KR20160054222A - Pharmaceutical composition for anti-oxidative or anti-inflammatory comprising leaf extract of alaskan ginseng - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for antioxidative or anti-inflammatory activities, comprising a leaf extract of Alaskan ginseng as an active ingredient. The composition comprising powder extracted from Alaskan ginseng leaves as an active ingredient reduces active oxygen inside a cell, and has excellent effects in preventing and treating the inflammation. In addition, the composition has no toxicity, and can be ingested in a form of a food.

Description

TECHNICAL FIELD The present invention relates to an antioxidant or anti-inflammatory pharmaceutical composition comprising an alaskan leaf extract as an active ingredient,

The present invention relates to a pharmaceutical composition and a health functional food which are capable of reducing intracellular active oxygen and preventing and treating inflammation by using an extract extracted from leaves of Alaskanicum as an active ingredient.

Due to the development of modern industrial society, humans enjoy economical leisure and cultural benefits, but life and death are directly or indirectly threatened due to environmental pollution and overeating. Therefore, interest in health of modern people is higher than ever before, and interest in functional foods or medicines with health maintenance and biorhythm control effect is increasing.

On the other hand, all aerobic organisms, including humans, utilize oxygen to conduct energy metabolism and have a self-defense function against the injury of reactive oxygen generated during the process. However, production of reactive oxygen, Oxygen toxicity is becoming increasingly recognized as a cause of various diseases such as degenerative diseases associated with aging and aging, as well as arteriosclerosis, hypertension, diabetes, arthritis, circulatory disorders, and cancer.

Oxygen, often called harmful oxygen, is a superoxide anion, which is the singlet oxygen produced by the reduction and oxidation of triplet oxygen (3O ₂ ), the safest form of oxygen, : O ₂ ^-), hydrogen peroxide _{(hydrogen peroxide, H 2 O 2} ) and hydroxyl radicals (hydroxyl radical, a radical (free radical), such as OH), they are proteins, DNA, enzymes, and cells, T- (T-cell ) And causes various diseases by damaging the factors of the immune system. In particular, the problem is that active oxygen attacks lipid peroxidation reaction by attacking unsaturated fatty acid which is a constituent of cell membrane, accumulates lipid peroxidation in the body, resulting in deterioration of biological function and induction of aging and adult diseases.

Therefore, studies on antioxidants, which are known as substances capable of regulating active oxygen, have been actively carried out, and it has been known that the lipid peroxidation inhibitor can directly induce oxidative damage in vivo, It has been extended to more active antioxidants such as free radical scavengers or preventive antioxidants that inhibit the radical and reactive oxygen production itself. Specific examples of the antioxidants include antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase, tocopherol, ascorbic acid, carotenoids, Glutathione and vitamin C, synthetic antioxidants tert-butylhydroxytoluene (BHT) and tert-butylhydroxyanisol (BHA), trace elements germanium (Ge) and selenium Many antioxidants have been developed and used. However, these conventional antioxidants have been limited in their use due to various problems such as toxicity, low activity, limitations of application, and adverse effects due to overdose.

In recent years, it has been known that phenylpropanoid compounds not only prevent various adult diseases but also have an effect on the treatment of rheumatoid arthritis of COX-2. Thus, domestic pharmaceutical companies are in the stage of development as a therapeutic agent for arthritis using natural products, .

Accordingly, studies for searching for a safer and stronger antioxidant from natural products or microbial metabolites have been actively carried out. Especially, since adult diseases are mostly metabolic diseases and the cause of disease is caused by the gradual progress of diseases caused by reactive oxygen species, drugs for prevention, such as preventive medicine and functional foods, that is, antioxidants There is also a growing interest in taking or consuming, such as vitamins or tea, at all times.

Korea Patent Publication No. 2010-0054386 Korean Patent Publication No. 2007-0078658

It is an object of the present invention to provide a pharmaceutical composition containing an extract extracted from leaves of Alaskanicum as an active ingredient to reduce intracellular reactive oxygen and prevent and treat inflammation.

Another object of the present invention is to provide a health functional food containing the extract extracted from the leaf of Alaskan canthus as an active ingredient.

In order to accomplish the above object, the pharmaceutical composition for antioxidant or antiinflammation of the present invention may contain a leaf extract of Alaskanthus as an active ingredient.

The leaf extract of Alaskanthus can be extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof.

The lower alcohol having 1 to 4 carbon atoms may be 20 to 80% methanol, ethanol, butanol or propanol.

Preferably, the alaskan maras leaf extract may be an ethanol-extracted extract.

The extraction temperature may be 15 to 30 ° C, and the extract of Alaskan marigold may be extracted 2 to 4 times.

The concentration of the alaskanthin leaf extract may be 0.001 to 20 mg / ml.

In order to achieve the above-mentioned other objects, the antioxidant or anti-inflammatory health functional food of the present invention may contain a leaf extract of Alaskan canthus as an active ingredient.

The leaf extract of Alaskantha can be an extract extracted with ethanol.

The health functional food may be selected from the group consisting of capsules, tablets, powders, granules, liquids, pills, flakes, pastes, syrups, gels, jellies and tablets.

The antioxidant or antiinflammatory pharmaceutical composition containing the extract of Alaskanthus leaf extract of the present invention as an active ingredient is effective in reducing intracellular reactive oxygen species, exhibiting excellent inflammation prevention and therapeutic effect, and being free from toxicity, have.

FIG. 1 is a graph showing (A) DPPH free radical scavenging activity and (B) ABTS free radical scavenging activity using extract powders prepared according to one embodiment of the present invention and a comparative example.
2 is a graph showing the reducing power side determined by using the extract powder prepared in accordance with the embodiment and comparative examples of the present invention.
FIG. 3 is a graph showing SOD-like activity using extract powder prepared according to one embodiment of the present invention and a comparative example.
FIG. 4 is a graph showing inhibition of lipid peroxidation measured using extract powder prepared according to one embodiment of the present invention and a comparative example.
5 is a graph showing (A) NO production, (B) iNOS inhibitory activity and (C) cell viability of the extract powder prepared according to an embodiment of the present invention.
FIG. 6 is a graph showing (A) IL-6 inhibitory effect and (B) TNF-alpha inhibitory effect of the extract powder prepared according to one embodiment of the present invention.

The present invention relates to a pharmaceutical composition and a health functional food which are capable of reducing intracellular active oxygen and preventing and treating inflammation by using an extract extracted from leaves of Alaskanicum as an active ingredient.

Hereinafter, the present invention will be described in detail.

The pharmaceutical composition for antioxidant or antiinflammation of the present invention contains a leaf extract of Alaskanicum as an active ingredient.

The alaskan ginseng (devil`s club, scientific name: Oplopanax horridus ) is native to the Americas, and is distributed mainly in southern Alaska, eastern Oregon, and Washington. It is a deciduous shrub, grows about 30-90 cm, and has a straight nature. It has yellow thorns, the leaves are crossed and bright green. Flowers are yellowish or greenish, and fruits are reddish.

In order to prepare the leaf extract of Alaskanthus, firstly, it is washed with water to remove the water of the alaskan marasifolia from which the foreign substance has been removed, and then dried and pulverized.

Next, the prepared alaskan marigold leaf powder and the extraction solvent are mixed at a weight ratio of 1: 8 to 12, preferably 1:10 to 12, at a temperature of 15 to 30 ° C, preferably 22 to 26 ° C, 48 hours, preferably 0.1 to 20 hours, twice or four times to prepare an extract.

Examples of the extraction solvent include water, lower alcohols having 1 to 4 carbon atoms, and mixed solvents thereof. The lower alcohol may be 20 to 80% methanol, ethanol, butanol or propanol. However, in the present invention, extraction using 70 to 80% of ethanol as an extraction solvent is preferable for the antioxidant and anti-inflammation It is preferable from the viewpoint that it can be described.

When the weight ratio of the alaskan marigold leaf powder to the extraction solvent is out of the above range, the effective ingredient of the alaskan marigold leaf may be extracted in the extract in a small amount.

If the extraction temperature is lower than the lower limit, antioxidant and anti-inflammatory effects can not be expected. If the extraction temperature is higher than the upper limit, antioxidative and anti-inflammatory effects may be poor.

In addition, it is preferable to repeat the extraction twice or four times in order to extract the active ingredient of the alaskan marigold leaf as much as possible at the time of extraction. The above-mentioned repeated extraction means extracting the alaskan leaf with the extractant solvent and adding the extractant and alaskanthus leaf to the extract. The extracted extract and the newly added mixture (alaskan leaf powder + Extraction solvent) is preferably mixed at a weight ratio of 1: 0.5-1 in order to extract as much as possible the active ingredient.

The alaskanthin leaf extract of the present invention is used at a concentration of 0.001 to 20 mg / ml. If the concentration is below the lower limit, antioxidative and anti-inflammatory effects can not be expected. If the concentration exceeds the upper limit, toxicity may be caused .

The leaf extract of Alaskanthus can be used as the extract itself or may be powdered for convenient use. The method for pulverizing the leaf extract of Alaskan is as follows: the extract is concentrated under reduced pressure to reduce its volume and then lyophilized by a freeze dryer to be powdered.

The pharmaceutical composition for antioxidant or antiinflammation comprising the alaskan marine leaf extract of the present invention as an active ingredient may contain 0.001 to 20% by weight, preferably 0.01 to 10% by weight, based on the total weight of the pharmaceutical composition, %.

The antioxidant or anti-inflammatory pharmaceutical composition may be formulated into various oral or parenteral dosage forms.

Examples of formulations for oral administration include tablets, pills, hard, soft capsules, liquids, suspensions, emulsions, syrups, granules and the like. These formulations may contain, in addition to the active ingredient, a diluent such as lactose, dextrose, (For example, silica, talc, stearic acid and magnesium or calcium salts thereof and / or polyethylene glycol), in addition to the active ingredient (s). The tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid Or a disintegrating or boiling mixture such as its sodium salt and / or an absorbent, a colorant, a flavoring agent and a sweetening agent. The formulations may be prepared by conventional mixing, granulating or coating methods.

Representative examples of formulations for parenteral administration include injectable preparations, and water, Ringer's solution, isotonic saline or suspensions may be mentioned as a solvent for the injectable preparation. The sterile, fixed oils of the injectable preparations may be used as a solvent or suspending medium, and any non-irritating fixed oils, including mono-, di-glycerides, may be used for this purpose. The injectable preparation may be a fatty acid such as oleic acid.

The dosage of the pharmaceutical composition for antioxidant or antiinflammation containing the alaskan marine leaf extract of the present invention as an active ingredient varies depending on the age, sex, weight and severity of the disease of the patient, The dosage is not more than 300 mg per kg body weight, preferably 100 to 200 mg per day, but not limited thereto.

The antioxidant or anti-inflammatory health functional food containing the alaskan maras leaf extract of the present invention as an active ingredient contains 0.0001 to 10% by weight of a leaf extract of Alaskan canola based on the total weight of the health functional food .

The health functional food may be in the form of a capsule, tablet, powder, granule, liquid, ring, flaky, paste, syrup, gel, jelly and jute for the purpose of supplementing nutrients that may be lacking in daily eating or supplementing functional raw materials useful in the human body And is easily manufactured and operated once.

In addition, the health functional food may contain at least one selected from the group consisting of glucose, citric acid, liquid oligosaccharide, corn syrup, soybean lecithin, butter, vegetable hardening oil, skim milk, sugar, margarine, salt, starch, And can be prepared by using commonly used components such as sodium bicarbonate and sugar ester.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention. Such variations and modifications are intended to be within the scope of the appended claims.

Example  One. Alaskan Fresh  Leaf extract

The leaves of Alaskan japonica were washed with water, and the water was removed and then pulverized with a freeze dryer. The prepared alaskanthin leaf powder and 70% ethanol were mixed at a weight ratio of 1:12, and the mixture was extracted at 25 ° C for 1 hour. To the extract, a mixture (Alaskan marigold leaf powder: 70% ethanol = 1: 12 weight ratio ) Was added and extraction was carried out at 25 DEG C for 1 hour. This procedure was repeated two more times to obtain an extract. At this time, the extract and the mixture (alaskan marigold leaf powder: 70% ethanol = 1: 12 weight ratio) are mixed at a weight ratio of 1: 1. The extract obtained by repeating the above three times was filtered and concentrated under reduced pressure to reduce the volume to 1/5. The concentrated extract of Alaskanthus sinensis leaf extract was pulverized with a freeze dryer to prepare alaskanthin leaf extract powder.

Comparative Example 1: Straw extract of alaskan

The same procedure as in Example 1 was carried out except that alaskan canthus stalks were used instead of alaskan canthus leaves to prepare alaskan canthus stem extract powder.

Comparative Example 2. Root Extract of Alaskan Origin

The same procedure as in Example 1 was carried out except that alaskan canthus root was used instead of alaskan canthus leaves to prepare alaskan canthus root extract powder.

<Test Example>

Test Example 1. Total polyphenol and flavonoid measurement

1-1. Total phenol content (mg GAE / g): After the sample is prepared with 10 mg / mL of extracted powder, add 2 mL of Folin-Ciocalteu phenol reagent (Sigma) to the sample, mix and react at room temperature for 5 minutes. 2 mL of 7% sodium carbonate was added to the reaction mixture, and the mixture was allowed to stand at room temperature for 1 hour and 30 minutes, and the absorbance was measured at 750 nm. At this time, gallic acid was used as the indicator material.

1-2. Total flavonoid content (mg QE / g) Measurement: After the sample was prepared with the extract powder of 10 mg / mL, the sample was mixed with 5% NaNO ₂ , left at room temperature for 5 minutes, mixed with 10% AlCl ₃ dissolved in water After reacting at room temperature for 6 minutes, NaOH was mixed and absorbance was measured at 510 nm. At this time, the indicator material was catechin.

Table 1 below shows the contents of (A) total phenol and (B) total flavonoids in the extract powder prepared according to the examples and comparative examples of the present invention ( p <0.05).

division Example 1 Comparative Example 1 Comparative Example 2 Extraction yield (%) 10.0 11.0 12.0 Total phenol content (mg GAE / g) 54.3 ± 0.3 ^a 16.3 + - 0.2 ^c 35.4 ± 0.0 ^b Total flavonoid content (mg QE / g) 5.2 ± 0.6 ^a 1.3 ± 0.1 ^c 2.0 ± 0.1 ^b

As shown in the above Table 1, the alaskan kenate leaf extract powder prepared according to Example 1 is superior to the alaskan kenate stem extract powder or alaskan kenate root extract powder prepared according to Comparative Examples 1 and 2, Phenol contents and total flavonoid contents were remarkably excellent.

Test Example  2. Antioxidant measurement ( DPPH  And ABTS free Radical Scatters  Measure)

Antioxidant activity was measured by the DPPH method and the ABTS method using an antioxidant such as vitamin C (1 mg / mL) as a comparative sample.

2-1. DPPH measurement (%): The antioxidative activity by reducing power was measured using a free radical called 2,2-Di (4-tert-octylphenyl) -1-picrylhydrazyl free radical (DPPH). DPPH was reduced by the test substance The degree of decrease in absorbance is compared with the absorbance of the blank test solution and the free radical scavenging rate is measured at a wavelength of 560 nm. As a 0.1 mM solution of DPPH (Aldrich Chem. Co., MW = 618.76), 61.88 mg was dissolved in methanol to make 100 mL. For the measurement, 0.15 mL of 0.1 mM of the extract is added to 0.15 mL of 0.1 mM DPPH solution in a 96-well plate, and incubation is started at 25 ° C for 10 minutes, and the absorbance St at 560 nm is measured. The blank test is carried out in the same manner as above using distilled water instead of the sample solution, and the absorbance Bt is measured. Also, the blank of the sample solution is measured by the same procedure using methanol instead of the 0.1 mM DPPH solution to determine the absorbance Bo.

The results were calculated by the following formula (1), and the results are shown in FIG.

[Equation 1]

St: absorbance at 560 nm after free radical scavenging of the sample solution

Bt: Absorbance at 560 nm after free radical scavenging of blank test solution

So: the absorbance at 560 nm before the reaction in the absence of free radicals in the sample solution

Bo: absorbance at 560 nm before the reaction in the absence of the free radical of the blank test solution

2-2. ABTS measurement (%): The ABTS radical scavenging experiment is one of the measurement methods of total antioxidant activity (TAA), in which a colorless reduced ABTS is oxidized to form a characteristic turquoise ABTS ^{O +} (ABTS radical cation) Method. This cyan ABTS ^{O +} reacts with the oxidizable substance to reduce the original colorless ABTS and oxidize the reacted substance, resulting in a decrease in absorbance and an antioxidant ability. 5 ml of 7 mM ABTS and 88 uL of 140 mM K2S2O8 were mixed and allowed to stand in the dark for 14 to 16 hours. The mixture was mixed with absolute ethanol at a ratio of about 1:88, and the absorbance of the control was adjusted to 0.7 ± 0.002 at 734 nm. solution. 50 uL of the sample solution and 1 ml of the ABTS solution were mixed, shaken for 30 seconds, reacted for 2.5 minutes, absorbance was measured at 734 nm, and ABTS radical scavenging activity was calculated according to the following equation.

&Quot; (2) &quot;

FIG. 1 is a graph (A) showing DPPH free radical scavenging activity and (B) ABTS free radical scavenging activity using extract powders prepared according to Examples and Comparative Examples of the present invention ( p <0.05).

As shown in FIG. 1, under the same experimental conditions, 1 mg / ml of ascorbic acid (Vit C) showed 98% DPPH activity and 99% ABTS activity, and the extract powder of Example 1 contained 99% DPPH (10 mg / ml) and 99% ABTS activity (5 mg / ml and 10 mg / ml), respectively.

On the other hand, the extract powder of Comparative Example 2 showed lower DPPH activity and ABTS activity than Vit C and the extract powder of Example 1.

The extract powder of Comparative Example 2 (5 mg / ml and 10 mg / ml) had slightly lower DPPH activity than the extract powder of Example 1 (10 mg / ml), and the extract powder of Comparative Example 2 10 mg / ml) was slightly lower than that of the extract powder of Example 1 (5 mg / ml and 10 mg / ml).

Test Example  3. Antioxidant measurement (reduction power measurement)

The reducing power of the extract powders prepared according to Examples and Comparative Examples was measured by slightly modifying the method of Kuda et al., Journal of Food Composition and Analysis, 18, pp625-633, 2005). 0.2 ml of 0.2 M phosphate buffer (pH 6.6) and 1% potassium ferricyanide (0.2 ml) were added to 0.2 ml of each sample, followed by reaction at 50 ° C for 20 minutes. Then, 0.2 ml of 10% trichloroacetic acid was added to each of the reacted samples, followed by centrifugation at 1,000 × g for 10 minutes. The supernatant (0.125 ml) was mixed with the same amount of distilled water (DW) and 0.02 ml of 0.1% ferric chloride (FeCl3) was added to a 96-well microplate (SPL, Suwon, Korea) , And a VERSA max microplate reader (Molecular Devices Corp., Sunnyvale, Calif.). The high absorbance of the reacted samples showed very high reducing power and the reducing power of the sample was calculated based on Vit C as the control group.

FIG. 2 is a graph showing reduction powers measured using extracted powders prepared according to Examples and Comparative Examples of the present invention ( p < 0.05).

As shown in FIG. 2, the reducing power at 0.01 mg / ml and 0.10 mg / ml of each extract was excellent in the order of Example 1> Comparative Example 2> Comparative Example 1, and 0.10 mg / ml of Example 1 It was confirmed that it has a higher reducing power than Vit C, which is a control group.

Test Example  4. Antioxidant measurement (Superoxidase Dismutase ( Superoxide dismutase ; SOD ) Similar activity measurement)

The SOD-like activity of extract powders prepared according to Examples and Comparative Examples was measured according to the method of Marklund S and Marklund G (1974). 2.6 ml of Tris-HCl buffer (50 mM tris + 10 mM EDTA, pH 8.5) and 0.2 ml of 7.2 mM pyrogallol were added to 0.2 ml of each sample solution, and reacted at 25 ° C for 10 minutes. Then, 0.1 ml of 1 M HCl was added to stop the reaction The amount of pyrogallol oxidized in the reaction solution was measured at 420 nm, and the SOD-like activity was shown by the following formula (3).

&Quot; (3) &quot;

FIG. 3 is a graph showing SOD-like activity using extract powders prepared according to Examples and Comparative Examples of the present invention ( p < 0.05).

As shown in FIG. 3, the control group, 0.01 mg / ml of tetramethylchromancarboxylic acid (Trolox) showed 99.8% SOD-like activity under the same experimental conditions, and at a concentration of 0.01 mg / ml and 0.10 mg / ml The extracted powders of Example 1 contained 17.5%, 97.2%, respectively; The extracted powders of Comparative Example 1 were 0% and 15.4%, respectively; The extracted powders of Comparative Example 2 showed similar activities of 13.4% and 95.2%, respectively, and 0.10 mg / ml of Example 1 showed similar activity to the control.

Test Example 5. Antioxidant measurement (measurement of inhibition of lipid peroxidation)

20 ml of linoleic acid suspension was prepared by mixing 1 ml of linoleic acid, 0.2 ml of tween 20, and 19.7 ml of deionized water. 0.2 ml of ascorbate (0.01%), 0.2 ml of ferrous sulfate (FeSO4, 0.01%), 0.5 ml of a phosphate buffer solution (0.02 M, pH 7.4) And 0.4 ml of the extract powder (IE) prepared in Example 1 were mixed and reacted at 37 ° C for 12 hours. 0.2 ml of trichloroacetic acid (TCA, 4%), 0.2 ml of butylated hydroxytoluene (BHT, 0.4%) and 2 ml of trivalenturic acid (TBA, 0.8%) were added to 2 ml of the reaction solution The mixture was allowed to stand at 100 ° C for 30 minutes, cooled, and extracted with 2 ml of chloroform. Optical density (OD) was measured at 532 nm. The lipid peroxidation inhibition (%) of the extract powder according to the present invention was measured using the measured optical density (OD), and the formula was as shown in the following formula (4).

&Quot; (4) &quot;

FIG. 4 is a graph showing inhibition of lipid peroxidation measured using extract powder prepared according to Examples and Comparative Examples of the present invention ( p < 0.05).

As shown in FIG. 4, 1 mg / ml of ascorbic acid (Vit C) showed 32% inhibition of lipid peroxidation under the same experimental conditions, 41% of the extract powder of Example 1 at the same concentration as the control group. 28% of the extracted powder of Comparative Example 1; The extract powder of Comparative Example 2 showed 38% inhibition of lipid peroxidation, and Example 1 and Comparative Example 2 showed superior lipid peroxidation inhibitory power to the control group.

Test Example  6. Anti-inflammatory measures (cytotoxicity, NO  Generation and iNOS  Measurement of inhibitory activity)

The anti-inflammatory test is carried out using the extracted powder of Example 1 having excellent antioxidant ability.

6-1. Cell viability (%) measurement: To perform the test in the range of the safety concentration of the sample, cytotoxicity was measured by modifying the method of Rochem et al. Cells (2 × 10 ⁵ cells / well) (RAW 264.7) were seeded in 24-well plates and cultured for 24 hours. Then, the medium was removed and 1 ml of the medium containing 3% of the serum in which the extract was dissolved was dispensed and cultivation was continued for 24 hours. After 24 hours, 0.25 mL of XTT-PMS solution (1 mg XTT and 10 g PMS / mL of MEM without phenol red) was added to each well and incubated for another 2 hours. The degree of cytotoxicity was determined by measuring the absorbance of formazan at 450 nm using a microplate reader. The medium was MEM containing 10% v / v fetal bovine serum (FBS), 0.5% (v / v) 50 g / ml streptomycin, 50 IU / ml penicillin, 0.125 g / ml fungizone, 3.024 g sodium bicarbonate And incubated at 37 ℃, 5% CO _{2 ,} 95% humidified air.

6-2. NO production measurement (%): In order to confirm the effect of the extract powder of Example 1 on the production of NO induced by LPS, experiments were carried out by the method described in the literature (Kim, JY et al , Isoliquiritigenin isolated from the roots of glycyrrhiza uralensis inhibits LPS-induced iNOS and COX-2 expression via the attenuation of NF-kappaB in RAW264.7 macrophages, Eur. J. Pharmacol., 584 (1), pp.175-184 , 2008). Cells (RAW 264.7) were seeded at a cell concentration of 5 × 10 ⁵ cells / well on a 24-well plate and the cells were treated with various concentrations (0, 6.25, 12.5, 25, 50 ug / ml) , Treated with 1 ug / ml of LPS and cultured for 24 hours. LPS was used as a positive control to induce NO production. Nitrite levels of the culture medium were determined using Griess reaction assay. Briefly, 100 uL of cell culture medium contains 100 uL of Griess reaction solution (1% w / v sulfanilamide containing 5% (v / v) phosphoric acid and 0.1% (w / v) naphtylethylenediamine -HCl), and the reaction was carried out at room temperature for 10 minutes. The absorbance was measured at 540 nm using a microplate reader (Perkin Elmer Cetus, Forster City, CA, USA). The medium was used as a blank in all experiments. The nitrite concentration of the sample was determined using a standard curve of the concentration of sodium nitrite.

6-3. Measurement of iNOS Inhibitory Activity: Whether the extract powder of Example 1 had an activity of inhibiting iNOS expression was confirmed by Western blotting. For the test method, RAW 264.7 cells were suspended in RPMI medium at a concentration of 1 × 10 ⁶ cells / ml, placed in a culture dish, treated with the extract powder of Example 1 at 37 ° C. in a 5% CO ₂ incubator After 1 hour, the cells were treated with 1 ug / ml LPS for 24 hours. As a control group, LPS-treated group and LPS-treated group were used. Cells were then harvested and Western blot was performed using anti-mouse iNOS antibody (BD Bioscience, USA, dilution 1: 1,000), and as a control to show no change in other proteins in the sample, anti-mouse Beta-actin antibody (Cell Signaling Technology, USA; dilution ratio 1: 1,000) was used.

FIG. 5 is a graph showing (A) NO production, (B) iNOS inhibitory activity and (C) cell viability of the extract powder prepared according to Example 1 ( ^# p <0.05, ^## p <0.01 vs. negative control * p <0.05, ** p <0.01 vs. LPS-treated positive control).

As shown in FIG. 5A, it was confirmed that NO production was suppressed as the concentration of extract powder of Example 1 increased. As a result, it was confirmed that the treatment with the extract powder of Example 1 showed a protective effect against cell damage caused by LPS-induced NO and could alleviate the inflammatory reaction.

As shown in FIG. 5B, it was confirmed that iNOS expression was increased in the group to which the extract powder of Example 1 was not administered. In the group to which the extract powder of Example 1 was administered, the concentration of the extract powder of Example 1 And the expression of iNOS was inhibited.

As shown in Fig. 5C, the extract powder of Example 1 was found to be low in cytotoxicity as a whole.

Test Example  7. Anti-inflammatory measure IL -6 and TNF - alpha  Measure)

The anti-inflammatory (immunological activity) of the extract powder of Example 1 was confirmed using macrophages (RAW 264.7). For this purpose, RAW 264.7 macrophages were treated with the extract powder of Example 1, and after 1 hour, 1 ug / mL of lipopolysaccharide (LPS) was treated for cell activation and cultured again for 24 hours. Then, cell culture medium was recovered and ELISA for IL-6 and TNF-alpha was performed. The IL-6 and TNF-alpha are known as inflammatory cytokines with various biological activities associated with normal defense responses and malignant inflammatory immune diseases such as septic shock, rheumatoid arthritis, and autoimmune diseases.

Figure 6 is a graph showing the extraction of the produced powder (A) The inhibitory effect of IL-6 and (B) TNF alpha-inhibiting effect according to the embodiment ^{1 (## p <0.01 vs. negative} control; * p <0.05 , ** p < 0.01 vs. LPS-treated positive control).

As shown in FIG. 6A, it was confirmed that IL-6 production was increased in the group treated with LPS alone compared to the group not treated with LPS. Production of IL-6 increased by LPS was observed according to Example 1 It was confirmed that the powder was inhibited by the prepared extract powder. Specifically, it was confirmed that when the concentration of the extract powder prepared in Example 1 was 100 ug / ml, the production of IL-6 was inhibited by about 25% or more.

As shown in FIG. 6B, it was confirmed that the TNF-alpha production was increased in the LPS-treated group compared to the LPS treated group, and the increased TNF-alpha production by the LPS was observed in Example 1 It was confirmed that the extraction powders prepared according to the present invention were inhibited by the extract powder. Specifically, it was confirmed that when the concentration of the extract powder prepared in Example 1 was 100 ug / ml, the production of TNF-alpha was inhibited by about 75% or more.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Preparation Example 1. Preparation of powder

The alaskanthin leaf extract powder obtained in Example 1    500 mg

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation Example 2. Preparation of tablets

300 mg of the alaskanthus leaf extract powder obtained in Example 1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

200 mg of the alaskanthus leaf extract powder obtained in Example 1

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation Example 4. Preparation of injection

600 mg of Alaskanthus leaf extract powder obtained in Example 1

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

It is prepared by the above-mentioned component content per ampoule according to the usual injection preparation method.

Formulation Example 5. Preparation of a liquid preparation

4 g of the alaskan marine leaf extract powder obtained in Example 1

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100g by adding purified water, To prepare a liquid agent.

Formulation example  6. Preparation of granules

1,000 mg of the alaskanthus leaf extract powder obtained in Example 1

Vitamin mixture quantity

Vitamin A Acetate 70

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2

Vitamin C 10 mg

Biotin 10

Nicotinic acid amide 1.7 mg

Folic Acid 50

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

The composition ratio of the above-mentioned vitamins and minerals is comparatively comparatively mixed with the granules according to the preferred embodiment. However, the blending ratio may be arbitrarily changed, and the above components are mixed according to the ordinary granule preparation method, Can be prepared and used in the manufacture of a health functional food composition according to a conventional method.

Formulation example  7. Manufacture of functional beverages

1,000 mg of the alaskanthus leaf extract powder obtained in Example 1

Citric acid 1,000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to the flask to obtain a total of 900 mL

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 L container, It is used in the production of the functional beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (13)

A pharmaceutical composition for antioxidant or antiinflammatory activity, which comprises a leaf extract of Alaskanicus as an active ingredient. The pharmaceutical composition for antioxidant or antiinflammatory activity according to claim 1, wherein the leaf extract of alaskanthin is extracted with water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The pharmaceutical composition according to claim 2, wherein the lower alcohol having 1 to 4 carbon atoms is 20 to 80% methanol, ethanol, butanol or propanol. The pharmaceutical composition for antioxidant or anti-inflammation according to claim 1, wherein the leaf extract of Alaskan is a ethanol extract. The pharmaceutical composition according to claim 1, wherein the extraction temperature is 15 to 30 占 폚. The pharmaceutical composition for antioxidant or antiinflammatory activity according to claim 1, wherein the extract of Alaskan marigold leaves is repeated 2 to 4 times. The pharmaceutical composition for antioxidant or anti-inflammation according to claim 1, wherein the concentration of the alaskanthin leaf extract is 0.001 to 20 mg / mL.  A health functional food for antioxidant or antiinflammatory characterized by containing leaf extract of Alaskan japonica as an effective ingredient. 9. The antifungal or anti-inflammatory health food according to claim 8, wherein the leaf extract of Alaskan is a ethanol extract. The health functional food for antioxidant or anti-inflammation according to claim 8, wherein the extraction temperature is 15 to 30 占 폚. [9] The antifoaming or anti-inflammatory health food according to claim 8, wherein the extract of Alaskan japonica leaf is repeated 2 to 4 times. [9] The antifoaming or anti-inflammatory health food according to claim 8, wherein the concentration of the alaskanthin leaf extract is 0.001 to 20 mg / mL. The health functional food according to claim 8, wherein the health functional food is selected from the group consisting of capsules, tablets, powders, granules, liquids, rings, flakes, pastes, syrups, gels,

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