

1) Experimental Materials and Test Methods

In this test, the atopy treatment composition according to the present invention was directly applied to the undiluted lotion, atopic skin improvement functional cosmetics, at room temperature, without additional preparation.

 The animals used in the experiment were New Zealand White Rabbits from Samtaco Bio Korea, and the rabbits used six females, 2.04-2.39 kg, aged 3-5 months. The rabbit is a species commonly used for ocular mucosal irritation tests, and there are relatively abundant test data to facilitate interpretation and evaluation of test results.

 The experimental animals were managed by setting temperature 21 ± 3 ℃, relative humidity 50 ± 10%, ventilation frequency 10 ~ 12 times / hr, lighting 12 hours (08: 00 ~ 20: 00), illumination intensity 200 ~ 300Lux. For feed, Purina Korea Co., Ltd. and drinking water were freely consumed.

The test group was configured as shown in Table 12 below. Test substance administration and method were calibrated each of the six test animals, and then 0.1 ml of the test substance was directly applied to the right eye and the left eye was treated as a control. After the eye drop, the upper and lower eyelids were held for about one second to prevent leakage of the test substance. Three of them were rinsed with about 20 ml of sterile physiological saline in the eyes to which the test substance was applied after 20 to 30 seconds, and the other three were not washed.

group Test substance castle Marisu Animal Number Dose
(Ml) Eye wash Non-clean Atopic Skin Improvement
functional cosmetics female 3 1-3 0.1 - Washing group Atopic Skin Improvement
functional cosmetics female 3 4 to 6 0.1 Sterile Physiological Saline

2) Observation and Inspection Items

At 1, 24, 48, and 72 hours after application of the test substance, changes in general symptoms, poisoning symptoms, and the presence of dead animals were observed. In addition, individual body weights were measured immediately before application of test substance and on days 1 and 3 after application. In addition, the irritation of cornea, iris and conjunctiva was observed at 1, 24, 48 and 72 hours after application of the test substance.

3) Eye irritation  Test results

Evaluation of the eye irritation response was carried out in accordance with the grading criteria of the ocular lesion, shown in Table 13 below.

(1) cornea (A) Turbidity: the degree of thickening of the eye (observing the thickest spots) ○ No purging or turbidity 0 ○ Turbidity is dispersed or concentrated (unlike normal transparency slightly slowed down), but the tip of Heungchae is clearly observed.
One ○ Translucent parts are easily observed, but the ends of the iris are slightly unclear 2 ○ It shows pearl color and the end of iris is not observed, and the size of pupil is barely observed.
3 ○ The cornea is opaque and the iris is not observed due to cloudiness. 4 (B) the range of clouded cornea ○ 1/4 or less (but not 0) 0 ○ more than 1/4 less than 1/2 One ○ more than 1/4 less than 3/4 2 ○ 3/4 or more up to 1 3 A × B × 5 Maximum = 80 (2) iris (A) reaction value ○ normal 0 ○ Significant wrinkles, congestion, swelling, and moderate congestion around the cornea, alone or mixed
One Appears and the iris responds to light (dull response is positive) 2 ○ No reaction to light, bleeding and mostly destroyed (some or all of the above symptoms)
3 A × 5 maximum = 10 (3) conjunctiva (A) redness (limited to bleeding conjunctiva and eye conjunctiva) ○ blood vessels are normal 0 Some blood vessels are clearly hyperemia One O Wide crimson hue, individual vessels not easily observed 2 ○ pale cerise 3 (B) conjunctival edema ○ does not swell 0 Slightly swelling than normal (including dialysis) One Significant swelling with partial abduction of the eyelid 2 ○ The swelling of the eyelids about half of the eyes closed 3 ○ The swelling of the eyelid about half or more eyes closed 4 (C) emissions ○ No emissions 0 Some discharge (except for small amounts observed in the inner tails of normal animals) One Emissions that wet the eyelashes and eyelids 2 Emissions that wet the eye area and the eyelashes and eyelids 3 Score (A + B + C) × 2 max = 20

To determine the degree of eye irritation, the individual ocular irritation icdex (IOI) was calculated at each observation point according to the grade of the ocular lesion. The highest value of the mean ocular stimulation index was calculated as the acute ocular irritation index (AOI). Table 14 is an eye irritation determination table and shows the acute eye irritation index obtained from the above results.

Degree Evaluation category 0 to 5 Nonirritant 5 to 15 Mild irritant 15-30 Moderate irritant 30 to 60 Severe irritant 60 to 80 Medium irritant 80-110 Maximum irritant

Eye irritation test results using the above evaluation criteria of eye irritation response is as follows.

Animals that died due to the application of the test substance during the trial and no unusual general symptoms were observed as shown in Table 15.

Animal No. Days after application Mortality 0 One 2 3 One - - - - 0/3 ² 2 - - - - 3 - - - - 4 - - - - 0/3 ² 5 - - - - 6 - - - -

As a result of measuring individual body weights immediately before application of the atopy treatment composition according to the present invention, 24 and 72 hours after application, no specific weight change was observed as shown in Table 16.

Group Animal No. Day (s) after application (kg) 0 One 3 T1
(No eye washed) One 2.39 2.41 2.51 2 2.08 2.09 2.21 3 2.17 2.19 2.36 T2
(Eye washed) 4 2.04 2.08 2.16 5 2.18 2.22 2.29 6 2.22 2.25 2.45 Mean
SD 2.18 2.21 2.33 0.12 0.12 0.13

8A to 11B are photographs showing the eyes of the non-washed and washed groups to which the atopy treatment composition according to the present invention is applied.

8A and 8B are eye pictures of the non-washed group 24 hours after applying the atopy treatment composition according to the present invention. 9A and 9B are eye photographs of the non-washed group at 72 hours after applying the atopy treatment composition according to the present invention. 10A and 10B are eye pictures of the washing group 24 hours after applying the atopy treatment composition according to the present invention. 11A and 11B are eye pictures of the washing group at 72 hours after applying the atopy treatment composition according to the present invention.

As a result of observing the application site with reference to FIGS. 8A to 11B, as shown in Table 17, slight redness of the conjunctiva was observed 1 hour after application of the test substance in all animals. Minor edema was observed. However, irritability of all individuals returned to normal within 24 hours. Therefore, the mean eye irritation index (MOI) at 1 hour was calculated as 2.67 in the non-washed group and 2.00 in the washed group, and the eye irritation of all subjects returned to normal within 24 hours. The non-clean group was 2.67 and the wash group was 2.00.

Group T1 (No eyewashed) T2 (Eye washed) Anima. No. One 2 3 4 5 6 Comea Degree of opacity (A) 1hr 0 0 0 0 0 0 24hr 0 0 0 0 0 0 48hr 0 0 0 0 0 0 72hr 0 0 0 0 0 0 Diffuse areas of Opacity (B) 1hr 0 0 0 0 0 0 24hr 0 0 0 0 0 0 48hr 0 0 0 0 0 0 72hr 0 0 0 0 0 0 Iris (C) 1hr 0 0 0 0 0 0 24hr 0 0 0 0 0 0 48hr 0 0 0 0 0 0 72hr 0 0 0 0 0 0 conjuncitiva Redness (D) 1hr One One One One One One 24hr 0 0 0 0 0 0 48hr 0 0 0 0 0 0 72hr 0 0 0 0 0 0 Edema (E) 1hr 0 0 One 0 0 0 24hr 0 0 0 0 0 0 48hr 0 0 0 0 0 0 72hr 0 0 0 0 0 0 Lacrima (F) 1hr 0 0 0 0 0 0 24hr 0 0 0 0 0 0 48hr 0 0 0 0 0 0 72hr 0 0 0 0 0 0 IOI 1hr 2 2 4 2 2 2 24hr 0 0 0 0 0 0 48hr 0 0 0 0 0 0 72hr 0 0 0 0 0 0 MOI 1hr 2.67 ²⁾ 2.00 ²⁾ 24hr 0.00 0.00 48hr 0.00 0.00 72hr 0.00 0.00

IOI (individual ocular irritation index) = (A × B × 5) + (C × 5) + (D + E + F) × 2

Mean ocular irritation index (MOI)

2) acute ocular irritation index (AOI) = the maximum value of MOI

In conclusion, the ocular mucosal application of atopic dermatological improvement functional cosmetics to New Zealand white female rabbits was determined to be a nonirritant with acute eye irritation index (AOI) of 2.67.

B) skin irritation test

 This test was conducted to investigate eye irritation of rabbits with atopic dermatological improvement functional cosmetics. According to Korean Food and Drug Administration Notification No. 2008-75, "Regulations on Screening of Functional Cosmetics," etc. The regulation was carried out in compliance with the regulations on management (December 1, 2006).

1) Experimental Materials and Experimental Methods

The animals used in the experiment were New Zealand White Rabbits from Samtaco Bio Korea, and the rabbits used six females, 2.04-2.39 kg, aged 3-5 months. The rabbit is a species commonly used for ocular mucosal irritation tests, and there are relatively abundant test data to facilitate interpretation and evaluation of test results.

 The experimental animals were managed by setting temperature 21 ± 3 ℃, relative humidity 50 ± 10%, ventilation frequency 10 ~ 12 times / hr, lighting 12 hours (08: 00 ~ 20: 00), illumination intensity 200 ~ 300Lux. For feed, Purina Korea Co., Ltd. and drinking water were freely consumed.

To apply the atopic therapeutic composition according to the present invention to the skin of the rabbit, depilation was performed on the day before administration, and divided into treatment sections and control sections on the rabbit skin. 12 shows treatment compartments and control compartments arranged on rabbit skin.

Referring to FIG. 12, 0.5 ml of the test substance was applied to the upper lice and the lice and the non-ice and the lice by using two places of abrasion sites and two non-abrasion sites per test animal, and then, once, respectively. Sterile distilled water was applied to the untreated control compartment. Here, two abrasions and skins were made by scratching the stratum corneum so that the stratum corneum was damaged but the dermis was not bleeding. After application, treatment sections were secured with a non-irritating tape and exposed for 24 hours. At the end of the exposure time of the test substance, the remaining test substance was gently cleaned with sterile distilled water.

2) Observation and Inspection Items

For 72 hours after application of the test substance, changes in general symptoms, intoxication symptoms and the presence of dead animals were observed. In addition, individual body weights were measured immediately before application of test substance and 24 and 72 hours after application. In addition, irritation such as erythema, crust and edema was observed 24 and 72 hours after application of the test substance.

3) Skin irritation test result

 Evaluation of skin reaction was performed according to Table 18 below.

(1) erythema and skin formation No erythema 0 Very mild erythema (only visible to the naked eye) One Pronounced erythema 2 Slightly severe erythema 3 Severe erythema (blush) and crust formation 4 Sum of total possible grades 4 (2) edema formation No edema at all 0 Very mild edema (visually discernible) One Mild swelling (obviously swollen to distinguish the margins) 2 Swelling of myopathy (when swelling about 1mm) 3 Severe edema (swelling more than 1 mm and extending beyond the exposed area) 4 Sum of total possible grades 4

Determination of the degree of irritation of the skin reaction results was followed by the commonly used method of calculating the Primary Irritation Index (P.I.I) of Draize, and the method of calculating P.I.I is shown in Table 19.

Degree Evaluation category 0 to 0.5 Nonirritant 0.6 to 2.0 Mild irritant 2.1 to 5.0 Moderate irritant 5.1 to 8.0 Strong irritant

The skin irritation test results using the evaluation criteria of the skin response are as follows.

In the case of general symptoms and mortality, no clinical symptoms, reflexes, or dead animals were observed in all treated animals as shown in Table 20 below.

Animal No. Days after application Mortality 0 One 2 3 One - - - - 0/6 ² 2 - - - - 3 - - - -

As a result of measuring individual body weights immediately before application of the test substance and 24 and 72 hours after application, no peculiar body weight change was observed as shown in Table 21.

Animal No. Days after application (kg) 0 One 2 3 One - - - - 2 - - - - 3 - - - -

Table 22 is a table showing the results of irritation determination according to P.I.I., Figures 13 and 14 are photographs showing the skin at 24 hours and 72 hours after the application of the atopy treatment composition according to the present invention.

Referring to Tables 22, 13 and 14, as shown in Table 22, weak erythema was observed in all subjects at 24 hours and at two test substance application sites at 48 hours. Primary Irritation Index (PII) was estimated at 0.50. Skin irritation returned to normal within 72 hours.

As a result of the test, the test substance was identified as Non Irritant by [PII] irritation when applied to the skin of New Zealand white rabbit.

Example  8. Atopic Controllability Analysis of the Compositions According to the Invention

In order to examine the effects of atopic controllability of the herbal medicines, which are the main ingredients in this project, the research team of the Environmental Engineering Department of Gwangju Institute of Science and Technology was commissioned as follows.

A) Test Analysis

i) Determination of the appropriate test concentration of the herbal extracts (cytotoxicity test)

ii) B-cell activity and IgE production inhibition test of herbal extract

iii) T-cell activity and cytokine production inhibition test of herbal extract

iv) To evaluate the effect of herbal extracts on the activity of atopy-derived immune cells

B-cell product assay; IgE production analysis

T-cell product assay: IL-4, IL-5, IL-13, IFN-g expression analysis

B) Test analysis form

 ① Determination of proper test concentration of herbal extract (Cytotoxicity test)

Before starting in vivo experiments, the effects of natural herbal extracts on IgE, one of the main features of atopy, are examined. It is important to determine the appropriate concentration that does not harm the cells before treating the herbal extracts and confirming their effects. To this end, various concentrations of extracts were treated on U266B1, a B cell line that secretes a large amount of IgE to be used in the experiment, to determine concentrations that do not affect cell viability through Trypan blue staining, MTT analysis.

② B-cell activity and IgE production inhibition test of herbal extracts; U266B1, B220 + B cell

In order to confirm the efficacy of natural herbal extracts in atopic diseases, the effects of the natural herbal extracts are first examined in vitro. Since IgE is widely known as an important factor in atopy, U266B1, which is a B cell line of humans that secrete much IgE, is used.

RPMI containing 15% of FBS is used to grow cells, increase cell numbers, and stimulate LPS and IL-4 at optimal B cell stimulation concentrations. At the same time, the extract was treated with the concentration determined through the toxicity test, and in the case of the control, the same amount of PBS was incubated for 72 hours, and then, the IgE concentration was collected from the culture medium through an ELISA (Enzyme linked immunosorbent assay). Check it. And in order to confirm the effect in a closer in vivo situation, B cells are isolated from the lymph nodes of mice and the experiment is conducted as described above. These results indicate indirectly the effects of herbal extracts on atopy.

③ T-cell activity and inhibition of important cytokine production of herbal extracts; CD4 + T cell

Next, we examine the effects of herbal extracts on the expression levels of the cytokines IL-4, IL-5, IFN-g, and IL-13, which are known as important mediators of atopy. To this end, CD4 + T cells were isolated from rat lymph nodes, treated with PMA and ionomycin (T cell activator) extracts, incubated together, RNA was isolated from cells, synthesized with cDNA, and mentioned above. The expression level of one cytokine is compared by real time PCR.

C) Application test of animal model of atopic dermatitis

 1) Induce animal models of atopic dermatitis

To determine the efficacy of noni and walnut colostrum natural herbal extracts in atopic diseases, first of all, DNCB and tick extracts were treated with BALB / c mice's ears and back to establish a disease model with symptoms similar to human atopic dermatitis. Induce.

15 is a schematic diagram conceptually illustrating an atopy induction model. Referring to Figure 15, atopy animal model derivation method is as follows.

First, 6-week-old BALB / c mice were divided into a control group and an atopy induction group, and then the control group was treated with the same amount of PBS as DNCB or tick, and the mice in the atopy-induced group received mite extract and DNCB in the ear once a week. Treat for 4 weeks. At this time, the ear is sterilized with gauze tape 5 times inside and outside of the ear to remove dust, dead skin and other foreign matter, and mite extract (Dematophasoides farinea) and 1% DNCB on both sides of both ears.

24 hours after disease induction, the thickness of the ear was measured using a type A dial caliper gauge, and blood for 2 weeks was prepared by bleeding the eye. When it was confirmed that atopic symptoms were induced, the induction was continued for 2 weeks, and after sacrifice, mice were sacrificed 24 hours after the last 4 weeks of induction to separate CD4 + T cellalc B220 + B cells from lymph nodes, and then treated with cytokines. Expression level, IgE level is confirmed in B-cells.

2) B-cell reactivity

As mentioned above, the most important factor in atopy is the concentration of IgE in serum. B cells are differentiated into plasma cells to secrete antibodies. In this case, isotype switching is caused by interleukin-4, and IgG is converted to IgE. In the atopy, Th2 cytokines such as interleukin-4, interleukin-5, and interleukin-13 play an important role, and this process increases IgE concentration. IgE can be largely divided into allergen-specific and specific species, and it is more important to lower these allergen-specific IgE concentrations because it is expected that there will be numerous allergens in atopy.

Next, B cells are isolated from lymph nodes around the atopic induction site and LPS / IL-4 stimulation is performed in vitro, and the degree of IgE secretion is compared.

3) T-cell reactivity measurement:

Atopy is known that Th2 cytokines (interleukin-4, interleukin-5, interleukin-13) play an important role in the development and progression of atopy, and recently, Th1 cytokines (interleukin-12, interferon) Gamma) is also known to play an important role in sustaining the symptoms. Based on this, the effect of the extract on the expression level of cytokines described above is examined to determine the mechanisms by which the extract can alleviate the symptoms of atopy. The degree of expression of atopy-related cytokines (IL-4, IL-5, IL-13, IFNg, TNFa, IL-12) after isolation of CD4 + T cells from lymph nodes around the ear, the site where atopy was induced Is confirmed by mRNA level using real-PCR.

D) Analysis content of test- In vitro analysis

i) Determination of the appropriate test concentration of the herbal extracts (cytotoxicity test): U266B1 cell line

U266B1, a B cell line that secretes a large amount of IgE, was treated with extracts of various concentrations to determine the concentration that does not affect cell growth through the WST-1 test. At this time, since the exact concentration of the herbal extract was not known, cell growth was confirmed by treating 0.5, 1, 5, and 10% herbal extracts of the medium based on the volume of the entire medium. After treating each mixture of 1 × 10 ⁶ U266B1 cells at different concentrations, the toxic effects of the herbal extracts on the cells were compared through a WST-1 test, a kind of MTT assay, after stimulation at 37 ° C. for 72 hours.

Figure 16 is a graph of toxicity results showing the relative value of toxicity when viewed as 100 treated with nothing in each cell. As can be seen in Figure 16, even when treated with 0.5% to 10% of the mixture mixture was confirmed that does not significantly affect the cell growth. Accordingly, it was decided to use 1%, 2.5%, and 5% herbal extracts in order to identify the effects of concentration in future experiments.

ii) inhibition of IgE production of herbal extracts; U266B1

Most patients with atopic dermatitis have high levels of immunoglobulin E (IgE) in their serum and activate mast cells due to IgE-mediated secretion of histamine, resulting in severe itching with inflammatory reactions. Will be displayed. In order to confirm the efficacy in the atopic disease of the herbal extracts according to the present invention, first, the degree of inhibiting the secretion of IgE in vitro using a cell line secreting a large amount of IgE.

In a RPMI containing 10% FBS, human B cell line U266B1 was raised and cell number was increased, and then stimulated through LPS (10ug / ml) and IL-4 (5ng / ml), which are optimal B cell stimulation concentrations. Cells were activated and class switching to IgE occurred. At the same time, the concentration determined by the toxicity test was 1%. 2.5% and 5% herbal extracts were treated, and in the case of controls, the same amount of ethanol was incubated for 72 hours, and then, the culture medium was collected, and the IgE concentration present therein was confirmed by ELISA (Enzyme linked immunosorbent assay). .

17 is a graph showing the ratio of IgE secretion according to the concentration of herbal extracts. Referring to FIG. 17, when the herbal extract was treated, it was confirmed that the concentration of IgE was reduced compared to the control, and the degree was reduced in proportion to the concentration when the control was 100%. In addition, although the degree of inhibition is not very large, it was confirmed that the degree of inhibition tends to be appeared depending on the concentration.

E) In vivo assay results

1) Establishment of atopic dermatitis animal model

i) Inducing animal models of atopic dermatitis

To determine the efficacy of noni and walnut colostrum natural herbal extracts in atopic diseases, DNCB and tick extracts were treated in BALB / c mice's ears to establish a disease model with symptoms similar to human atopic dermatitis. Induced.

18 is a schematic diagram conceptually illustrating an atopy induction model. Referring to Figure 18, atopic animal model derivation method is as follows.

First, 6-week-old BALB / c mice were divided into a control group and an atopy induction group, and then the control group treated with the same amount of PBS as DNCB or tick. Mice in the atopy induction group were treated with ear mite extracts and DNCB once a week for 4 weeks. At this time, the ear is sterilized with gauze tape 5 times inside and outside of the ear to remove dust, dead skin and other foreign matter, and mite extract (Dematophasoides farinea) and 1% DNCB on both sides of the ear. 24 hours after atopic induction, the thickness of the ear was measured by using a type A dial caliper gauge, and blood for 2 weeks was prepared by bleeding eyes and preparing serum for ELISA. If it is confirmed that atopic symptoms are induced, then continue induction for 2 weeks and continue to observe the symptoms of atopy.

24 hours after the last 4 weeks of induction, the mice were killed to separate CD4 + T cells from lymph nodes to compare cytokine expression levels, and CD19 + B cells were isolated and stimulated with LPS and IL-4 to check IgE levels.

ii) induction of atopic symptoms

Four weeks later, the ear thickness of normal and atopic dermatitis rats was measured and IgE levels in serum, which is an important factor in atopic dermatitis, were examined to determine whether atopic symptoms were well induced.

19 is a photograph showing the ear thickness of normal rats and atopy-induced rats, Figure 20 is a graph showing the concentration of IgE in fluorescence.

19 and 20, it can be seen that the ears of the atopic dermatitis-induced mice are relatively swollen, thicker, harder, and keratinous. In addition, when IgE concentration in serum was confirmed by ELISA, it was confirmed that the concentration was increased about 13 times in atopic dermal rats compared to normal rats.

2) Effect of Herbal Extracts on Atopic Symptoms

i) B cell: IgE secretion inhibitory activity

The most important factor in atopy is the concentration of IgE in serum. B cells are differentiated into plasma cells to secrete antibodies. At this time, class switching occurs due to interleukin-4 (IL-4), and IgG is converted to IgE. Therefore, CD19 + B cells were isolated from lymph nodes of atopy-induced rats and treated with LPS and IL-4 while medicinal extracts were treated with 1%, 2.5%, and 5% for 72 hours before they were present in the supernatant. IgE concentration was measured by ELISA.

21 is a graph showing the concentration of IgE according to the concentration of the herbal extract, it can be seen that the concentration of IgE is reduced compared to the control when treated with the herbal extract. This degree of inhibition can be seen to increase with increasing the concentration of herbal extracts.

ii) T cells: effect on cytokine expression

Atopy is known that Th2 cytokines (interleukin-4, interleukin-5, interleukin-13) play an important role in the development and progression of atopy, and recently, Th1 cytokines (interleukin-12, interferon) Gamma) is also known to play an important role in sustaining the symptoms. Based on this, the effect of the extract on the expression level of cytokines was examined to determine the mechanisms by which the extracts can alleviate the symptoms of atopy.

After CD4 + T cells were isolated from lymph nodes around the ear, the site where the atopy was induced, the extracts were treated with 0% to 5% in vitro and treated with PMA + ionomycin for 6 hours, followed by atopy related cytokines. The expression level of Caine (IL-4, IL-5, IL-10, IL-13, IFNg, TNFa) was confirmed by mRNA level using real-PCR.

22 is a graph showing the effect of the herbal extracts on the cytokine expression level of CD4 + T cells. Referring to FIG. 22, the mRNA levels of IL-5, IL-10, and TNFa, among the six cytokines screened, were reduced by the herbal extract and showed a concentration-dependent tendency.

F) conclusion

The effects of atopy controllability of natural herbal extracts consisting of noni and colostrum colostrum were investigated through in vitro and in vivo atopic models.

Herbal extracts are not toxic to cells (up to a concentration of 0-10%) and effectively inhibit the secretion of IgE, an important factor in atopy, not only in cell lines but also in B cells in atopic conditions. Confirmed.

To determine the effect on T cells and to determine more precise mechanisms of action, the effects of cytokine expression were found. Herbal extracts lowered the levels of IL-5, IL-10, and TNFa among various cytokines. Seems to look.

This confirms that the herbal extract has a potential for atopic control.

Example  9. Atopic improvement efficacy test of atopic skin improver according to the present invention

In order to measure the improvement effect of the herbal composition for improving atopic dermatitis according to the present invention, 10 infants and 6-year-old children with atopic dermatitis were tested by applying to the affected area twice a day for 2 months. After the application for 2 months, the efficacy of improvement was quantified into five levels of satisfaction, satisfaction, don't know, dissatisfaction, and very dissatisfaction.

Table 23 shows the results of the self-evaluation of the clinical trial.

name gender Occur
Time Hospital treatment
experience Extremely
dissatisfaction dissatisfaction I do not know satisfied very good OO south child U √ OO female 08/10 U √ Park OO south 2 months after birth radish √ OO south 3-4 years old U √ OO Choi south 4 years old U √ Park OO south √ OO female 4 years old U √ Sim OO female 3 years old U √ OO south newborn baby U √ Hwang OO female 15 months after birth U √ Sum 0 0 2 5 3 (0.0%) (0.0%) (20%) (50%) (30%) 80% / 100%

As shown in the clinical trial self-assessment results, eight out of ten participants (80%) were satisfied. In addition, during the treatment period of 20 days on average, the cure rate is 76%, which is very high, and satisfactory for most clinical participants.

Figures 23a to 23k is a photograph of each part of the human body before the application of the herbal composition of the present invention, Figures 24a to 24k correspond to each of the Figure 23a to 23k, each part of the human body after the application of the herbal composition of the present invention It is a photograph.

Claims (16)

An atopic therapeutic composition comprising an alginic acid oligomer, a tangerine peel extract, a noni extract, and a vortex extract as active ingredients. The method of claim 1,
When the total weight of the atopic therapeutic composition is 100 parts by weight, the alginic acid oligomer is 10 to 20 parts by weight, characterized in that the atopic treatment composition. The method of claim 2,
15 to 25 parts by weight of the tangerine extract, 5 to 15 parts by weight of the Warsong extract, 3 to 7 parts by weight of the Noni extract is characterized in that the atopic treatment composition. The method of claim 3,
The atopic therapeutic composition is an atopic therapeutic composition, characterized in that the emulsion form comprising an aqueous layer and an oil phase layer. The method of claim 4, wherein
The tangerine peel extract, the Noni extract, and the Wasong extract are atopic treatment composition, characterized in that dissolved in the aqueous layer. The method of claim 4, wherein
The oily layer comprises at least one of jojoba oil, evening primrose oil, camellia oil, and shea butter. The method of claim 1,
An atopic therapeutic composition further comprising an emulsifier or thickener. The method of claim 7, wherein
The emulsifier or thickener is an atopic therapeutic composition, characterized in that the olive oil wax or cetyl alcohol. The method of claim 1,
An atopic therapeutic composition comprising at least one of aloe extract, fish extract, glycerin, silkamino acid, and menthol. Preparing shredded seaweed or kelp;
Adding acid to the seaweed or kelp;
Adding a base to the mixture and subjecting it to heat treatment;
Adding ethanol to the mixture; And
Filtering the mixture and drying the filtered solution to obtain alginic acid. Extracting alginic acid from seaweeds;
Low molecular weight of the alginic acid to form an alginic acid oligomer; And
A method for preparing an atopy composition comprising mixing a tangerine peel extract, a noni extract and a vortex extract to the alginic acid oligomer. The method of claim 11,
The alginic acid is extracted by any one of an acid hydrothermal treatment method, an alkaline hot water extraction method, an acid / alkaline hot water extraction method, and a high-temperature pressure treatment method. The method of claim 11,
The acid number heat treatment method
Preparing shredded seaweed or kelp;
Adding acid to the seaweed or kelp;
Adding a base to the mixture and subjecting it to heat treatment;
Adding ethanol to the mixture; And
Filtering the mixture and drying the filtered solution to obtain alginic acid. The method of claim 11,
The mixing step
Dissolving the alginic acid oligomer in water;
Adding the tangerine peel extract, the noni extract and the wasong extract to a solution in which the alginic acid oligomer is dissolved to form an aqueous phase layer;
Forming an oily layer comprising oil; And
Mixing the aqueous layer and the oily layer to form an emulsion. The method of claim 14,
The forming of the emulsion may include emulsifying half of the aqueous layer by emulsifying the oily layer, and then emulsifying the remaining half of the aqueous layer by adding the emulsified oily layer and the aqueous layer to emulsify. . The method of claim 14,
In the method of forming the emulsion, a half of the oil phase layer is emulsified by mixing the water phase layer, and the remaining half of the oil phase layer is added to the emulsified oil phase layer and the water phase layer to prepare a predetermined atopic treatment product. Way.

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