KR102110510B1 - Phytosome comprising Centella asiatica extracts and the method preparing thereof - Google Patents

Abstract

The present invention relates to an anti-atopic dermatitis effect of a phytosome containing a centella asiatica (phytosome) and a manufacturing method thereof and the phytosome containing the centella asiatica extract, a phytosome containing a centella extract according to the present invention Silver has excellent stability and sealing efficiency, and has an effect of significantly improving anti-atopic dermatitis activity compared to using the extract itself as the extract is used in the form of a phytosome.

Description

Phytosome comprising Centella asiatica extracts and the method preparing thereof

The present invention relates to an anti-atopic dermatitis effect of a phytosome containing a centella asiatica (phytosome) and a preparation method thereof and a phytosome containing the centella asiatica extract.

Atopic dermatitis (AD) is a chronic inflammatory skin disease caused by the infiltration of inflammatory cells such as mast cells, eosinphils, and T lymphocytes. Is caused by several genetic factors, environmental factors, and immunological abnormalities. AD caused by allergen increases IL-4, IL-5 and IL-13 secretion of Th2 cells and increases serum IgE production. Increased Th2 cytokines in the acute and chronic AD stages directly affect epidermal keratinocytes, which are inflammatory skin lesions that infiltrate immune cells. Produces pro-inflammatory cytokines. Therefore, inflammation and activation of immune cells play an important role in the development of AD.

Keratinocytes are involved in the inflammatory response based on the production of pro-inflammatory cytokines that play an important role in AD. Stimulation of epidermal keratinocytes by TNF-α and IFN-γ induces the expression of various inflammatory mediators. Therefore, suppression of activation of these cytokines and keratinocytes may be effective in treating skin diseases.

Nuclear factor kappa B (NF-κB) is a factor that plays an important role in regulating inflammatory responses in AD and various immune responses such as AD. Facilitated translocation of NF-κB can exacerbate allergic inflammation by increasing the production of inflammatory cytokines and chemokines. Therefore, compounds that inhibit NF-κB activation can be applied as therapeutic agents for allergic diseases. Cycloporine and tacrolimu (FK506) are potent immunosuppressive agents and have been used to treat AD. In addition, various methods have been reported for pharmacological control of AD containing steroid and topical administration of NF-κB inhibitors, and NF-κB inhibitor IMD-0354 suppresses abnormal proliferation of mast cells to reduce allergic reactions. There are research results. Therefore, activation of NF-κB is important for the progression of allergic inflammation.

On the other hand, phytosomes are drugs used to improve vesicular drug delivery systems in which lipoid membranes and phospholipid molecules are bound and linked. Durg carriers. Phytosomes are liposome vesicles formed by the interaction of hydrogen bonds between the phospholipids of the lipid membrane and the phytomolecules of plants, and deliver the skin during transdermal and dermal delivery of the skin. It plays a role in improving the permeability of therapeutic agents. Phytosome complexes increase the skin absorption rate of phytomolecules for regulating skin physiology, and accordingly, attention has been paid to human body systemic function through the skin as well as potential utilization as a cosmetic raw material.

Although various pharmacological effects of the extract of Cypress have been reported, many studies have not been conducted on its phytosome formulation and anti-dermatitic effect. Thus, the present inventors have developed a phytosome (Centella asiatica phytosome; CA phytosome) containing the extract of Cypress, confirmed that the stability and drug encapsulation efficiency of CA phytosome according to the present invention is excellent, and its anti-dermatitis effect and mechanism of action Confirmed to complete the present invention.

Sung JE, Lee HA, Kim JE, Go J, Seo EJ, et al. Lab Anim Res. 2016; 32 (1): 34-45. Shinomol GK, Muralidhara and Bharath MM. Recent Pat Endocr Metab Immune Drug Discov. 2011; 5 (1): 33-49.

An object of the present invention is to provide a phytosome containing Centella asiatica extract.

Another object of the present invention is to provide a method for producing the phytosome containing the centella extract.

In order to achieve the object of the present invention as described above,

The present invention is a step of preparing a first solution by dissolving Centella asiatica extract in at least one first solvent selected from the group consisting of 50 to 100% by volume of ethanol aqueous solution, 50 to 100% by volume of methanol aqueous solution and chloroform. (Step 1);

Preparing a second solution by dissolving the phospholipid in 50 to 100% by volume of an ethanol aqueous solution (second solvent) (step 2); And

Mixing and reacting the first solution and the second solution, followed by concentration and powdering (step 3);

It provides a phytosome containing a centella asiatica extract characterized in that it is prepared by a method comprising a.

In addition, the present invention provides a method for producing a phytosome containing the extract of Centella asiatica.

The phytosome containing the titrated extract of Centella asiatica according to the present invention is very excellent in stability and encapsulation efficiency, and as the extract is used in the form of a phytosome, anti-atopic dermatitis compared to using the extract itself There is an effect that the activity is significantly improved.

1 is a view showing the effect of LPS-treated on RAW 264.7 macrophages, NO production, iNOS and COX-2 and NF-κB DNA binding activity according to CA phytosome treatment; (A) Quantitative analysis results of nitrite induced by LPS using Griess reaction, (B) Western blot analysis results for iNOS and COX-2 protein expression, (C) NF-κB DNA in RAW 264.7 cells by EMSA As a result of binding activity analysis, (D) Western blot analysis results for p50, p65, IκBα and p-IκBα protein expression.

Hereinafter, the present invention will be described in detail.

Bottleweed ( Centella asiatica ) Contains extract Phytosome ( phytosome )

The present invention is a step of preparing a first solution by dissolving Centella asiatica extract in at least one first solvent selected from the group consisting of 50 to 100% by volume of ethanol aqueous solution, 50 to 100% by volume of methanol aqueous solution and chloroform. (Step 1);

Preparing a second solution by dissolving the phospholipid in 50 to 100% by volume of an ethanol aqueous solution (second solvent) (step 2); And

Mixing and reacting the first solution and the second solution, followed by concentration and powdering (step 3);

It provides a phytosome containing a centella asiatica extract characterized in that it is prepared by a method comprising a.

In the phytosome containing the centella extract according to the present invention, the first solvent may be an aqueous ethanol solution of 60 to 80% by volume, and the second solvent may be an aqueous ethanol solution of 85 to 99% by volume. Preferably, the first solvent is an ethanol aqueous solution of 65 to 75% by volume, the second solvent may be an ethanol aqueous solution of 90 to 99% by volume, the first solvent is an 69 to 71% by volume ethanol aqueous solution, and the second More preferably, the solvent is an aqueous ethanol solution of 94 to 96% by volume.

In general, as a reaction solvent in the manufacturing process of phytosomes, aprotic solvent is selected from the group consisting of dioxane, acetone, methylene chloride and ethyl acetate. However, in one embodiment of the present invention, one or more solvents selected from the group consisting of methanol, ethanol, and chloroform are used as reaction solvents, respectively, and the solution (the first solution is) And a second solution).

The phytosome containing the centella extract according to the present invention may contain 30 to 50 parts by weight of the centella extract as compared to 100 parts by weight of the phytosome. Preferably it may be contained in 30 to 35 parts by weight, more preferably 33 to 34 parts by weight. In one embodiment of the present invention, it was confirmed that the phytosome contained a quantitative extract of centella (TECA) in a weight ratio of 33.5%.

Centella asiatica (L.) Urban (syn. Hydrocotyl asiatica L.) belongs to the family Apiaceae, one of the plants used in dermatology, Gotu kola, Indian pennywort Also known as. The medicinal herb grows in tropical regions of Asia, Oceania, Africa, and America, and the medicinal herb is used to improve the cognitive ability of skin degenerative diseases and neurodegenerative diseases such as gastric ulcer, gastric mucosal lesions and anxiety, as well as peeling epidermis It has also been found to be useful in the treatment of skin diseases such as burns, hypertrophic scars or eczema, and sinus lesions, and is also useful in improving microcirculation in chronic venous insufficiency. Centella extract is registered as a cosmetic ingredient (Bylka, Znajdek-Awizen, Studzinska-Sroka, & Brzezinska, 2013) in the International Nomenclature of Cosmetic Ingredients (INCI).

The bottled grass extract according to the present invention may be obtained by extracting from nature using an extraction method known in the art, and the extract defined in the present invention is extracted from the bottled grass using an appropriate solvent, for example, It may be an extract obtained by extracting a bottled grass containing all of the crude extract, a polar solvent soluble extract, a non-polar solvent soluble extract, or a titrated extract of Centella asiatica (TECA). The centella extract is preferably a quantitative extract of centella (TECA) comprising 25 to 35 parts by weight of asiatic acid and 25 to 35 parts by weight of madecaffeic acid compared to 40 parts by weight of asiaticoside, more preferably 40 parts by weight of asiaticoside It may be a quantitative extract of centella composed of 30 parts by weight of asiatic acid and 29 to 30 parts by weight of madec acid.

As a suitable solvent for extracting the extract of the medicinal herb extract, any of pharmaceutically acceptable organic solvents may be used, and water or an organic solvent may be used, but is not limited thereto. For example, purified water, methanol (methanol) ), Alcohols having 1 to 4 carbon atoms, acetone, ether, benzene, chloroform, including ethanol, propanol, isopropanol, butanol, etc. ), Ethyl acetate, methylene chloride, hexane and cyclohexane can be used alone or in combination. Preferably, one or more solvents selected from the group consisting of ethanol, ethyl acetate and hexane can be used.

As the extraction method, any one of methods such as hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, water vapor distillation, ultrasonic extraction, elution, and compression can be used. In addition, the desired extract may be further subjected to a conventional fractionation process, or may be purified using a conventional purification method. There is no limitation in the method for producing the extract of the medicinal herb of the present invention, and any known method can be used.

For example, the centella extract contained in the composition of the present invention may be used as it is while the primary extract extracted by the solvent extraction method described above is centrifuged, filtered, concentrated and freeze-dried and stored in a refrigerator compartment, and the primary extract is used. It may be prepared in powder form by additional processes such as distillation under reduced pressure and freeze drying or spray drying. In addition, the primary extract is further purified using various chromatography such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. You can also get

Therefore, in the present invention, the extract of coleoptera is a concept that includes all of the extracts, fractions and refinements, their dilutions, concentrates or dried products obtained at each stage of extraction, fractionation or purification.

In the phytosome containing the centella extract according to the present invention, the phospholipid may be phospholipid selected from the group consisting of phosphatidylcholine, phophatidylethanolamine and phosphatidylserine, preferably May be phosphatidylcholine, particularly preferably phosphatidylcholine from soy. The phytosome according to an embodiment of the present invention generally has an advantageous economical effect by using phosphatidylcholine derived from soybean, which is much cheaper than the reagent-grade phosphatidylcholine used for phytosome production.

In the phytosome containing the centella extract according to the present invention, the step 1 or step 2 may further include the step of removing the insoluble material by using the first solution or the second solution using filter paper.

In step 3, the centella extract and phospholipids may be mixed in a molar ratio of 1: 1 to 3, and the concentration is the cost of the solvent (aliphatic hydrocarbon), lyophilization, freeze drying, and spray drying. , May be concentrated (evaporated) by a method such as vacuum concentration, vacuum drying, and distillation.

Bottleweed ( Centella asiatica ) Contains extract Phytosome  Manufacturing method

The present invention is also a first solution by dissolving Centella asiatica extract in one or more first solvents selected from the group consisting of water, 50 to 100% by volume of ethanol aqueous solution, 50 to 100% by volume of methanol aqueous solution and chloroform. Manufacturing step (step 1);

Preparing a second solution by dissolving the phospholipid in 50 to 100% by volume of an ethanol aqueous solution (second solvent) (step 2); And

Mixing and reacting the first solution and the second solution, followed by concentration and powdering (step 3);

It provides a method for producing a phytosome containing a centella asiatica extract.

In the manufacturing method according to the present invention, the first solvent may be an aqueous solution of ethanol of 60 to 80% by volume, and the second solvent may be an aqueous solution of ethanol of 85 to 99% by volume. Preferably, the first solvent is an ethanol aqueous solution of 65 to 75% by volume, the second solvent may be an ethanol aqueous solution of 90 to 99% by volume, the first solvent is an 69 to 71% by volume ethanol aqueous solution, and the second More preferably, the solvent is an aqueous ethanol solution of 94 to 96% by volume.

In the manufacturing method according to the present invention, the phytosome containing the centella extract may contain 30-50 parts by weight of the centella extract compared to 100 parts by weight of the phytosome. Preferably it may be contained in 30 to 35 parts by weight, more preferably 33 to 34 parts by weight.

In the manufacturing method according to the present invention, the extract of centella asiatic acid comprises 25 to 35 parts by weight of asiatic acid and 25 to 35 parts by weight of madecaic acid compared to 40 parts by weight of asiaticoside (titrated extract of Centella asiatica; TECA) Can be Preferably, it may be a quantitative extract of centella, consisting of 30 parts by weight of asiatic acid and 29 to 30 parts by weight of madecaffeic acid compared to 40 parts by weight of asiaticoside.

In the manufacturing method according to the present invention, the phospholipid may be phospholipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, preferably phosphatidylcholine ( phosphatidylcholine), particularly preferably phosphatidylcholine from soy.

In the manufacturing method according to the present invention, the step 1 or the step 2 may further include the step of removing the insoluble material by using the first solution or the second solution using a filter paper.

In the manufacturing method according to the present invention, in step 3, the centella extract and phospholipids may be mixed in a molar ratio of 1: 1 to 3, and the concentration is the cost of the solvent (aliphatic hydrocarbon), lyophilized It may be concentrated (evaporated) by a method such as (lyophilization, freeze drying), spray drying, vacuum concentration, vacuum drying, and distillation.

In the present invention, a phytosome (CA phytosome) containing a titrated extract of Centella asiatica improves the symptoms of dermatitis of the ears and back induced by phthalic anhydride (PA) treatment in a rat model, It suppresses iNOS (inducible nitric oxide synthase) and COX-2 (cyclooxygenase-2) expression induced by PA or LPS, release of TNF-α, IL-1β and IgE in the blood, and production of NO (nitric oxide), LPS It was experimentally demonstrated that it reduces the NF-κB DNA binding activity induced by p50, p65, IκBα and p-IκBα protein expression (see Experimental Examples 3 to 9). In addition, it was experimentally proved that CA phytosomes containing the extract of Centella extract showed similar or better effect in a smaller dose than when using the Classified extract of Centella (see Experimental Example 10). Therefore, the phytosome containing the centella extract according to the present invention can be usefully used for preventing, treating or improving atopic dermatitis.

Hereinafter, the present invention will be described in more detail by examples. It will be apparent to those skilled in the art that these examples are merely for more specifically describing the present invention, and the scope of the present invention is not limited to these examples.

< Manufacturing example  1> Bottleweed  Preparation of quantitative extract

After collecting the centella asiatica collected in the oven at 50 ° C., it was powdered using a milling machine. The powdered bottle paste (1 kg) was extracted at room temperature for 3 days using 4 L of 75% (v / v) ethanol, and this was repeated three times in total. The extract was subjected to a filtration process using a deep filter coated with activated carbon (depth-filter), and the separated precipitate and filtrate fractions were respectively concentrated under reduced pressure at 80 ° C. The precipitate fraction was dried at a temperature of 50 ° C. to make an asiaticoside powder, so that the yield was 0.12% (w / w) of the dried screen. Subsequently, the filtrate fraction was hydrolyzed at 80 ° C. with an alkali solution containing sodium hydroxide (1%, w / v), and then concentrated, precipitated and dried in the same manner as the above method. The yield of the genin powder containing asiatic acid and madecassic acid obtained from the filtrate fraction was 0.18% (w / w) of the above-ground portion of the dried centella. The asiaticoside powder and the genin powder extract were mixed to prepare a titrated extract of Centella asiatica (TECA). The components of the quantitative extract of centella (TECA) are composed of 40% by weight of asiaticoside, 30% by weight of asiatic acid, and 29-30% by weight of madecaic acid (Table 1). All solvents used were commercial grade, and were purchased from Dongkuk Pharmaceutical (Chungbuk, Korea).

extract ingredient Ingredient content (% by weight) Centella quantitative extract
(Titrated extract of Centella asiatica; TECA) Asiaticoside 40 Asiatic acid 30 Madecassic acid 29-30

< Example  1> Bottleweed  Containing extracts Phytosome  Produce

Phosphatidylcholine derived from soybeans in bulk form used to prepare health foods on the market for the production of CA phytosome (CA phytosome) contained in the phytosome formulation of the extract of corticulum (TECA) Was purchased, CA phytosomes were prepared by reacting about 1 mole of phosphatidylcholine and 1 mole of Centennial extract in ethanol.

Specifically, the first solution was prepared by dissolving 13.6 g of Centrifugal Extract (TECA) of Preparation Example 1 using 150 ml of 70% ethanol (first solvent) by shaking at room temperature to remove insoluble substances with Whatman filter paper. . 8.2 g of the soybean-derived phosphatidylcholine powder was dissolved using 120 ml of 95% ethanol (second solvent), and then, similarly, a second solution was prepared by removing insoluble substances with Whatman filter paper. After mixing the first solution and the second solution, the mixture was mixed 10 times for 30 seconds using a high-speed mixer, and then distilled and removed by distilling ethanol at room temperature so that the volume of the solution was about half of the initial volume using a vacuum distiller. The solution was again transferred to a plate made of steel use stainless (SUS) to have a thickness of about 5 mm, and then all liquid components were removed using a vacuum freeze dryer. When using a vacuum freeze dryer, after cooling to minus 20 degrees for the first 3 hours, vacuum was used for over-night. The solids from which the liquid component was removed were collected with a spatula and ground in a mortar for 3 minutes to finally obtain a CA phytosome (centella asiatica phytosome; CA phytosome). CA phytosomes were sealed and used at room temperature.

At this time, when comparing and comparing the quantitative extract (TECA) produced in the Preparation Example 1 as a standard substance, the CA phytosome of the same weight as the TECA standard substance has a 33.5% weight ratio compared to the standard TECA substance (TECA) ).

< Comparative example  1 to 6> Bottleweed  Containing extracts Phytosome  Produce

In the same manner as in Example 1, a CAP phytosome containing the extract of medicinal herb (TECA) in a phytosome formulation was prepared, but the types of the first solvent used for the preparation of the first solution were different as shown in Table 2 below. Comparative Examples 1 to 6 were prepared.

1st solvent Second solvent Comparative Example 1 Distilled water 95% ethanol Comparative Example 2 50% ethanol 95% ethanol Comparative Example 3 95% ethanol 95% ethanol Comparative Example 4 100% ethanol 95% ethanol Comparative Example 5 70% methanol 95% ethanol Comparative Example 6 chloroform 95% ethanol

< Experimental Example  1> CA Phytosome  Encapsulation rate and stability analysis

1-1. CA according to the reaction solvent Phytosome Encapsulation rate  evaluation

In order to evaluate the encapsulation efficiency and drug content of quantitative extract (TECA) in CA phytosome according to the difference of the first solvent, the CA phytosome of Example 1 and Comparative Examples 1 to 6 was centrifugal filter device (Amicon centrifugal filter device) , molecular weight cut off [MWCO], 10000 Da, Millipore, Billerica, MA, USA), and then filtered by ultrafiltration, it was measured by HPLC. 20 μl of the filtered sample was injected into a C18 column (Sepax BR-C18, 5 μm 120 mm 4.6 × 150 mm). As a mobile phase, a mixture of acetonitrile and water in a ratio of 1: 1 was used, and a flow rate of 1.0 ml / min was used. In order to detect unenclosed centellar extract (TECA), 500 μl of the phytosome formulation was filtered for 30 minutes at 14,000 rpm through centrifugal ultrafiltration. The content of the extract of centella (TECA) in the filtrate was measured using HPLC with UV monitoring at 227 nm. The encapsulation efficiency and extract content were calculated using the following formula and expressed as a percentage as shown in Table 3 below.

Encapsulation efficiency (%) = ((Whole weight of TECA used in manufacturing phytosomes-weight of TECA not enclosed in phytosomes) / (weight of all TECAs used in phytosome production)) × 100

TECA content (%) = ((Whole weight of TECA used when preparing phytosomes-weight of TECA not encapsulated in phytosomes) / (weight of prepared CA phytosomes)) × 100

1st solvent Encapsulation efficiency (%) TECA content (%) Comparative Example 1 Distilled water 40.3 9.6 Comparative Example 2 50% ethanol 67.5 16.7 Example 1 70% ethanol 95.6 33.5 Comparative Example 3 95% ethanol 80.2 26.2 Comparative Example 4 100% ethanol 74.8 19.6 Comparative Example 5 70% methanol 51.9 16.0 Comparative Example 6 chloroform 48.3 13.4

As a result, it was found that when using ethanol rather than using distilled water, methanol, or chloroform, the encapsulation efficiency and TECA content of CA phytosomes were remarkably excellent. In particular, 70% ethanol as the first solvent and 95% ethanol as the second solvent It was confirmed that the encapsulation efficiency and TECA content of Example 1 in which CA phytosomes were prepared were highest.

1-2. CA according to the reaction solvent Phytosome  Storage stability evaluation

In order to evaluate the storage stability of CA phytosomes according to the difference of the first solvent, the CA phytosomes of Examples 1 and Comparative Examples 1 to 14 were stored in a constant temperature bath controlled at a relative humidity of 70% ± 5 and a temperature of 25 ° C. Immediately after production, after a week, 1 month, and 6 months elapsed, the particle size was measured using a particle measuring device, and precipitation and separation were observed through an optical microscope (MEIJI VCC-6574A, SANYO). The particle size was expressed as a percentage as shown in Table 4 below by calculating the ratio (%) of the particle size after one week, one month, and six months to the particle size (100%) immediately after manufacture.

1st solvent Immediately after manufacturing (%) A week later(%) After 1 month (%) 6 months later(%) Comparative Example 1 Distilled water 100.0 120.4 138.4 212.4 Comparative Example 2 50% ethanol 100.0 117.2 122.5 197.8 Example 1 70% ethanol 100.0 100.2 100.9 102.1 Comparative Example 3 95% ethanol 100.0 103.0 110.8 143.5 Comparative Example 4 100% ethanol 100.0 106.7 115.3 172.7 Comparative Example 5 70% methanol 100.0 110.5 128.4 196.9 Comparative Example 6 chloroform 100.0 115.3 132.1 204.4

As shown in Table 4, it can be seen that the CA phytosome of Example 1 according to the present invention has excellent storage stability because the particle size does not increase significantly over time. Also, as a result of visual observation, precipitation and separation The phenomenon did not appear.

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2-1. Gel electrophoresis migration analysis

Gel electromobility shift assay (EMSA) was performed according to the manufacturer's instructions (Promega, Madison, WI, USA). Briefly, 1 × 10 ⁶ cells were washed twice with 1x PBS, then 1 ml of PBS was added and the cells were scraped off and collected in a cold Eppendorf tube. Cells were centrifuged at 15,000 g for 1 minute and the resulting supernatant was removed. Solution A [10 mM HEPES, pH 7.4, 10 mM potassium chloride (KCl), 1.5 mM magnesium chloride (MgCl ₂ ), 0.5 mM dithiothreitol, 0.2 μg / ml phenylmethylsulfonyl fluoride, 1 Μg / ml pepstatin A, 1µg / ml leupeptin, 10µg / ml soybean trypsin inhibitor, 10µg / ml aprotinin and 0.5% Nonidet P-40 ] Was added to the pellet at a 2: 2 ratio (v / v) and incubated on ice for 10 minutes. After centrifuging the cells at 15,000 g for 7 minutes, the supernatant was used as a cytosolic extraction (nuclear extract). Solution C (Solution A + 20% glycerol, 0.2 mM EDTA and 420 mM NaCl) was added to the pellets at a ratio of 2: 1 (v / v), vortexed on ice for 20 minutes, and centrifuged at 15,000 g for 15 minutes. Separated. The oligonucleotide sequence for NF-κB was 5'-AGT TGA GGG GAC TTT CCC AGG C-3 '. The consensus oligonucleotide was end-labeled at 37 ° C. for 10 minutes using T4 oligonucleotide kinase and [ ³² P] ATP. ³² P- labeled oligonucleotide ⁽³² P-labeled oligonucleotide) to the nuclear extract (10μg) and after reaction at room temperature for 20 minutes, gel loading buffer was added to each reaction and the 1㎕ nondenaturing (6% nondenaturing gel gel ), And electrophoresis was performed until 3/4 of the gel was dyed. The gel was dried at 80 ° C. for 1 hour and exposed to the film overnight at 70 ° C. The relative density of protein bands was scanned by density measurement using MyImage (SLB, Seoul, Korea) and quantified by Labworks 4.0 software (UVP Inc., Upland, CA, USA).

2-2. Cell culture

Murine macrophage cell RAW 264.7 cells were purchased and used from the Korea Cell Line Bank (Seoul, Korea). RAW 264.7 cells were grown in a DMEM medium containing 10% FBS, penicillin (100 units / ml) and streptomycin sulfate (100 μg / ml) at 37 ° C., 5% CO ₂ , humidified atmosphere. Cells were incubated with various concentrations (5, 10 or 20 μg / ml) of CA phytosomes or positive chemicals, and then treated with 1 μg / ml of LPS to stimulate for 24 hours or 1 hour. At this time, CA phytosomes of various concentrations dissolved in ethanol were added together with LPS. The final concentration of ethanol used was less than 0.05%. Cells were treated with 0.05% ethanol as vehicle control.

2-3. Nitrite quantitative analysis

The nitric oxide (NO) concentration was determined by measuring the level of nitrite in the cell culture medium. RAW 264.7 mouse macrophages were seeded in a 6-well plate (1 × 10 ⁶ cells / well) with 2 ml of cell culture medium and cultured for 24 hours. The old culture medium is discarded and replaced with a new medium to keep the cells. RAW 264.7 macrophages were pretreated with various concentrations of CA phytosomes (5, 10 and 20 μg / ml). LPS (1 μg / ml) was treated for 24 hours for all samples except the control group to induce nitrite production in RAW 264.7 macrophages. Then, 100 μl of the Griess reagent [0.1% NED, 1% sulfanilamide, 2.5% phosphoric acid] was added to 100 μl of the collected supernatant and incubated for 10 minutes at room temperature and dark conditions. The nitrite level was confirmed by measuring absorbance at a wavelength of 540 nm using a microplate reader, and the NO concentration was determined by comparison with a standard curve.

2-4. Statistics processing

The experiments of the present invention were performed in triplicate, and all experiments were repeated three or more times with similar results. Data are expressed as mean values ± standard deviation. Statistical treatment was performed using the Student's t test at the following significance levels: * p <0.05 (significance level between the experimental group and the untreated control), #p <0.05 (significance level between the experimental group and the PA treatment group) .

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<Experimental Example 3> Effect of CA phytosome on LPS-induced NO production and iNOS and COX-2 expression in RAW 264.7 cells

By measuring the nitrite released from the culture medium by the Griess reaction using the method for quantitative analysis of nitrite of Experimental Example 2-3, the effect of CA phytosomes on NO production induced by LPS in RAW 264.7 cells was confirmed. When LPS and CA phytosomes (5, 10, 20 μg / ml) were treated together for 24 hours, the nitrite concentration increased by LPS was significantly decreased depending on the CA phytosome concentration (FIG. 1A). In addition, in order to determine whether CA phytosome inhibits NO production through inhibition of gene expression related to NO production, iNOS expression was confirmed by Western blot analysis, and since iNOS can be regulated by COX-2, COX- Western blot analysis was also performed on 2 expressions. Cells showed very low levels of iNOS and COX-2 protein expression in the unstimulated state (control), but after 24 hours of LPS (1 μg / ml) treatment, expression increased significantly in response to LPS, and CA phyto Some (5, 10, 20 μg / ml) treatment reduced the concentration of iNOS and COX-2 increased by LPS in RAW 264.7 cells in a concentration-dependent manner (FIG. 1B).

<Experimental Example 4> Effect of CA phytosomes on NF-κB DNA binding activity in RAW 264.7 cells

Activation of NF-κB plays an important role in iNOS and COX-2 induction by LPS or other inflammatory cytokines. Thus, the present inventors confirmed that CA phytosomes can inhibit NF-κB activation in LPS-activated RAW 264.7 cells. As a result of the previous experiment, the LPS treatment time for NF-κB activation to the maximum was found to be 1 hour, and RAW 264.7 cells were co-treated with LPS and CA phytosomes for 1 hour each. A nuclear extract was prepared from the co-treated cells, and NF-κB DNA binding was analyzed through EMSA of Experimental Example 2-1. In RAW 264.7 cells, LPS induced potent NF-κB binding activity, which was significantly inhibited in a concentration-dependent manner by simultaneous treatment with CA phytosomes (FIG. 1C). In addition, the present inventors confirmed that CA phytosomes can inhibit not only activation of NF-κB but also translocation and IκB phosphorylation of the NF-κB subunit. As a result, CA phytosomes inhibited LPS-induced NF-κB transcription and DNA binding activity in a concentration-dependent manner. As with the inhibitory effect on NF-κB activity, it was confirmed that the nuclear translocation of p65 and p50 was inhibited concentration-dependently, and phosphorylation of IκB-α by LPS was also suppressed concentration-dependently by CA phytosomes (FIG. 1D).

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So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will appreciate that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

Claims (11)

Preparing a first solution by dissolving a centella asiatica extract in an aqueous ethanol solution (first solvent) of 68 to 72% by volume (step 1);
Preparing a second solution by dissolving the phospholipid in 95% by volume of an aqueous ethanol solution (second solvent) (step 2); And
Mixing the first solution and the second solution and then concentrating and pulverizing them (step 3);
Method for producing a phytosome containing a centella asiatica extract, characterized in that it is prepared by a method comprising a. delete delete delete According to claim 1,
The phytosome is a manufacturing method characterized in that it contains 30 to 50 parts by weight of Centella extract compared to 100 parts by weight of phytosome. According to claim 1,
The method of manufacturing the extract is characterized in that it contains 25 to 35 parts by weight of asiatic acid and 25 to 35 parts by weight of madecaic acid compared to 40 parts by weight of asiaticoside. According to claim 1,
The phospholipid is a phosphatidylcholine (phosphatidylcholine) manufacturing method characterized in that. According to claim 1,
In step 3, the medicinal extract and phospholipids are prepared by mixing in a molar ratio of 1: 1 to 3. delete delete delete

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