KR20090088151A - Antiallergic material from ixeris dentata - Google Patents

Abstract

A Petasites japonicas extract or Petasites japonicas fermentation of anti-allergic or anti-inflammatory material is provided to have an effect of suppressing degranulation in a RBL-2H3 (Rat mast cell line) without side effect. A Petasites japonicas extract for anti-allergic or anti-inflammatory material is obtained by using alcohol, decompressing the extract and culturing with Lactobacillus. The Lactobacillus is Lactobacillus acidophilus (KCTC 3168), Bifidobacterium breve (KCCM 10097) or Bifidobacterium longum (KCCM 100493). A method for producing Petasites japonicas fermentation comprises: a step of dipping a Petasites japonicas in 5% of salt water for one night; a step of adding Lactobacillus and fermenting at 4-15‹C for 1-15 days; and a step of adding 1L of water and extracting, decompressing, and freeze-drying at 90‹C for two hours.

Description

Butterbur extract or butterbur fermentation as anti-allergic or anti-inflammatory substance {Antiallergic material from Ixeris dentata}

      The present invention relates to coltsfoot extract or coltsfoot fermentation as an antiallergic or anti-inflammatory substance. According to the present invention, there can be provided an anti-allergic or anti-inflammatory substance having an antiallergic effect but little side effects.

Allergic patients are increasing every year due to the complexity of society, the development of industry and civilization, environmental pollution, stress, and dietary changes. In 1980, less than 1% of allergic patients, including atopic dermatitis, increased to more than 5% in the 2000s, and more than 10% of potential patients were estimated.

An allergy refers to a phenomenon in which an alien substance (antigen, Allergen), which enters the body from outside the body, exhibits a unique and modified reaction that has not occurred until now. At this time, foreign substances causing symptoms such as anaphylaxis, asthma and inflammation are called allergens and the resulting diseases are called allergic diseases or hypersensitivity reactions. The cause of allergy is excessive immune response of the living body resulting from antigen antibody reaction, and is classified into type 1-4 according to reaction time and presence of complement involvement.

In type 1 reaction, IgE antibody binds to antigen and binds to receptors on mast cell or cell membrane of basophils in the blood and degranulates, resulting in the transfer of chemicals such as histamine and cytokines into cells. It is liberated outside, causing smooth muscle contraction, capillary permeability, and gastric acid secretion. At this time, a systemic or local reaction occurs depending on the invasion route of the antigen. For example, symptomatic differences such as anaphylactic shock, bronchial asthma, urticaria and hay fever appear. Diseases caused by this type 1 reaction are closely related to genetic causes and are also referred to as atopic diseases. This type 1 reaction develops quickly after antigens invade the body. So it is also called immediate allergy.

Type 2 reactions cause an allergic reaction in which cell membranes in vivo become antigens due to infectious diseases, drugs, genetic characteristics, etc., which bind to antibodies and proceed with lysis of complement cells. For example, a baby whose Rh (-) blood type is pregnant with a baby whose Rh (+) blood type is pregnant, inadequate transfusion, autoimmune hemolytic anemia, hemolytic anemia by a drug, granulocytopenia, thrombocytopenic purpura, and the like.

Type 3 reactions are allergic reactions in which antigen-antibody conjugates formed by antigen-antibody reactions are deposited on blood vessel walls or tissues to activate complement and inflammatory reactions, thereby impairing blood vessels or tissues. Examples include erythema, lymphadenopathy, arthralgia, arthritis, nephritis, acute glomerulonephritis following streptococcal infection.

Type 4 reactions are chronic inflammatory diseases and are sometimes referred to as delayed allergic reactions since the onset of reaction becomes worse after 48 hours. Type 4 reactions are caused by a number of active factors that are released from sensitized T lymphocytes by the reaction of sensitized T lymphocytes with antigens. For example, chronic inflammation.

The most common hypersensitivity reactions among these allergic diseases are type 1 hypersensitivity reactions such as atopy and asthma and type 4 hypersensitivity reactions such as chronic inflammation. For example, 80% to 90% of patients with atopic allergic dermatitis have elevated serum IgE, especially when accompanied by respiratory atopy (allergic rhinitis or asthma). In about 85% of patients with atopic dermatitis, a skin reaction test or RAST (radioallergosorbent test) test shows positive IgE for food and inhaled allergens. If the serum IgE is increased, it is known that atopic disease, which is a type 1 hypersensitivity reaction, does not stop and causes type 4 hypersensitivity reactions such as chronic dermatitis. Therefore, type 1 and type 4 hypersensitivity reactions are very close allergic reactions and are an important key for treating allergic diseases.

Allergic reactions are not constant depending on the type of antigen, the type of antibody, and the constitution of the individual, and are known to have high genetic predisposition. Allergic development is known to influence genetic factors in addition to acquired factors such as climate, air pollution, infection, and stress.

To improve allergic disorders, take a shower or bath to remove allergens (such as house dust and mites) from your skin and avoid ingesting allergens. Nevertheless, drug therapy is used when allergic diseases occur. As corticosteroids, steroids, antihistamines, immunosuppressants, etc., which have excellent anti-inflammatory effects, are used. Of these, steroids can cause side effects such as skin atrophy, vasodilation, discoloration, and purpura. Antihitamins are likely to cause sleepiness, transient effects, and immunosuppressive agents such as kidney failure. In addition, desensitization or interferon may be used for treatment.

Despite the recent advances in the development of allergic diseases and the development of radical immunology, many advances have been made in identifying and treating the causes of allergic diseases. However, the development of drugs for treating allergic diseases has not been fully developed. . None of the drugs that have been developed so far can cure allergies, and the symptoms are expected to improve, but side effects are large.

Therefore, there is an urgent need for the development of herbal drugs with anti-allergic effects but little side effects.

On the other hand, the homegrown butterbur ( Petasites) japonicus , Japanese butterbur) is a seasonal vegetable used in spring and early summer. It is also bitter, so it is used after it is soaked in water before cooking. However, there are a large amount of polyphenol compounds, which are browning substances, and anti-inflammatory and anti-cerebral ischemic effects have been reported.

The present invention is to solve the problems as described above is to provide a substance having excellent anti-allergic effect by using the herbal drug butterbur.

In order to solve the above problems, the present invention provides a butterbur extract as an antiallergic or anti-inflammatory substance.

In another aspect, the present invention provides a butterbur fermentation product characterized in that cultured by putting the coltsfoot extract and lactic acid bacteria as an antiallergic or anti-inflammatory material.

Also, marinated overnight in 5% brine, rinsed with water, washed with water and soaked in 2% brine, and then wetted with Lactobacillus. acidophilus (KCTC 3168), Bifidobacterium breve (KCCM 10097) or Leuconostoc Mesenteroides (KCTC 1093) strain and put into fermentation at 4-15 degrees for 1-15 days and then put again 1 liter of water to extract for 2 hours at 90 degrees to provide a butterbur fermentation, characterized in that concentrated under reduced pressure and lyophilized.

According to the present invention, there can be provided an anti-allergic or anti-inflammatory substance having an antiallergic effect but little side effects.

[Production of Sample]

1. Butterbur Extract Manufacturer

500 g of butterbur was poured into 2.5 L of water or alcohol (ethanol or methanol), extracted at 90 ° C for 2 hours, and concentrated under reduced pressure.

2. Method of preparing butterbur fermentation product (1)

In distilled water 100㎖ coltsfoot extract 0.1 -10g (preferably 1g) and lactic acid bacteria of Lactobacillus acidophilus (KCTC 3168), Bifidobacterium breve (KCCM 10097) or Bifidobacterium 1 g of longum (KCCM 100493) wet weight was added and incubated at 37 ° C. (15-40 ° C. suitable) for 3 days (possibly 1-7 days) and lyophilized.

3. Method of preparing butterbur ferment (2)

Wash 500 grams of butter (water) with water, soak in 5% brine overnight, wash with water and soak in 2% brine. Lactobacillus Lactobacillus Here acidophilus (KCTC 3168), Bifidobacterium breve (KCCM 10097) or Leuconostoc mesenteroides (KCTC 1093) strains are fermented in 1 g wet weight for 1-15 days (preferably 3 days) at 4-15 degrees. 1 liter of water was added thereto, extracted at 90 degrees for 2 hours, concentrated under reduced pressure, and lyophilized.

[Measurement of the Effect]

(1) Degranulation inhibitory effect (antiallergic effect) of RBL-2H3 cells

Eagle's medium modified with Dulbeccos containing RBL-2H3 cells (Rat mast cell line) with 1% fetal bovine serum and L-glutamine Eagle's medium (hereinafter referred to as DMEM) was incubated in a 37 ° C., 5% CO ₂ incubator with moisture, and the cells with adherence were suspended using trypsin-EDTA solution, separated, recovered, and used for the experiment. RBL-2H3 cells were dispensed into 5 wells of 5 × 10 ⁵ cells / well in 24 wells, and then added to the mouse single cell phosphorus (monoclonal), 0.5 μg / ml of IgE, followed by culturing in an incubator for 12 hours. The cells were washed with 0.5 ml of siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH, pH 7.2), followed by 0.16 ml of shiraganian buffer (5.6 mM glucose ( glucose), 1 mM CaCl ₂ , 0.1% BSA was added) and incubated at 37 ° C. for 10 minutes. Thereafter, 0.04 ml of the coltsfoot extract and coltsfoot fermentation according to the present invention were added to each of 0.04 ml, followed by 20 minutes, followed by 0.02 ml of antigen (1 µg / ml of DNP-BSA (dinitrophenol-bovine serum albumin)) Cells were activated for a minute and then centrifuged at 2000 rpm for 10 minutes to transfer 0.025 mL of the supernatant to 96 wells. 1mM of p-NAG 0.025ml of p-NAG, prepared by adding p-nitrophenyl-N-acetyl-β-D-glucosaminide to 0.1M citrate buffer pH 4.5, was incubated at 37 ° C for 60 minutes, and then 0.1M Na ₂ CO ₃ 0.2 ml of NaHCO ₃ was added to stop the reaction, and the absorbance was measured at 405 nm using an ELISA reader.

(2) Passive cutaneous anaphylaxis model

IgE serum against Dinitrophenol-bovine serum albumin (DNP-BSA) was injected into the back of 10 μg of anesthetized mice (males 20-25 g) with physiological saline and sensitized manually. Fermentation products were dissolved or suspended in saline and administered orally or intraperitoneally one hour prior to challenge. After 48 hours, 0.2 ml of physiological saline containing 0.2 mg of DNP-HSA and 1.6 mg of evans blue was injected into the tail vein, and after 30 minutes, the amount of evans blue pigment leaked on the back was measured.

Cut the IgE administration site (1x1 cm) of mice and the like into a test tube, add 0.7 ml of 1N-KOH, incubate overnight at 37 ° C, and 0.6N phosphoric acid in this test tube. 4 ml of acetone mixture (5:13) were added, followed by shaking and filtration to measure the extracted pigment at 620 nm.

(3) small animal model

BALB / c mice (compound inducer compound 48/80) and ICR mice (Histamine test) were removed from the back of the cervix and left for 5 minutes in one group in an observation box (24x22x24cm), and the sample was dissolved in saline. Or suspended and intraperitoneally injected 1 hour before the induction drug, or orally administered 6 hours before. A pruritic inducer (50 μg of compound 48/80) was injected intravenously, one by one in an observation box, and then recorded under an unattended condition with 8-mm video (SV-K80, Samsung) for 1 hour and counted practicable behavior. However, it is admitted that scratching the injection site with the hind feet is not permitted.

(4) biochemical analysis using RT-PCR

After RBL-2H3 cells were added with Tri reagent (Qiagen, USA) and left at room temperature for 5 minutes, chloroform was added and left for 10 minutes. Supernatant was taken after centrifugation at 7,500 rpm. Isopropyl alcohol was added to the supernatant, and the supernatant was removed after centrifugation at 14,000 rpm. Washed with 70% ethanol, the supernatant was removed and dried at room temperature for 20 minutes. DEPC treated water was added and absorbance was measured at 260 nm. The absorbance 1 was 40 mg / ml of RNA and the amount of RNA was calculated.

Pre-mixture and reverse primer were placed in a sterile tube treated with 0.1% DEPC and reacted at 72 ° C for 5 minutes, and then immediately transferred to ice, and the forward primer and distilled water treated with 0.1% DEPC were added to AccuPower RT-PCR Premix tube at 42 ° C. After cDNA synthesis reaction for 60 minutes, reverse transcriptase was inactivated for 5 minutes at 94 degrees, and PCR was performed according to the following table PCR conditions. The primer used was 20 pmol and the final concentration was 1 pmol.

PCR products were identified by agarose gel electrophoresis with ethidium bromide (EtBr). Agarose gel was 1.5% gel and 0.5xTBE was used as the buffer.

[Table 1] PR-PCR reaction conditions

(5) Measurement of TNF-a and IL-4

RBL-2H3 cells (3 × 10 ⁶ cells) were incubated in a 5% CO ² incubator at 37 ° C with moisture using DMEM, suspended cells were suspended by trypsin-EDTA solution and isolated. It was recovered and used for the experiment. RBL-2H3 cells were dispensed in 5 wells of 5 × 10 ⁵ cells / well in 24 wells, and then 0.5 μg / ml of IgE was added to the monoclonal cells of rats, and cultured in an incubator for 12 hours. The cells were washed with 0.5 ml of Shiraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH, pH7.2) and then 0.16 ml of Shiraganian buffer (5.6 mM glucose, 1 mM CaCl ₂ , 0.1% BSA was added), and then incubated at 37 ° C. for 10 minutes in an incubator. Thereafter, 0.04 ml of the coltsfoot extract and coltsfoot fermentation according to the present invention were added to each of 0.04 ml, followed by 20 minutes, followed by 0.02 ml of antigen (1 μg / ml of DNP-BSA (dinitrophenol-bovine serum albumin)) at 40 ° C. Cells were activated for a minute and then centrifuged at 2,000 rpm for 10 minutes to transfer 0.025 ml of the supernatant to 96 wells, and IL-4 and TNF-a were measured by Endogen Elisa kit (USA).

(6) Immunoblot

RBL-2H3 cells (3 × 10 ⁶ cells) were incubated in a 5% CO ₂ incubator at 37 ° C with moisture using DMEM, suspended cells were suspended by trypsin-EDTA solution and isolated. It was recovered and used for the experiment. RBL-2H3 cells were dispensed in 5 wells of 5 × 10 ⁵ cells / well in 24 wells, and then 0.5 μg / ml of IgE was added to the monoclonal cells of rats, and cultured in an incubator for 12 hours. The cells were washed with 0.5 ml of siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH, pH 7.2), followed by 0.16 ml of shiraganian buffer (5.6 mM glucose ( glucose), 1 mM CaCl ₂ , 0.1% BSA was added) and incubated at 37 ° C. for 10 minutes. Thereafter, 0.04 ml of coltsfoot extract and coltsfoot fermentation according to the present invention were added thereto, followed by 20 minutes, followed by 0.02 ml of antigen (1 μg / ml of DNP-BSA (dinitrophenol-bovine serum albumin)) for 40 minutes at 37 ° C. After activating the cells, centrifuge for 10 minutes at 2,000 rpm and add 0.1 ml of hypotonic buffer (10 mM Tris-HCl, 1.5 mM MgCl ₂ , 1 mM DTT, 0.1% NP-40, pepstatin 5μg / ml, aprotinin 5μg / ml) After suspension, it was left on ice for 15 minutes. After centrifugation for 15 minutes at 4 ℃ and 12,000rpm, the supernatant cytosol fraction was taken and again precipitated by 0.1ml extraction buffer (10mM Tris-HCl, 50mM KCl, 300mM NaCl, 1mM DTT, pepstatin 5μg / ml, aprotinin 5μg / ml) After addition, the mixture was left on ice for 30 minutes after suspension, and centrifuged at 4 ° C and 12,000 rpm for 30 minutes, and the supernatant nuclear fraction was taken. After cell lysate (40μg) was electrophoresed on 10% SDS polyacrylamide gel and transferred to PVDF (Millipore) membrane, the membrane was blocked with 2% skim milk solution dissolved in PBS for 1 hour at room temperature, again 1% skim milk solution The primary antibody diluted 1: 500 was immersed for 2 hours and shaken slowly. The primary antibody diluted solution was discarded, washed twice with a washing solution (0.05% PBS-tween) for 10 minutes, and the secondary antibody diluted 1: 5000 in 1% skim milk solution was slowly shaken for 1 hour. The secondary antibody dilution solution was discarded as above, washed three times with a washing solution three times, and detected using an enhanced chemilluminescence system (PIERCE).

<Experiment Result>

The inhibitory effect on anakilasis and pruritus was measured for Butterbur Extract (PJ) and Butterbur Ferment (FPJ) (Table 2).

Both coltsfoot extract and coltsfoot fermentation according to the present invention showed the effect of inhibiting anaphylaxis and pruritus effect. But the butterbur fermentation showed better antipruritic and anti-anaxilaxic effects than the butterbur extract.

[Table 2]

Compared with the water extract of the butterbur and the alcohol extract, the effect of inhibiting anaphylaxis and pruritus was better in the alcohol extract having a high extraction rate such as polyphenol (Table 3). Caffeic acid and chlorogenic acid were isolated from the alcohol extracts by searching for active ingredients. HPLC analysis showed that chlorogenic acid contained 0.7% and caffeic acid 0.3% in butterbur. Both chlorogenic acid and caffeic acid inhibited pruritus and PCA responses (Table 3).

Table 3

In order to elucidate the antiallergic mechanism of butterbur, the effect of degranulation inhibition of RBL-2H3 cells was measured. As a result, alcohol extract showed better degranulation inhibitory effect than water extract. In addition, caffeic acid chlorogenic acid also inhibited degranulation. Among them, caffeic acid showed the best effect.

In addition, the effects on the productivity of inflammatory cytokines and IgE production-induced cytokines in RBL-2H3 cells were measured for the coltsfoot extract, coltsfoot fermentation and its isolated components according to the present invention. Butter fermentation of 2g / kg in mice was not able to observe sudden side effects such as diarrhea, abnormal behavior, abnormal organs (Fig. 1, 2).

In addition, the effect of the anti-inflammatory effect of Butterbur alcohol extract and its main component on carragenan-induced inflammation in the mouse (Fig. 3) was measured. In the carragenan-induced inflammation of air pouch, an inflammatory marker of air pouch, both the extract and its components, caffeic acid and chlorogenic acid, showed inhibitory effects. In addition, it was confirmed that the production of protein, PGE2 and inflammatory cytokine TNF-a inhibits.

1 is a graph showing the results of measuring the effect on the productivity of inflammatory cytokines and IgE production-induced cytokines in RBL-2H3 cells for butterbur extract, butterbur fermentation and its isolated components according to the present invention.

Figure 2 is a graph showing the involvement of protein expression of TNF-α and IL-4 for butterbur extract, butterbur fermentation and its isolated components according to the present invention.

3 is The anti-inflammatory effect of the butterbur alcohol extract and the main ingredient according to the present invention is a graph showing the result of measuring the effect on carragenan-induced inflammation in mice.

Claims (8)

Butterbur extract as anti-allergic or anti-inflammatory substance. The method of claim 1, The coltsfoot extract is a coltsfoot extract, characterized in that using alcohol. The method according to claim 1 or 2, Butterbur extract, characterized in that the extract is concentrated under reduced pressure. As an anti-allergic or anti-inflammatory substance, coltsfoot fermentation, characterized in that cultured with the coltsfoot extract and lactic acid bacteria according to claim 1 or 2. As an anti-allergic or anti-inflammatory substance, the coltsfoot fermentation product characterized by incubating the coltsfoot extract and lactic acid bacteria according to claim 3. The method of claim 4, wherein The lactic acid bacteria is Lactobacillus acidophilus (KCTC 3168), Bifidobacterium breve (KCCM 10097), or Bifidobacterium Butter fermentation, characterized in that the longum (KCCM 100493). The method of claim 5, wherein The lactic acid bacteria is Lactobacillus acidophilus (KCTC 3168), Bifidobacterium breve (KCCM 10097), or Bifidobacterium Butter fermentation, characterized in that the longum (KCCM 100493). Marinate coltsfoot in 5% brine overnight, wash with water, and soak in 2% brine to fully wet Lactobacillus acidophilus (KCTC 3168), Bifidobacterium breve (KCCM 10097) or Leuconostoc Mesenteroides (KCTC 1093) Strain put into fermentation at 4-15 degrees for 1-15 days, 1 liter of water was added to this again extracted at 90 degrees for 2 hours, concentrated under reduced pressure and lyophilized.

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