KR20180021012A - the natural composite with the fuction of antioxidation and antiphlogistics - Google Patents

Abstract

The present invention relates to a medicinal composition having antioxidant and anti-inflammatory effects, and more specifically, to a composition having antioxidant and anti-inflammatory effects, which is extracted from a mixture of Angelica gigas NAKAI fine roots, Caesalpinia sappan L., Lindera strichnifolia Fernandez-Villar roots, and paeony, and is harmless to liver and kidney; and to a pharmaceutical composition comprising the same. The present invention provides a natural composition having antioxidant and anti-inflammatory effects, which is extracted from a mixture of Angelica gigas NAKAI fine roots, Caesalpinia sappan L., Lindera strichnifolia Fernandez-Villar roots, paeony, Cyperus rotundus Linne rhizome, Prunus persica Batsch seeds, Carthamus, licorice, and cinnamon. In addition, the present invention provides a natural composition having antioxidant and anti-inflammatory effects, which is extracted from a mixture comprising: 100 parts by weight of Angelica gigas NAKAI fine roots; 50-80 parts by weight of Caesalpinia sappan L.; 50-80 parts by weight of Lindera strichnifolia Fernandez-Villar roots; 50-80 parts by weight of paeony; 50-80 parts by weight of Cyperus rotundus Linne rhizome; 30-70 parts by weight of Prunus persica Batsch seeds; 30-70 parts by weight of Carthamus; 20-50 parts by weight of licorice; and 20-50 parts by weight of cinnamon. In addition, the present invention provides a pharmaceutical composition comprising the composition having antioxidant and anti-inflammatory effects.

Description

Natural compositions with antioxidant and anti-inflammatory properties,

The present invention relates to a pharmaceutical composition which is effective for antioxidant and antiinflammation. More specifically, it relates to a composition which is extracted from a mixture of Angelica keiskei koidz., Jojoba, miso, and anti-inflammatory drugs and which is harmless to liver and kidney and has antioxidant and anti- ≪ / RTI >

The oxidation of the human body is due to free radicals, and the free radical theory was proposed by D. Harman in the mid-1950s. Free radicals are produced by the action of leukocyte phagocytosis, the action of electron transport system, myeloperoxide (MPO) during the production of ATP in mitochondria, ultraviolet rays, tobacco, normal metabolic processes, stress, pollutants and bacteria When the radicals remain in the body due to the cause, they cause various problems such as wrinkle formation, skin cancer, cell death, rheumatoid arthritis, atopic dermatitis, acne and the like through cell destruction, connective tissue cleavage and cross- . Superoxide dismutase (SOD), catalase, vitamin E, vitamin C, and ubiquinol, which are natural antioxidants (radical scavenging agents), exist in the human body to remove free radicals. The antioxidant system begins to decline gradually due to age, pollution, ultraviolet rays, stress, etc., and the antioxidant system is disabled, leading to an increase in free radicals in the body.

The increased radical destroys collagen (collagen), elastin, and hyaluronic acid, which are connective tissues of the dermis, causing skin settlement (wrinkles), oxidizing the lipid part of the cell membrane and destroying the cell, , Skin cancer and the like. In addition, this radical participates in the spontaneous oxidation reaction during the melanin formation process, and causes the cause such as stain, freckles, and wrinkles.

In addition, the reduction of inflammation can be measured by a reduction in the density of inflammatory cell types, such as monocytes or astrocytes, neutrophils, mast cells, and basophils. The reduction of inflammation can also be measured by measuring neutrophil activity (Jones et al., 1994). Factors such as frequency of mast cell degranulation or measurement of histamine levels or levels of reactive oxygen species can also be used as a measure of inflammation reduction. Levels of inflammation may also be determined by PCR using specific genes such as interferon-alpha, -beta and -gamma, tumor necrosis factor-alpha, interleukin 1 beta, -2, -4, -5, -6, -8, -18, -23, -27, CD4, CD28, CD80, CD86, MHCII, and iNOS. Measurement of tissue and / or patient fluid, such as plasma proinflammatory cytokines, may be a measure of inflammation reduction.

Such treatment of inflammation or the spread of a medicament having an anti-inflammatory effect has been problematic in that it induces toxicity to its own cells in the case of human or in the case of medicine.

Therefore, the development of anti-inflammatory agents using natural materials is greatly promoted.

The composition disclosed in Japanese Patent Application Laid-Open No. 10-2010-0000728 (composition for external application for antiinflammation or wound healing containing herbal medicine extracts, hereinafter referred to as "prior art") refers to a composition comprising at least one herbal medicine extract of chrysanthemum, An antiinflammatory composition containing at least one of the extracts of Chrysanthemum morifolium extract, Quercus mongolica extract, Quercus gamma extract or Chrysanthemum morifolium extract and a mixture of the herbal medicine extract and Fogue extract as an active ingredient, Composition "

The above prior art and prior art still show toxicity to cells and cause side effects. Therefore, the present invention provides a natural extract composition having antioxidant and anti-inflammatory functions that exclude the toxicity of cells to the maximum.

In addition, the above-mentioned prior art and prior art still show toxicity to liver and kidney, causing side effects. Thus, the present invention provides a natural extract composition having antioxidant and anti-inflammatory functions without influence of liver and kidney do.

The present invention also provides a natural extract composition having antioxidant function and anti-inflammatory function

In order to solve the above problems and needs,

The present invention provides a natural composition having antioxidative and antiinflammatory properties extracted from a mixture of Angelica keiskei koidz., Jojoba, miso, anti-arabic medicine, herbal supplement, doensein, safflower, licorice and cinnamon.

The present invention also relates to a method for preparing a herbal composition comprising 50 to 80 parts by weight of a root, 50 to 80 parts by weight, 50 to 80 parts by weight, 30 to 70 parts by weight, 30 to 70 parts by weight of safflower, 50 to 80 parts by weight, 20 to 50 parts by weight of licorice and 20 to 50 parts by weight of cinnamon are mixed to provide a natural composition having antioxidative and antiinflammatory properties.

The present invention also provides pharmaceutical compositions comprising the above antioxidant and anti-inflammatory compositions.

The natural composition according to the present invention shows remarkable efficacy in antioxidative and anti-inflammatory properties compared to the prior art and prior art.

In addition, the composition having antioxidative and anti-inflammatory effects according to the present invention shows remarkably high antioxidative and anti-inflammatory functions without showing any toxicity to the liver and kidney.

FIG. 1 is a photograph showing a remarkable decrease in the size of the wound of a wound caused by administering the natural composition according to the present invention. FIG.
FIG. 1B is a graph showing a significant decrease in the size of wound of a wound caused by administering the natural composition according to the present invention. FIG.
FIG. 2 is a graph showing the leukocyte production amount of the wound-induced rats administered with the natural composition according to the present invention.
FIG. 3 is a graph comparing neutrophil production to wound-induced white blood cells of a rat by administering the natural composition according to the present invention.
FIG. 4 is a graph comparing the amount of lymphocytes produced in wound-induced white blood cells of a rat by administering the natural composition according to the present invention.
FIG. 5 is a graph comparing the amount of mononuclear cells produced in wound-induced leukocytes of a subject by administering the natural composition according to the present invention.
FIG. 6 is a photograph of the amount of MMP-1, 2, and 9 expressed in the wound tissue of wound-induced rats administered with the natural composition according to the present invention.
FIG. 6B is a graph showing the results of measuring the amount of MMP-1, 2, 9 expressed in the wound tissue of wound-induced rats by administering the natural composition according to the present invention.
FIG. 7 is a photograph showing the staining of wounded skin tissues induced by wounding by administering the natural composition according to the present invention. FIG.

BRIEF DESCRIPTION OF THE DRAWINGS FIG.

The present invention provides a composition having antioxidative and antiinflammatory properties extracted from a mixture of Angelica keiskei koidz., Jojoba, miso, red pepper, herbal extract, safflower, safflower, licorice and cinnamon.

Preferably, 50 to 80 parts by weight of leek, 50 to 80 parts by weight of lees, 50 to 80 parts by weight of the leaching agent, 50 to 80 parts by weight of rosin, 30 to 70 parts by weight of rosin, 30 to 70 parts by weight of safflower, 20 to 50 parts by weight of licorice and 20 to 50 parts by weight of cinnamon are mixed to provide an antioxidant and anti-inflammatory composition.

The present invention further enhances the antioxidant and anti-inflammatory effects by adding an auxiliary composition to the above-mentioned composition.

The above-mentioned auxiliary composition means a mixture of evening primrose, willow and tangerine.

In the present invention, 5 to 20 parts by weight of evening primrose, 10 to 30 parts by weight of willow and 10 to 30 parts by weight of tangerine fruit may be further added to the natural composition, based on 100 parts by weight of Angelica gigas.

Therefore, the present invention further preferably comprises 50 to 80 parts by weight of lees, 50 to 80 parts by weight of lees, 50 to 80 parts by weight of the leaching agent, 50 to 80 parts by weight of roots, 30 to 70 parts by weight of roots, 30 to 70 parts by weight of safflower, 20 to 50 parts by weight of licorice, 20 to 50 parts by weight of cinnamon, 5 to 20 parts by weight of evening primrose, 10 to 30 parts by weight of willow and 10 to 30 parts by weight of tangerine fruit, To provide a composition that is effective for anti-inflammation.

The present invention relates to a natural composition (hereinafter referred to as "DG") extracted from the above raw materials, and exhibits remarkable effects on antioxidative and anti-inflammatory properties.

The present invention also provides a pharmaceutical composition comprising a composition (DG) effective for the above-mentioned antioxidant and anti-inflammation.

The above pharmaceutical composition means a drug for the prevention and treatment of antioxidative function and anti-inflammatory, which means that the natural composition (DG) of the present invention is included therein.

The above-mentioned Angelica sp. Of the present invention refers to a cut-off of only the tail portion of the Angelica gigas. I think that it makes the blood (blood) run well.

The above-mentioned jojoba of the present invention is also called firewood, red-eyed tree, red-purple, and its scientific name is Caesalpinia sappan L.

It grows in the tropics and grows about 5 to 9 meters high. There are small thorns on the trunk. There is a pear (pore), though it is a little greenish gray. The leaves are double-headed leaves, and the feathers are round and round. Yellow flowers bloom in May-June and fruit in September-October.

In the oriental medicine, it has the efficacy of the blood, blood, old blood, blood and anal excretion. It is mainly used for symptoms such as wife's hemorrhagic abdominal pain (腹气 气血 心 腹痛), pneumoconiosis (经 closed), postpartum hemorrhagic abdominal pain (postpartum umbilical abdominal pain), tetanus, Representative prescriptions include: Jojok-tang, fire-fighting sound, fire-fighting mountain, and toxic mountain.

The above-mentioned omeprazole of the present invention is the root of Lindera strichnifolia Fernandez-Villar (camphora and Lauraceae). Lindera aggregata Kosterm. Of CP is the same species as this species.

It is fusiform with roots and slightly curved. It has a length of 10 ~ 15cm and a diameter of 1 ~ 2.5cm. Outer surface is yellowish brown to brown with vertical wrinkles and sparse roots. The vagina is hard and not easily broken, and the broken face is powdery. The cross section is yellowish white to light yellowish brown when viewed with a magnifying glass, the waterline is radial, the ring is visible, and the color of the center is relatively dark. There is a fragrance, and the taste is a bit small, very refreshing.

The above-mentioned agent of the present invention is a perennial perennial herb which grows in a height of 40 to 80 cm, and its roots are fusiform and thick. Because it turns red when it cuts roots, it is called an effective medicine. Radical leaves are divided into 1-2 upper right and upper leaves are deeply divided into 3 pieces. Small leaf is lanceolate or elliptical, egg-shaped with dark greenish surface and no hair. Petioles and veins are reddish brown. Flowers bloom from May to June, and there are various varieties such as white and red, attached to the end of main stem. The petals are about 8 ~ 11 pieces, are obovate, and the length is about 5㎝. There are 3 ~ 5 varieties in the nest. Origin originates in about 33 species in Europe, temperate Asia, and North America.

The above-mentioned perfume of the present invention is a root stem of Cyperus rotundus Linne (Chrysanthemum Cyperaceae), and the root is removed. The brown and fur-like fibers in the nodules are not removed, and they are called curls. The removed ones are called incense rice, and they are called fragments.

The above figure of the present invention is a medicinal product made from seeds of Prunus persica Batsch or Prunus persica Franchet varidiana Maximowicz (Korea, Japan). China has a copy of Prunus davidiana Franchet with peach trees, but it should have the same scientific name as our country.

The abovementioned safflower of the present invention means 'korthom' or 'dye' in Arabic as Carthamus and used as a raw material of reddish paper used in marriage in the past.

The stem is upright, the base is lignified, and the upper part is tapered with many branches. It is very soft and hairs-free, has a thin vertical groove with white green, and is easy to break when mature. Leaves are alternate and wide lanceolate, length is 3 ~ 5 ~ 9cm width is 1 ~ 3. 5 cm. It is rigid and has almost no petiole, but its edges are sharp serrated. Flowers bloom from July to August, look like thistles, yellow with reddish color and one at the end of the branch.

The licorice of the present invention is called ural licorice and curly licorice, and grows in Russia (Siberia), Iran, Afghanistan, Pakistan, China (Gansuk province, Shin Kangsung), Mongolia, and cultivated in Korea. European licorice is distributed in Southern Europe, Central Asia and China. As a variant of European licorice, there are Russian licorice, Persian licorice, and Iran licorice, but not medicinal.

The cinnamon of the present invention is a medicinal substance made of bark of Cinnamomum japonicum SIEB. Which is an evergreen tree belonging to the camphoraceae family.

The dried bark of cinnamon is curved or half-dry, forming an irregular piece. The thickness is usually 3mm, the color of the outer part is blackish brown or dark red, and there are spots like grayish white flowers. The inside is dark red to reddish. The outer layer of the cut surface is grayish brown.

Mountain areas are mainly native to southern China, southern Japan, Vietnam, Cambodia and Thailand. The active ingredients are volatile essential oils of about 1%, including Phellandrene, Eugenal, and Methyleugual.

In the present invention, it is preferable to dry and use the above-mentioned material, but it is also possible to use it without drying.

In the present invention, as a method of extracting a composition by mixing the above-mentioned materials, a method may be used in which water is mixed with a mixed material, followed by immersion and heating to obtain an extract.

The amount of water used may be 100 to 20,000 parts by weight based on 100 parts by weight of the raw material mixed with the whole material, but is not limited thereto.

In the present invention, 100 parts by weight of a mixture obtained by pulverizing and mixing the above materials is placed in 500 to 2000 ml of 80% ethanol [alcoholic solution] and refluxed for 1.5 to 4 hours. The filtrate is concentrated and concentrated under reduced pressure on a rotary vacuum evaporator And can use it.

Further, the concentrated solution may be lyophilized with a freeze dryer to obtain a concentrated powder.

The above-mentioned concentrated powder may be dissolved in distilled water.

Preferably, 50 to 80 parts by weight of leek, 50 to 80 parts by weight of lees, 50 to 80 parts by weight of the leaching agent, 50 to 80 parts by weight of rosin, 30 to 70 parts by weight of rosin, (Hereinafter referred to as DG) obtained by mixing 30 to 70 parts by weight of safflower, 20 to 50 parts by weight of licorice and 20 to 50 parts by weight of cinnamon with antioxidative and anti-inflammatory effects.

The present invention shows that the composition is effective for antioxidant and anti-inflammation through the following examples.

<Examples>

1. Preparation of Extraction Compositions

(1) Prepare a mixture of 12 g of Angelica gigas corn, 8 g of marigold, 8 g of red ginseng, 8 g of red ginseng, 8 g of curd seeds, 6 g of doenjang, 6 g of safflower, 4 g of licorice and 4 g of cinnamon.

(2) 2 g of evening primrose, 3 g of willow and 3 g of tangerine fruit are mixed with 12 g of Angelica gigas, 8 g of joan, 8 g of red ginseng, 8 g of red ginseng, 8 g of red ginseng, 8 g of red ginseng, 6 g of doenjang, 6 g of safflower, 4 g of licorice and 4 g of cinnamon .

[The amount of the raw material is the amount of two herbal medicines for obtaining a natural composition]

The raw materials prepared above were pulverized and mixed, and the mixture was refluxed for 3 hours in 1000 ml of 80% ethanol [ethanol]. The filtrate was concentrated under reduced pressure on a rotary vacuum evaporator.

The concentrated solution was lyophilized with a freeze dryer to obtain 20.09 ~ 12.00 g of powder. The powder was dissolved in distilled water and used for the study.

The powder may be diluted differently according to the purpose of the experiment, and generally 1 g of the powder may be dissolved in 50 to 500 ml of distilled water.

In the present invention, the concentrated solution or powder thus obtained is called a natural composition (DG) as described above, and the following animal experiment was conducted.

<Experiment>

A. Male Wistar Rat (170-200g), 6 weeks old, was purchased from Korea Bio Co., Ltd. The animals were allowed to refine with a 2-week stabilization period, and a sufficient amount of general solid feed (Purina, Korea) was fed until the sacrifice day and a sufficient amount of water was fed while free-flowing. The conditions of the animal room were 22 ± 2 ℃ for conventional system, 200-300 Lux for 12 hours in a day, and all lights were blocked for 12 hours. This experiment was conducted in accordance with the animal ethics code of the Daejeon National University Experimental Ethics Committee (Animal Use Ethics Committee Approval No. DJUARB 2013-027). The contents and amounts per kg of general diets are as follows (Table 1).

The Components of Conmal Diet Components Percentage (%) Crude protein 20.0 Crude fat 4.5 Crude fiber 6.0 Crude calcium oxide 7.0 Calcium 0.5 Phosphorus 1.0 Total amount 39.0

B. Classification of experiment group

The experimental group consisted of a control group with natural treatment, DG group treated with DG extract alone, Terra group treated with tetramycin (Oxytetracycline HCL 5 mg + Polymaxin B Sulfate 10000 IU), DG + Terra group treated with DG extract and tetramycin Four groups and six animals were categorized, and each animal was housed in a cage.

1. Liver function test

(1) To determine the activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in serum, blood was collected by cardiac puncture after the end of the experiment. Blood was solidified at room temperature for 30 minutes, centrifuged at 3,000 rpm for 15 minutes, and serum was separated and measured by a biochemical automatic analyzer using the principle of JSCC UV method.

(2) ALT, an index component of liver function measurement, was 28.3 ± 1.7 U / L in the control group, 32.7 ± 5.4 U / L in the DG administration group, 29.5 ± 2.4 U / L in the Terra administration group and 32.0 ± 3.7 U / L in the DG + / L. AST was 97.5 ± 6.0 U / L in the control group, 107.3 ± 9.5 U / L in the DG group, 97.5 ± 7.8 U / L in the Terra group and 101.7 ± 8.9 U / L in the DG + Terra group.

2. New Function Test

(1) New function tests

To measure creatinine and blood urea nitrogen (BUN) activity in the serum, blood was collected using the cardiac puncture method at the end of the experiment. Blood was solidified at room temperature for 30 minutes, centrifuged at 3,000 rpm for 15 minutes, and serum was separated. The content of creatinine was measured by a biochemical automatic analyzer using the principle of Creatinine Jaffe Method and the content of BUN by the principle of kinetic UV assay for urea / urea nitrogen.

(2) Effect on creatinine concentration

Serum creatinine levels were 0.53 ± 0.02 ㎎ / ㎗ in the control group, 0.57 ± 0.03 ㎎ / ㎗ in the DG group, 0.53 ± 0.02 ㎎ / ㎗ in the Terra group and 0.57 ± 0.02 ㎎ / ㎗ in the DG + Terra group

(3) Effect on Blood urea nitrogen (BUN) concentration

Serum BUN concentrations were 16.42 ± 1.43 ㎎ / ㎗ in the control group, 17.85 ± 1.35 ㎎ / ㎗ in the DG treated group, 17.20 ± 0.50 ㎎ / ㎗ in the Terra treated group and 17.22 ± 0.60 ㎎ / ㎗ in the DG + Terra treated group appear.

3. Antioxidant efficacy measurement

(1) Total polyphenol content measurement

1) Total polyphenol content was measured by Gutfinger 's method. 0.5 ml of 50% Foiln-Ciocalteu's phenol reagent was added to 1 ml of the extracted sample solution, followed by reaction at room temperature for 3 minutes. 1 ml of a saturated solution of Na ₂ CO ₃ and 7.5 ml of distilled water were added to the reaction solution, and the mixture was allowed to stand for 30 minutes. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was taken and absorbance was measured at 760 nm. The content of total phenol was determined according to the calibration curve using gallic acid as a reference material. GAE (gallic acid equivalent) / g was used as a measurement unit.

2) The total polyphenol content of DG extract was determined as 57.55 ± 1.70 mg / g as gallic acid as a standard.

(2) Measurement of total flavonoid content

1) Total flavonoid content was determined by applying the method ^24), such as Nieva Moreno. 10 ml of aluminum nitrate, 0.1 ml of 1 M potassium acetate and 4.3 ml of 80% ethanol were added to 0.5 ml of a mixture of 0.1 ml of each sample and 0.9 ml of 80% ethanol, and the mixture was allowed to stand at room temperature for 40 minutes. The absorbance was measured at 415 nm, The content was determined from a standard curve prepared using quercetin.

2) Total flavonoid content in DG extract was measured as quercetin as a standard, and the total flavonoid content was 14.30 ± 1.75 mg / g.

(3) Measurement of DPPH radical scavenging ability

1) In the free radical scavenging activity test, 150 μl of a 0.2 mM DPPH solution dissolved in the alcohol was mixed with 100 μl of a DG extract (1, 10, 100, 1,000 ug / ml) by using a stable free radical DPPH , Incubated at 37 ° C for 30 minutes, and then absorbance was measured at 517 nm. In the control group, water was added in place of the sample solution, and a correction value was obtained by adding a solvent instead of the DPPH solution. The free radical scavenging ratios were calculated according to the following [Table 2].

Erase rate (%) = ( Absorbance of the control group - Absorbance of the sample added group ) × 100 Absorbance of control group

2) DPPH removal rate was 7.6 ± 0.2% at 1 ㎍ / ㎖ concentration, 10.3 ± 1.3% at 10 ㎍ / ㎖ concentration, 59.3 ± 1.5% at 100 ㎍ / ㎖ concentration and 93.2 ± 1.0% at 1,000 ㎍ / ㎖ concentration And increased in concentration - dependent manner.

(4) Measurement of ABTS radical scavenging ability

1) The ABTS assay method was modified according to the previously reported method for 96 well plate. DG extract was diluted to a final concentration of 1, 10, 100, 1,000 (㎍ / ㎖). The ABTS solution was diluted with 7.4 mM ABTS (2,2azinobis- (3ethyl benzothiazoline6-sulfonic acid) mM potassium persulphate was prepared and allowed to stand in a dark place for one day to form a cation (ABTS +). Then, the absorbance was measured at 734 nm to dilute the absorbance value to 1.5 or less. 150 μl of diluted ABTS + solution and DG extract 5 μl each of them were mixed, reacted at room temperature for 10 minutes, and the absorbance was measured at 734 nm. The antioxidant capacity of the control group was determined by using percentage of ABTS radical scavenging ability. (Table 3)

Erase rate (%) = (1- (Absorbance of the sample-added group) ) × 100 Absorbance of control group

2) The ABTS scavenging rate was 3.4 ± 3.0% at 1 ㎍ / ㎖ concentration, 5.9 ± 0.5% at 10 ㎍ / ㎖ concentration, 31.4 ± 4.1% at 100 ㎍ / ㎖ concentration and 92.5 ± 0.8% at 1,000 ㎍ / ㎖ concentration And the radical scavenging ability was increased in a concentration dependent manner.

(5) Measurement of intracellular ROS activity

cells/well이 되게 분주하였다. 24시간 동안 배양 한 후, DG 주정 추출물을 1, 10, 100(㎍/㎖)의 농도로 처리하고, LPS 1 ㎍/㎖을 처리하여, 다시 24시간 동안 37℃, 5% CO

배양기에서 배양하였다. 배양 후, 1,200 rpm에서 5분간 원심 분리하여 모은 세포를 차가운 PBS로 2회 세척한 후, DCF-DA 10 μM이 되도록 첨가하여 15분 동안 빛이 차단된 상온에서 염색하였다. 염색 후 차가운 PBS를 넣어 1,200 rpm에서 5분간 원심 분리한 다음 상청액을 제거하고 다시 PBS 400 ㎕를 부유시켜 유세포 분석기(Flow cytometer, Becton Dickinson, Franklin Lakes, NJ USA)를 이용하여 형광강도의 세기에 따른 변화를 분석하였다.1) 2 ', 7'-dichlorofluorescin diacetate (DCF-DA) was used to measure ROS in Raw 264.7 cells. Raw 264.7 cells were plated in 1.5-well plates

cells / well. After 24 hours of incubation, the DG extract was treated at a concentration of 1, 10, 100 (ug / ml), treated with 1 μg / ml of LPS,

Lt; / RTI &gt; After incubation, the cells were centrifuged at 1,200 rpm for 5 minutes, washed twice with cold PBS, added with DCF-DA 10 μM, and stained for 15 minutes at room temperature. After staining, cold PBS was added and centrifuged at 1,200 rpm for 5 minutes. After removing the supernatant, 400 μl of PBS was suspended and analyzed by flow cytometer (Becton Dickinson, Franklin Lakes, NJ USA) The changes were analyzed.

2) The amount of ROS produced was 87.1 ± 9.9% at the concentration of 1 ㎍ / ㎖, 84.0 ± 6.3% at the concentration of 10 ㎍ / ㎖, 100.0 ± 6.3% at the concentration of 1 ㎍ / ㎍ / ㎖ concentration and 53.1 ± 5.5%, respectively, and showed significant (* p <0.05, *** p <0.001) scavenging ability at the concentration of 10 and 100 ㎍ /

4. Anti-inflammatory efficacy measurement

(1) Measurement of total nitric oxide (NO) production inhibitory effect

1) NO concentration was measured by using Griess Reagent System. Raw 264.7 cells were cultured in 96 well plates at 10 ⁴ cells / well and cultured for 24 hours. The DG extract was treated at a concentration of 1, 10, and 100 μg / ml and treated with 1 μg / ml of LPS , And cultured for another 24 hours. 50 μl of N1 buffer was treated with 10 μl of N2 buffer at room temperature for 10 min, and 50 μl of N2 buffer was added to each well. After reacting for 10 min, absorbance was measured at 540 nm. The concentration of NO in the culture medium was determined using the standard curve of concentration of nitrite standard.

2) Effect on NO production

The amount of NO produced was 95.7 ± 1.2% at the concentration of 1 ㎍ / ㎖, 93.7 ± 1.1% at the concentration of 10 ㎍ / ㎖, 100 ㎍ / ㎖ at the concentration of 10 ㎍ / ㎖, (86.1 ± 2.5%) at the concentration of 100 μg / ㎖, and decreased (** p <0.01) at the concentration of 100 ㎍ / ㎖

(2) Measurement of cytokine production amount

1) Raw 264.7 cells were plated on 12-well plates at a density of 1.5 × 10 ⁵ cells / ml, cultured for 24 hours, treated with DG extract at concentrations of 1, 10, and 100 μg / ug / ml. After culturing for 24 hours, cell culture medium was collected, and IL-1β, IL-6, and TNF-α contained in the culture were measured using a custom-made 4-plex cytokine Milliplex panel.

2) Effect on IL- 1β production in RAW 264.7 cells

The amount of IL-1 β production was 36.2 ± 2.1 pg / ㎖ in the control group, 13.2 ± 1.2 pg / ㎖ in the normal group, 30.2 ± 2.2 pg / ㎖ in the 1 ㎍ / ㎖ group and 22.0 ± 2.4 pg in the 10 ㎍ / ( P <0.05, **: p <0.01, ***: p <0.001) at the concentration of 100 μg / ml and 19.6 ± 1.6 pg / .

3) Effect on IL-6 production in RAW 264.7 cells

The amount of IL-6 produced was 8729.2 ± 651.1 pg / ㎖, 2.8 ± 1.6 pg / ㎖ in normal group, 6323.2 ± 382.3 pg / ㎖ in 1 ㎍ / ㎖ of DG administration group and 4851.0 ± 610.4 pg / ㎖ in 10 ㎍ / At the concentration of 100 ㎍ / ㎖, it was 4727.7 ± 449.7 pg / ㎖, which showed a significant decrease (** p <0.01, *** p <0.001) at all concentrations compared with the control group.

4) Effect on TNF- α production in RAW 264.7 cells

The amount of TNF-α produced in the control group was 1686.0 ± 198.4 pg / ㎖, 251.6 ± 33.7 pg / ㎖ in the normal group, 1375.1 ± 88.6 pg / ㎖ in the 1 ㎍ / ㎖ group and 1341.7 ± 92.2 pg / Ml and 1004.5 ± 127.8 pg / ㎖ at 100 ㎍ / ㎖, respectively, and decreased at all concentrations (* p <0.05, ** p <0.01) compared with the control group.

5. Measurement of gene expression of wound-induced and wound area and wound factors

(1) Wound induction and experimental group classification

The rats were anesthetized using anesthetics (Zoletil 0.5 ml + rum poo 0.1 ml) and the skin of the back was cleaned. The epidermal skin was cleaned with alcohol, and a square with a size of 2 × 2 cm ² was formed in the middle of the hip joint with the spine as a reference. The skin was removed to the upper side of the fascia using surgical scissors to induce wounding. The experimental group consisted of a control group with natural treatment, DG group treated with DG extract alone, Terra group treated with tetramycin (Oxytetracycline HCL 5 mg + Polymaxin B Sulfate 10000 IU), DG + Terra group treated with DG extract and tetramycin Four groups and six animals were categorized, and each animal was housed in a cage.

(2) Test solution administration

After the induction of wounding, 2 ml (200 mg / kg) was orally administered at 10:00 am for each week for 3 weeks for each experimental group from the next day. The teramycin was administered for 2 weeks, 2 weeks, and 3 weeks. The dose of DG alcohol extract was calculated on the basis of a mouse body weight of 300 g in a single dose of 60 kg of adult body weight per adult.

(3) Wound area measurement

1) In order to measure the effect of mixed medication with medicinal herbs and topical medicines on wound healing, the wound area was measured for 1 week, 4 days, 8 days, 11 days, 15 days, 18 days, and 22 days for a total of 3 weeks . The measurement method was averaged using a Digimatic caliper.

2) After the end of the experiment, the wound area was decreased by 80.2% in the control group, 93.3% in the DG group, 91.7% in the Terra group and 93.8% in the DG + Terra group. (* P <0.05, ** p <0.01, *** p <0.001) in the DG, Terra and DG + Terra treated groups at 10, 14, 1 and 1B).

The Change of Wound Size in DG Extract and Terra- myCin Groups Control DG Terra DG + Terra One 100.0 ± 30.5 100.0 + - 22.5 100.0 ± 20.6 100.0 + - 23.2 4 96.2 ± 21.5 95.3 ± 22.5 93.7 ± 23.0 96.2 ± 25.2 7 82.8 ± 17.9 69.6 ± 22.1 74.5 ± 19.3 63.2 ± 17.0 10 52.3 ± 5.9 37.9 ± 7.2 ** 38.7 8.3 * 32.9 ± 13.5 * 14 36.7 ± 2.1 14.1 ± 3.6 *** 14.9 ± 4.7 *** 13.7 ± 6.8 *** 17 24.3 ± 3.0 9.3 ± 2.3 *** 10.4 ± 3.2 *** 9.9 ± 4.9 *** 21 19.8 ± 7.0 6.7 ± 1.4 ** 8.3 ± 1.8 ** 6.2 ± 2.9 **

(4) Influence on immune cells in blood

1) At the end of the final experiment, 10 ㎖ of heart blood was collected with EDTA - treated tubular syringe and neutrophil, monocyte, lymphocyte distribution, AST, ALT, creatinine and BUN were measured. The number of hemocyte counts was measured by a cardiac puncture method using an automatic hemocytometer (MS9-5, MELET SCHLOESING, France) according to the Fonio method using Minos-ST.

2) Effect on total leukocyte production

Total leukocyte production was 5.98 ± 1.85 Thous / uL in the DG group, 9.73 ± 2.15 Thous / uL in the Terra group, and 6.13 ± 1.32 Thous / uL in the DG + Terra group when the control group production was 11.78 ± 1.29 Thous / (** p <0.01) in the DG and DG + Terra groups compared to the control group (Figure 2).

3) Effect on neutrophil production

The ratio of neutrophils to leukocytes was 2.28 ± 0.55% in the DG group, 2.97 ± 0.19% in the Terra group, and 1.87 ± 0.46% in the DG + Terra group, respectively, when the control group production was 2.80 ± 1.01% DG group, and DG + Terra group (Fig. 3).

4) Influence on lymphocyte production

The proportion of lymphocytes to leukocytes was 90.4 ± 8.72% in the control group, 95.43 ± 1.24% in the DG group, 93.38 ± 3.20% in the Terra group and 97.77 ± 0.77% in the DG + Terra group, , Terra, and DG + Terra (Fig. 4).

5) Effect on Monocyte Production

The ratio of mononuclear cells to leukocytes was 0.30 ± 0.14% in DG group, 0.30 ± 0.19% in Terra group and 0.25 ± 0.11% in DG + Terra group, respectively, when the control group production was 1.45 ± 0.61% (* P < 0.05) in all treatment groups (Fig. 5).

(5) Influence on inflammatory cytokines and factors

1) A luminex instrument was used to measure inflammatory cytokines in serum. Blood was collected and incubated for 30 minutes at room temperature. After centrifugation at 3,000 rpm for 15 minutes, serum was separated to measure IL-1β, IL-6 and TNF-α.

2) Effect on IL- 1β production

As a result of measuring the IL-1 β production in the serum, the control group is 106.0 ± 9.7 pg / ㎖, DG group is 89.1 ± 9.5 pg / ㎖, Terra group are 85.3 ± 9.8 pg / ㎖, DG + Terra group are 83.7 ± 9.9 pg / ㎖, which was significantly decreased (* p <0.05) in the Terra and DG + Terra groups compared to the control group.

3) Effect on IL-6 production

The amount of IL-6 produced in the serum was 916.3 ± 137.7 pg / ㎖ in the control group, 871.7 ± 67.2 pg / ㎖ in the DG treated group, 812.2 ± 69.1 pg / ㎖ in the Terra treated group and 805.5 ± 61.0 pg / ( P <0.05) in the group treated with Terra and DG + Terra compared to the control group.

4) Effect on production of TNF-α

The amount of TNF-α produced in the serum was 66.4 ± 11.1 pg / ㎖ in the control group, 50.4 ± 9.6 pg / ㎖ in the DG group, 47.3 ± 6.7 pg / ㎖ in the Terra group and 45.2 ± 6.1 pg / ( P <0.01, *** p <0.001) compared to the control group.

(6) Measurement of gene expression of a wound factor

1) RNA extraction

RT-PCR was performed to determine the expression of wound factors expressed in the dorsal skin of the rat. After separating the skin tissue, the tissue was disrupted with a homogenizer and 1,000 μl of RNAzolB was added, and 200 μl of chloroform (CHCl ₃ ) was added and mixed again for 15 seconds. The mixture was centrifuged at 13,000 rpm, and 400 μl of the supernatant was recovered. The supernatant was mixed with 400 μl of isopropanol, and the mixture was slowly shaken and left on ice for 15 minutes. After centrifugation at 13,000 rpm, the cells were washed with 80% EtOH and dried in a vacuum pump for 1 minute to extract RNA.

2) Reverse transcription polymerase chain reaction

The reverse transcription reaction was performed using a mixture of RT premix kit (reaction buffer, dNTPs mixture, RNase inhibitor, stabilizer, oligo dT ₁₅ primer) and final volume of distilled water treated with diethyl pyrocarbonate (DEPC) 20 &lt; / RTI &gt; The 20 μl reaction mixture was mixed well, centrifuged at 2,000 rpm for 5 seconds, and reacted for 60 minutes at 45 ° C in a heating block to synthesize first-strand cDNA. M-MLV RT was inactivated at 95 ° C for 5 minutes and the synthesized cDNA was used for polymerase chain reaction (PCR).

3) RT-PCR

RT-PCR was performed by adding each sample and primer to a mixture containing 250 mM dNTPs mix, RT buffer (10 mM Tris-HCl, pH 9.0, 30 mM KCl, 1.5 mM MHCl ₂ ) . MMP-1 at 55 ° C for 2 minutes, 95 ° C for 10 minutes, 60 ° C for 15 seconds, MMP-2 at 61 ° C for 2 minutes, 95 ° C for 10 minutes, 60 ° C for 15 seconds, MMP- At 95 ° C for 10 minutes, at 60 ° C for 15 seconds, at GAPDH at 57 ° C for 2 minutes, at 95 ° C for 10 minutes, and at 60 ° C for 15 seconds. (Table 3). After electrophoresis on 1% agarose gel, the band of each gene was visualized by UV spectroscopy to reveal RNA expression.

The Sequences of Pimers in Used This Study Primer F / R Sequences MMP-1 F GAG ACG TGG ACC GAC AAC AG R AGG CCC ATA TAA AGC CTG GA MMP-2 F TGG CGA GTA CTG CAA GTT CC R GTA AGA GGT GCC CTG GAA GC MMP-9 F CCT GCA GTG CCC TTG AAC TA R TCC AAC AAG AAA GGA CAG CG GAPDH F GTT ACC AGG GCT GCC TTC TC R CAC CCC ATT TGA TGT TAG CG

4) Effect on gene expression

The expression levels of MMP-1, 2, and 9 in the wounded skin tissue were decreased in all of the DG, Terra, and DG + Terra groups compared to the control group in the MMP-1 but significantly (* p <0.05) Decrease occurred only in DG group. ( P <0.05) in MMP-2 and DG, Terra, and DG + Terra, respectively. In MMP-2, DG, Terra and DG + (** p < 0.01) (Figure 6 and Figure 6b)

(7) Histopathological examination

1) Anesthetized with ether, blood was drawn through cardiac puncture and wound tissue was extracted. The tissues of each experimental group were fixed in 10% neutral formalin for 48 hours. After washing for 12 hours in tap water flowing through the fixed tissues, the tissue fixation solution was completely removed. For the dehydration of the tissues, dehydration was performed in order of increasing concentration from 60% to 100% alcohol, and the xylene was subjected to a transparent process and then a paraffin block was prepared. The prepared blocks were cut to thickness of 3 ~ 4 ㎛ using a microtome, deparaffinized and functionalized, and stained with hematoxyline and eosin (H & E).

2) Histopathological changes

Hematoxylin & Eosin staining of wounded skin tissue revealed collagen deposition, thickening of skin re - epithelium, chronic inflammation and increased cell fusion. Collagen deposition and chronic inflammation were decreased in the DG, Terra, and DG + Terra groups compared with the control group. Skin re-epithelization was decreased in the Terra group and DG + Terra group, (Fig. 7).

Thus, the present invention provides a natural composition having high antioxidative and anti-inflammatory functions and a pharmaceutical composition containing the natural composition without toxicity to liver function and renal function.

The present invention is very useful in an industry that produces, processes, distributes and sells functional foods, medicines, etc. that perform antioxidative and anti-inflammatory treatment and prevention functions using herbal medicines and herbs.

Claims (2)

A natural composition having antioxidative and anti-inflammatory effects extracted from a mixture of Angelica gigas Nakai, Jojoba, Opium, Cinnamon, Herbarium, Doenjang, Safflower, Licorice, Cinnamon, Evening Primrose, Willow and Tangja.
A pharmaceutical composition comprising the composition of claim 1 effective for antioxidant and anti-inflammation.

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