KR20150075497A

KR20150075497A - Persicaria thunbergii extract for allergic diseases and process for preparation thereof

Info

Publication number: KR20150075497A
Application number: KR1020130163520A
Authority: KR
Inventors: 최시영
Original assignee: 최시영
Priority date: 2013-12-26
Filing date: 2013-12-26
Publication date: 2015-07-06

Abstract

The present invention relates to an anti-allergic extract derived from a natural substance and uses thereof. According to the present invention, provided are a Persicaria thunbergii extract having anti-allergic effects by blocking a secretion of an allergy inducing material, a pharmaceutical composition comprising the same as an active ingredient, and a health functional food which can be used in developing a functional cosmetic, a food, and a medicine for inhibiting onset of allergy. A secretion of an allergy inducing material in mast cells is fundamentally blocked in comparison with conventional treatment for mainly recovering a symptom, thus can be effectively used as a selective functional additive for food or the like as well as a cosmetic and a drug with minimized side effects.

Description

TECHNICAL FIELD [0001] The present invention relates to persimmon extracts having an antiallergic effect and a method for producing the same,

More particularly, the present invention relates to an extract of Persicaria thunbergii having an antiallergic effect by blocking the secretion of an allergen-inducing substance and a pharmaceutical composition containing it as an active ingredient, It is about food.

The present invention relates to the antiallergic effect of the outbreak extract of Persicaria thunbergii . The outbreak extract of the native plant, which is a native plant, contains the degranulation (histamine secretion) of allergen-causing mast cells and the production of tumor necrosis factor and interleukin- And its efficacy was reversible. This is for antiallergic functional cosmetics, foods or medicines using this extract by showing excellent allergy inhibitory effect in local animal model of allergy.

Mast cells are widely known as the major causative agents of various allergic diseases, namely allergic rhinitis, allergic dermatitis, asthma, food allergy, and anaphylactic shock. These cells have a receptor for IgE, an allergenic antibody on the cell surface (FcεRI), which is stimulated by allergenic substances (called antigens or allergens) and causes various allergies such as histamine Secrete the substance to the outside of the cell.

Most of the allergic drugs currently used in clinical practice are drugs that alleviate allergic symptoms, and research in pharmaceutical companies is proceeding in that direction. In order to overcome these disadvantages, the present invention aims to develop a therapeutic agent for allergy which does not have side effects such as antihistamines used in patients by blocking the secretion of allergen-inducing substances in mast cells.

The gourd according to the present invention is a perennial herb according to the present invention. It is native to Korea and also lives in the Far East of China, Japan, and Russia. Buckwheat-like fruit grows, and it has been cultivated as a wild plant for eating and making food similar to handmade bread, but now it is a weed. It grows up in a group of water, reaching a height of 60 to 100 centimeters. The stem is clustered, there are no hairs, and the thorns are down. Leaves are alternate phyllotaxis, 4 ~ 10cm long, 3 ~ 7cm wide, with both sides sideways, lanceolate long and pointed. Flowers bloom from August to September at the end of a branch with a lot of densely gathered reddish and white. I also put a blockade of pollen on the ground with my own pollen. Fruits are stained with water. The efficacy in the private sector is to treat the symptoms that come from weakness of the spleen, which regulates the energy, stops the pain, turns yellowish, has no strength, is not appetite, and is empty. It also acts as a diuretic to urinate well. The main treatment is gastrointestinal illness, indigestion, sore thighs and thighs, and it is known in the private sector that it attaches to the bruises on the leaves. However, the research on the antiallergic effect on the extract of tortoiseshell is not known at all.

Accordingly, the present inventors have investigated the distribution of native plant extracts that are naturally occurring in the rivers around the residence area and have experimented with their antiallergic activity. Among them, the inventors of the present invention invented that the extract of mugwort suppresses the secretion of allergens from mast cells, Animal model has excellent antiallergic effect, and the present invention has been completed in order to apply this substance to humans.

On the other hand, prior art related to the antiallergic composition is Korean Patent Laid-Open No. 10-2005-0109958 (composition for antiallergic use), Korean Patent Laid-open No. 10-2009-0088151 (antiallergic or anti- Fermented products).

It is an object of the present invention to provide a method for inhibiting the secretion of allergen-releasing substances secreted from mast cells that cause discomfort to humans or cause various allergic diseases such as atopic dermatitis, allergic rhinitis and asthma, To provide antiallergic materials which are minimized in side effects and maximized in effectiveness.

In order to achieve the above object, the present invention provides a water or alcohol extract of Persicaria thunbergii having an antiallergic effect by blocking the secretion of an allergen-inducing substance.

In addition, the present invention provides a pharmaceutical composition for preventing or treating an allergic disease comprising, as an active ingredient, a water or alcohol extract of Persicaria thunbergii having an antiallergic effect by blocking secretion of an allergen-inducing substance.

The present invention also provides a health functional food for preventing or alleviating allergies comprising water or an alcohol extract of Persicaria thunbergii having an antiallergic effect by blocking the secretion of an allergen-inducing substance.

(1) washing and pulverizing dried persimmon ( Persicaria thunbergii ); and (2) heating the corn crushed by the above step (1) in a hot water bath to obtain a coriander extract; and (3) Filtering the elongated extract obtained in step (2) to obtain a supernatant and concentrating it; And (4) freezing the concentrate obtained in the step (3) and freeze-drying the concentrate to obtain a powder, wherein the secretion of the allergen inducing substance is blocked, thereby producing an antiallergic effect of Persicaria thunbergii to provide.

The present invention also provides a method for producing an alcoholic extract, comprising the steps of: (1) immersing Persicaria thunbergii in a lower alcohol and extracting it with an ultrasonic extractor to obtain an alcoholic extract; and (2) concentrating the extract of the extracts obtained in step (1). And (3) freezing the concentrate obtained in the step (2) and freeze-drying the concentrate to obtain a powder, wherein the secretion of the allergen inducing substance is blocked to thereby produce an antiallergic effect of persicaria thunbergii extract to provide.

According to the present invention as described above, the extract of the extract of the present invention suppresses the induction of allergy in an animal model of allergenic degranulation (histamine secretion) action of allergen-inducing mast cells at a low concentration, so that functional cosmetics , And natural extracts that are easy to develop into foods and pharmaceuticals. Therefore, unlike conventional treatment for symptom recovery, it can be usefully used as a selective functional additive for foods and the like as well as cosmetics and medicines whose side effects are minimized by blocking the secretion of allergens in mast cells.

Brief Description of the Drawings Fig. 1 is a graph showing a concentration-dependent inhibitory effect of an extract of Fusarium oxysporum (Fusarium oxysporum) on the degranulation (histamine secretion) by an allergen-inducing antigen in mast cells (RBL-2H3 cells)
FIG. 2 shows the results of inhibitory effects of the extract on the tumor-induced necrosis factor (TNF-α) and interleukin-4 (IL-4) expression, which are inflammatory cytokines, in mast cells stimulated by the antigen.
Fig. 3 shows experimental results on the reversibility of the inhibitory effect on mast extract extract mast cells.
Fig. 4 shows the results of Western blotting before the functional groups of the extract.
FIG. 5 is a graph showing the inhibitory effect of the antler extract according to the present invention on a mast cell-mediated locally allergic animal model.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Example 1: Preparation of extracts

The inventors of the present invention prepared extracts of tortoiseshell extracts using hot water and an organic solvent extraction method in order to obtain tortoise extract having excellent antiallergic effect.

(1) Hot water extraction method

In order to prepare the hot - water extract of the ginseng topping, the dried ginseng tops were washed with distilled water and finely ground with a grinder. The finely ground ginseng was heated in a hot water bath to obtain extracts. At this time, 1.0 g of water was added to 100 g of the starting material, and the mixture was heated at a temperature of 100 - 120 ° C for 2 - 3 hours to extract the water. A cooler was installed to maintain the constant temperature and prevent the loss of steam. The resulting extract was filtered to obtain a supernatant, which was concentrated using a concentrator. The frozen concentrate was frozen and lyophilized at -80 ° C to prepare an extract powder of extract.

(2) Organic solvent extraction method

In order to prepare organic solvent extracts of the stem, fruit or leaf, the extracts were immersed in low alcohol and extracted with an ultrasonic extractor for 2 - 3 weeks to obtain an alcohol extract. Only the supernatant was separated, and the foreign substances were removed by filtering the gauze. 95% ethanol was used as the alcohol. The alcohol extract obtained by the above process was concentrated using a concentrator, freezed and lyophilized at -80 ° C to prepare an extract powder of a cornstarch extract.

Experimental Example 1: Inhibitory effect of extracts from the extracts of allergens on mast cells

The present inventors searched for the suppressive effect of the extract from the extract of Example 1 on the secretion of allergen-inducing substances from mast cells inducing allergy in the body. RBL-2H3 cells were cultured in minimal medium supplemented with glutamine, antibiotics and 15% fetal bovine serum. For secretion experiments, 200,000 cells per well were incubated with 25 ng / ml DNP-specific IgE in a 24-well incubator after collection by trypsin. The next day, cells were washed with PIPES buffer (25 mM PIPES, pH 7.2, 159 mM NaCl, 5 mM KCl, 0.4 mM MgCl 2, 1 mM CaCl 2, 5.6 mM glucose, and 0.1% BSA) Pre-culture. After preincubation, the antigen is stimulated to a final concentration of 25 ng / ml. The degree of secretion of allergen inducers is determined by the amount of p-nitrophenol in p-nitrophenyl-acetyl-β-D-glucosaminidedbfo, the activity of hexosaminidase, a marker of degranulation secreted in the medium.

As shown in FIG. 1, the secretion of allergen from RBL-2H3 mast cells by the antigen was significantly inhibited from the low concentration (30 占 퐂 / ml) .

Experimental Example 2: Inhibitory effect on the cytokine production on mast cells of the extract

(TNF-α) and interleukin-4 (IL-4), which are well-known as cytokines secreted from allergen-related mast cells. Were used for RT-PCR. First, RBL-2H3 cells stimulated in 6 well plates were lysed with trizol reagent (Invitrogen). Transfer the dissolved sample to a 1.5 ml E-tube, add 200 μl of chloroform, centrifuge at 12,000 g for 20 minutes, mix the supernatant with isopropanol (1: 1) The RNA was precipitated for more than one hour. The RNA was then centrifuged at 12,000 g for 20 minutes to completely precipitate the RNA. The RNA was washed once with ethanol and dissolved in 50 μl of DEPC-water to obtain total RNA. The polymerase chain reaction (PCR) was repeated 30 times at 94 ° C. for 45 seconds, 55 ° C. for 45 seconds, 72 ° C. for 60 seconds. Primer is forward 5'-CAC CAC GCT CTT CTG TCT ACT GAA C-3 ', reverse 5'-CCG GAC TCC GTG ATG TCT AAG TAC T-3', IL-4 is forward 5'- CCG ATT ATG GTGTAA TTT CCT ATG CTG-3 ', reverse 5'-GGC CAA TCA GCA CCT CTC TTC CAG-3', Rat GAPDH forward 5'-GTG GAG TCT ACT GGCGTC TTC- -CCA AGG CTG TGG GCA AGG TCA-3 '.

As a result, as shown in Fig. 2, the production of tumor necrosis factor (TNF-a) and interleukin-4 (IL-4) started to be suppressed at a concentration of 30 ㎍ / / Ml), the inhibition on cytokine production was evident.

Experimental Example 3: Reversible effect of inhibition of mast cell activation

As shown in FIG. 3, when the antigens were treated with the antimicrobial extract for 30 minutes, the cells were washed 5 times with the medium before stimulation with the antigen, (Allergic substance secretion) inhibition was restored. These results suggest that the inhibition of the secretion of allergen - inducing substances from mast cells by the antigens of the extracts of Tallarum canis is reversible. This inhibition of reversible mast cell degranulation suggests that there is no side effect of the extracts due to their irreversible nature.

Experimental Example 4. Studies on the functional groups of mugwort extract on mast cells

The inventors of the present invention searched for the inhibitory effect on the activation of Syk-LAT among the various signal transduction pathways that cause allergy in mast cells inducing allergy in the body. RBL-2H3 cells were seeded at a density of 1.0 × 10 ⁶ per well on a 6-well plate and cultured in MEM medium containing 20 ng / ml DNP-specific immunoglobulin E for 12 hours to sensitize the cells. Cells were stimulated with antigen and washed twice with ice-cold PBS. Cells were washed twice with Lysis buffer (20 mM HEES, pH 7.5, 150 mM NaCl, 1% Nonidet p-40, 10 % Glycerol, was dissolved in 60 mM octyl- β -glucoside, 10 mM NaF, 1 mM Na 3 VO 4, 1 mM phenylmethylsulfonyl fluoride, 2.5 mM nitrophenylphosphate, 0.7 ㎍ / ㎖ pepstatin, and a protease-inhibitor tablet). Next, centrifugation was carried out at 13,000 rpm for 5 minutes, and the supernatant was mixed with 2x laemmli sample buffer and protein denaturation was performed at 100 ° C for 5 minutes. The protein samples thus obtained were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 120 V for 1 hour and 30 minutes using 10% or 12% polyacrylamide gel (polyacrylamide: bis acrylamide = 29: And transferred to a nitrocellulose membrane (Schleicher & Schuell, BA85) for 1 hour and 30 minutes at 300 mA. The cells were blocked with 5% skim milk or 5% BSA in TBS-T (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for one hour and then incubated with each specific antibody Lt; / RTI > Then, HRP (horseradish peroxidase) conjugated monoclonal antibody or polyclonal antibody was reacted for 1 hour, then the remaining nonspecific antibody was washed away with TBS-T, and X-ray was detected using an ECL detection kit (Amersham Pharmacia Biotech) ray film. As shown in Fig. 4, the phosphorylation degree of Syk and LAT began to decrease at a concentration of 30 ㎍ / ㎖ or higher, and protein phosphorylation was almost inhibited at 300 ㎍ / ㎖.

Experimental Example 5. Detection of antiallergic effect in locally allergic animal model

Balb-c mice 0.5 g of immunoglobulin E was injected intradermally into one ear and sensitized for 12 hours. After sensitization, 5% arabic gum suspension was orally administered orally. One hour later, 250 μl of antigen solution (1 mg antigen + 5 mg of Evans blue / ml PBS solution) was injected through the tail vein. After 30 minutes of injection, the mice 'ears were removed and extracted in formaldehyde at 63 ° C for 12 hours. The absorbance of the extracted Evans Blue was measured at 620 nm to confirm the antiallergic effect. The results are summarized in FIG.

As shown in FIG. 5, the extracts of Tortoiseshell showed a significant effect on the allergic reaction by the antigen at 100 mg / kg or more, and 1000 mg / day showed the same effect as the commercial cetirizine drug used as the control agent. From these results, it can be confirmed that the extract of Gomari extract of the present invention shows a high inhibitory effect even in an allergic animal model induced by the antigen.

Having described specific portions of the present invention in detail, it will be apparent to those skilled in the art that this specific description is only a preferred embodiment and that the scope of the present invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

Water or alcohol extract of Persicaria thunbergii with antiallergic effect by blocking secretion of allergen.

A pharmaceutical composition for preventing or treating an allergic disease, which comprises, as an active ingredient, a water or alcohol extract of Persicaria thunbergii having an antiallergic effect by blocking the secretion of an allergen-inducing substance.

A health food for preventing or improving allergies comprising water or an alcohol extract of Persicaria thunbergii having an antiallergic effect by blocking the secretion of allergen-inducing substances.

(1) washing and pulverizing dried tallgrass ( Persicaria thunbergii );
(2) heating the corn crushed by the step (1) in a hot water bath to obtain a coriander extract;
(3) filtering the extract from the extract obtained in the step (2) to obtain a supernatant and concentrating; And
(4) A method for producing Persicaria thunbergii extract having an antiallergic effect by blocking the secretion of an allergen causing substance, which comprises freezing the concentrate obtained in the step (3) and freeze-drying the powder to obtain a powder.

(1) immersing the dried persimmon ( Persicaria thunbergii ) in a lower alcohol and extracting it with an ultrasonic extractor to obtain an alcoholic extract;
(2) concentrating the extract obtained from step (1) above; And
(3) freezing the concentrate obtained in the step (2) and lyophilizing the concentrate to obtain a powder, and isolating the secretion of the allergen causing substance, thereby producing an allergic persimmon extract ( Persicaria thunbergii ).