WO2016021847A1

WO2016021847A1 - Oral anti-atopic composition comprising rice prolamin

Info

Publication number: WO2016021847A1
Application number: PCT/KR2015/007448
Authority: WO
Inventors: 안봉환; 양성렬; 정영도; 최기오; 김성진; 장미선; 윤현중; 김현우; 송동업
Original assignee: 전남대학교산학협력단
Priority date: 2014-08-05
Filing date: 2015-07-17
Publication date: 2016-02-11
Also published as: KR20160017607A

Abstract

The present invention relates to an oral anti-atopic composition comprising rice prolamin as an active ingredient. The composition of the present invention has an excellent anti-atopic efficacy and has high stability by comprising protein of commercial food as an active ingredient, and thus can be broadly used.

Description

Oral anti-atopic composition containing rice framine

The present invention relates to an oral composition containing rice framine as an active ingredient and exhibiting anti-atopic activity. Background

Atopic dermatitis is a skin eczema disease that is accompanied by chronic recurring severe itching. It is a typical allergic disease with allergic asthma and allergic rhinitis. Until the 1970s, only 3% of children under 6 years of age were reported, but recently, children 20¾> and 1 ~ 3% occur in adults. Atopic dermatitis has recently increased.

The principle of treatment for atopic dermatitis is to remove the cause and triggers to prevent the onset and exacerbation of symptoms, and to keep the skin tone and clean with proper bath and moisturizers. In order to prevent secondary skin infections, topical steroids, topical calcineurin inhibitors, antihistamines, immunomodulators, antiviral agents, etc. are used appropriately. However, patients with no acute reaction or severe atopic dermatitis can take special treatment such as phototherapy, appropriate UV treatment, immunosuppressant such as interferon gamma, cyclosporine, and intravenous injection of immunoglobulin in consideration of patient's symptoms and circumstances. have.

However, atopic dermatitis, such as atopic dermatitis is not easy to cure, it requires a long period of treatment, so long-term treatment is required to develop a drug that is easy to take with less side effects. On the other hand, the present inventors have reported that the glutinous rice extract exhibits an excellent anti-inflammatory effect on the intestinal tissue (Ahn et al., Republic of Korea Patent Application

10— 2013-0008148, 2013).

In addition, the present inventors have reported that the external preparations containing the rice extract and tea extract as an active ingredient shows an excellent dermatitis improvement effect in the mouse model (Ahn et al., Korean Patent No. 10-1477863, 2014). ―

^"However, research on the effect on atopic dermatitis when orally ingested chamssal extract in relation to the study, not yet, and further also not yet been studies on the anti-(抗) atopic chamssal active material contained in the extract. Disclosure of the Invention

Challenge to be solved

An object of the present invention is to provide a composition for the treatment and prevention of atopic diseases. More specifically, the subject of the present invention is rice. To confirm the protective effect of the extract against atopic diseases, to isolate and confirm the active ingredient in the rice extract, to provide a useful therapeutic agent for the treatment and prevention of atopic diseases. Solution to problem

In order to solve the above problems, the present inventors examined the effects of glutinous rice water extract on atopic diseases and studied a substance showing effective anti-atopic activity even by oral administration in the extract of oatmeal, oatmeal or The present invention was completed by discovering that rice frramine protein contained in tem rice is the main ingredient exhibiting such anti-atopic activity. Accordingly, the present invention provides an oral composition having almost no side effects even with long-term administration and having excellent anti-atopic activity. That is, the present invention relates to the following.

1. Oral anti-atopic composition comprising rice prolamin (prolamin) as an active ingredient.

2. The oral antiatopic composition according to 1 above, which comprises at least 0.01% of rice prolamin.

3. The oral antiatopic composition according to 1 above, wherein the rice prolamin is glutinous rice framine or spicy rice framine.

4. The oral anti-atopic composition according to 1 above, wherein the rice frramine is formulated in a form contained in rice extract.

5. A method of preventing or treating atopy comprising administering a therapeutically effective amount of rice frramine.

6. Use of rice prolamin for the preparation of oral anti-atopic composition. Effects of the Invention

The composition according to the present invention exhibits excellent anti-atopic efficacy in atopic dermatitis mouse model, and includes a protein of rice, a commercial food, as an active ingredient, which has almost no side effects even when taken for a long time, and thus can be widely used. . Detailed description for carrying out the invention

Rice prolamin used in the composition of the present invention is contained in rice Protein is composed of polypeptides of molecular weight of about 13kDa, 16kDa, lOkDa, of which 13kDa type is dominant.

Rice framine, the active ingredient of the anti-atopic composition of the present invention, can be purified from glutinous or spicy rice.

In order to measure the anti-atopic activity of the composition of the present invention, 2,4-dinitrochlorobenzene was injected into BALB / c mice's skin while ingesting a feed containing sesame water extract or purified rice frramine for 6 weeks. (DNCB) was applied to induce atopic dermatitis and the effect of intake of rice extract and rice framine on dermatitis development.

As a result, the rice extract and rice framine showed distinct anti-atopic efficacy, and in the case of rice framine, the framine from oat rice and framine from spicy rice showed similar anti-atopic effect. In addition, heating rice framine at 10 CTC for 30 minutes did not reduce its anti-atopic activity. Based on the above experimental results, the present invention was completed to provide an oral anti-atopic composition containing rice prolamin as a main ingredient.

The composition may be formulated in an oral dosage form, and the composition for oral administration of the present invention may contain a pharmaceutically acceptable carrier. Oral dosage forms include solid dosage forms such as tablets, capsules, powders, and granules, and can also be formulated as liquids.

The concentration of rice frramine contained in the composition is preferably 0. 2¾ or more.

Brief description of the drawings

1 is a rice extract (seam rice extract or administered to BALB / c mice) Rice Prolamin) uptake and DNCB treatment schedule are summarized.

Figure 2 shows that the concentration of rice seed extract inhibits atopic dermatitis symptoms induced by DNCB in mice.

Figure 3 shows that the glutinous rice water extract inhibits the decrease in skin barrier function induced by DNCB in mice in a concentration-dependent manner.

Figure 4 shows that glutinous rice water extract inhibits the increase in serum IgE concentration induced by DNCB in mice in a concentration-dependent manner.

Figure 5 is an electrophoresis (SDS-PAGE) of the framine fraction purified from the rice and spicy rice powder.

Figure 6 shows that the concentration of rice prolamin inhibits atopic dermatitis symptoms caused by DNCB in mice.

FIG. 7 shows that the increase in serum IgE concentration induced by DNCB in mice inhibits intake dependent on rice intake.

FIG. 8 shows that glutinous rice framine and spicy rice framine inhibit the symptoms of atopic dermatitis induced by DNCB in mice to a similar degree, and the dermatitis inhibitory activity does not decrease even when they are heated for 30 minutes. FIG. 9 shows the suppression of the increase in serum IgE concentration induced by DNCB in mice to a similar level between glutinous rice and spicy rice framine and their inhibition in locrc.

Inhibition of IgE concentration increase inhibitory activity did not decrease even after 30 minutes of heating.

Best Mode of Invention Hereinafter, the present invention will be described in more detail with reference to the following examples and test examples. However, the following Examples and Test Examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto. Example 1 Preparation of White Rice Extract

Distilled water weighed five times was added to 600 g of glutinous rice powder (class name: Baek Okchal) and shaken three times for 30 seconds with a blender. After shaking, the mixture was centrifuged at 1,300 X g for 10 minutes to obtain a supernatant, and then lyophilized to use as a rice seed extract. The above process was repeated five times to obtain 235.8 g of the rice extract from the true rice powder 3. Okg. Example 2. Preparation of Rice Pramine

Pramine protein was extracted by slightly modifying the method described by Ju et al. (J Food Sci. 66, 229-232, 2001) from the powder of real rice (variety name: Baekokchal) and spicy rice (variety name: Saenuri). That is, 1 kg of rice powder was degreased with 4 ᅳ e of nucleic acid and dried in a hood for 24 hours. The degreased rice powder was suspended in 5% NaCl solution 4, shaken for 4 hours, and centrifuged at 3,000 X g for 30 minutes to remove the albumin-globulin fraction from the upper layer. The precipitate was again suspended in 5% NaCl of 4 and the albumin ᅳ globulin removal process was repeated. Thereafter, the precipitate was suspended in 70% ethanol 3 and shaken for 4 hours to extract framine and centrifuged for 30 minutes at 3,000 × g to obtain an upper fraction of framine. The precipitate was resuspended in 3 £ h ethanol and the framine extraction process was repeated. Ethanol was removed from the extracted prolamin fraction under reduced pressure and centrifuged to obtain a precipitate. The precipitate was sufficiently dissolved in 50 mM 25 mM NaOH solution, centrifuged, and then ethanol was added to ethanol. The concentration was 70% and neutralized to pH 7 using Γ7Μ acetic acid. To this solution was slowly added three volumes of cold acetone to precipitate the protein and centrifuged it. The protein precipitate was dissolved in 25 mM NaOH-70% ethanol again and precipitated twice with acetone. The final isolated protein precipitate was dialyzed in distilled water, lyophilized and stored at 4 ^° C. A portion of this was dissolved in 25 mM NaOH and bovine serum albumin was used as the standard protein. Protein quantification was performed using BCA kit (Thermo Scient ific, Rockford, IL, USA). As a result, 1.36g and 1.05g of the prolamin fraction was purified from 1kg of raw rice and spicy rice powder, respectively, and the content of solid component protein was 96.8%, 95 and 4%, respectively. The purified framine fractions were used as true rice framine and spicy rice framine, respectively. Example 3 Heat Treatment of Rice Pramine

0.5 g each of the real rice framine and spicy rice prolamin prepared in Example 2 were suspended in distilled water 50 and heated in boiling water for 30 minutes, and then cooled and lyophilized to be used in subsequent experiments.

Experimental Example 1. Protective effect of rice extract on DNCB-induced atopic dermatitis in mice

1) experimental method

(1) animals and reagents

Six-week-old female BALB / c mice were purchased from a central laboratory animal (Seoul, Korea) and were fed freely with water and feed while grown under 12-hour night-day cycles, constant temperature, and humidity conditions. Forage products from SAFE Lab Diets (Augy, France) R03 was prepared as a powder, and the glutinous rice extract and rice frramine powder prepared in Example 1-3 were respectively 1—10% and 0.01-0. 1% was added to the mice and fed a new daily feed. DNCB was purchased from Sigma-Aldr i ch (St. Loui s, MO, USA) and used in acetone-olive oil (4: l / v: v) mixture. _,

(2) Atopic dermatitis protective effect measurement

1 summarizes rice extract (rice extract or rice frramine) intake and DNCB treatment schedule. In other words, the experimental animals were fed with rice extract from 4 weeks before DNCB treatment, and then continued to consume the feed during 2 weeks of DNCB treatment. One day before the start of DNCB treatment (-1 days), the mice and the like were depilated using an electric epilator. DNCB treatment was performed using a patch with 1 cm ² gauze attached. At the start of DNCB treatment (day 0) and after 3 days (+3 days), 1% DNCB Ιθ was added to the depilated back for 2 days, respectively, 7 days after the start of DNCB treatment (+7 days) and 10 days after On (+10 days) 0.2% DNCB 100 ^ was added to the gauze and attached to the epilated back for one day. 14 days after the start of DNCB treatment (+14 days), the dermatitis on the site of DNCB treatment was visually observed, and the plaque, edema, abrasion, and keratin were evaluated by three points as follows. To the extent indicated.

0 points ： There is no symptom

1 point ： Weak symptom

2 points ： Slightly severe symptoms

3 points ： Severe symptoms (3) transdermal moisture loss (TEWL) measurement

In order to observe the effect of atopic dermatitis on skin barrier function and the effects of rice extracts, transdermal moisture loss was measured, which was measured by Tewameter TM300 (Courage and 14 days after DNCB treatment (+14 days) under forane anesthesia. Khazaka Electronic GmbH, Koln, Germany).

(4) Determination of Serum Iranunoglobulin E (IgE) Concentration

Since the degree of atopic dermatitis is known to be related to serum IgE concentrations, we observed changes in serum IgE concentrations according to rice extract intake in DNCB treated mice. For this purpose, blood was collected by cardiac puncture under anesthesia 14 days after DNCB treatment (+14 days), and serum was separated and stored at -80 ^° C for use. Serum IgE concentration was measured by sandwich ELISA using a kit (eBioscience, San Diego, CA, USA).

(5) Statistical Analysis

The measurement results were expressed as mean 土 standard deviation, and the significance test was performed according to one-way variance analysis (one ᅳ way AN0VA) and Scheffe test.

2) test result

(1) Influence of Sesame Rice Extract on DNCB-Induced Atopic Dermatitis Add the glutinous rice extract prepared in Example 1 to 1, 3 and 10%, respectively, to feed and to atopic dermatitis induced by DNCB while ingested in mice. Affectionate The impact was measured. In order to evaluate the effects on atopic dermatitis in more detail, not only the symptoms of dermatitis, but also the deterioration of skin barrier function accompanying dermatitis and the increase of serum IgE concentration in atopic diseases were observed. The experimental results are shown in FIGS. 2, 3 and 4. The number of mice in each experimental group was 6, and ^* p <0.05 and * ^* p <0.01 indicate the difference of difference with respect to the DNCB control group.

As shown in FIG. 2, the mice were treated with DNCB and exhibited severe dermatitis symptoms. At this time, when the intake was added to the feed, the symptoms of dermatitis caused by DNCB were suppressed depending on the amount of the extract. The feed added with 1% of the rice extract did not show a significant inhibitory effect, but the feed added with 3-10% showed a significant inhibitory effect. In addition, as shown in Figure 3, the treatment of mice with DNCB significantly increased the transdermal moisture loss, which means that the skin barrier function was significantly reduced. Water loss was suppressed concentration dependently. In addition, mice as shown in FIG.

As a result of treatment with DNCB, the serum IgE concentration increased significantly, and ingestion of rice bran extract inhibited the increase of serum IgE concentration by DNCB depending on the intake amount.

As a result, atopic dermatitis is remarkably generated by DNCB treatment in mice, and ingestion of glutinous rice water extract can significantly inhibit the occurrence of atopic dermatitis.

(2) Effect of True Rice Pralamin on DNCB-Induced Atopic Dermatitis First, the rice prolamin fraction purified in Example 2 was extracted from Lae'H. Electrophoresis (SDS-PAGE) was performed according to the method (Nature 192, 680-682, 1970). FIG. 5 shows the results of electrophoresis performed by adding 20% and 10 // g of real rice framine and spicy rice framine to 15% polyacrylamide gel. These samples show a major polypeptide band with a molecular weight of about 13 kDa, which is consistent with the structural properties of rice frramine. Therefore, it was confirmed that the sample prepared from the real rice and spicy rice in Example 2 is a highly purified framine protein.

The purified glutinous glutamine was added to feeds at 0.01, 0.03, and 0.01%, respectively, and fed to mice to determine the effects of DNCB-induced atopic dermatitis and serum IgE levels. The experimental results are shown in FIGS. 6 and 7, respectively. The number of mice in each experimental group was 6, and ^* p <0.05 and "ρ <0. () 1 indicate the difference of difference with respect to the DNCB control group.

As a result, as shown in FIG. 6, it is 0.01-0. At 1% concentration, the symptoms of dermatitis caused by DNCB were suppressed in a concentration dependent manner. In addition, as shown in FIG. 7, ingestion of boiled rice prolamin inhibited the increase of serum IgE concentration by DNCB depending on the intake amount.

As a result, it can be seen that the intake of alum dermalin in place of the sesame seed extract in mice significantly inhibits the occurrence of atopic dermatitis caused by DNCB.

(3) Comparison of Anti-Atopy Activity and the Heat Resistance Test of Anti-Atopy Activity between Korean Rice Pramine and Spicy Rice Pramine

According to Ahn et al. (Korean Patent No. 10-0963339, 2010), rice framine protects gastric mucosal damage caused by harmful factors such as ethane. It was reported that framine from refined rice showed higher specific activity (speci fic act ivi ty) than pro-lamine purified from thale rice. Therefore, in the present invention, the anti-atopic activity of real rice framine and spicy rice prolamin was compared.

To this end, the effects of the atopic dermatitis and serum IgE concentrations induced by DNCB were measured by adding 0.05% of lamb rice framine and spicy rice prolamin prepared in Example 2 to the feed and ingested in mice. . In addition, in order to observe the heat resistance of these rice frramine, as in Example 3, the real rice framine and spicy rice frramine were heated at 100 ^° C for 30 minutes, and then added to the feed to 0.05%, respectively, and the mice The effects of DNCB-induced atopic dermatitis and serum IgE levels were measured. The above results are shown in FIGS. 8 and 9. The number of mice in each group was 6 and ρ <0. () 1 indicates the difference between the DNCB control and the difference.

As shown in FIGS. 8 and 9, not only glutinous rice framine but also spicy rice framine significantly inhibited the symptoms of necrosis caused by DI B and increased serum IgE concentration at 0.05% concentration. .

In addition, even when heated to 100 ^° C for 30 minutes, both the rice promamine and spicy rice prolamin maintained anti-atopic activity before heating.

The above results suggest that both the real rice framine and the real rice framine have similar anti-atopic activity, and the activity is highly resistant to high fever such as cooking process.

Claims

Claims Oral anti-atopic ancestors comprising rice prolamine (prol amin) as an active ingredient.

The oral anti-atopic composition according to claim 1, which comprises at least 0.01¾ »of rice frramine.

The oral anti-atopic composition according to claim 1, wherein the rice frramine is a real rice prolamine or a rice frulamine.

The oral anti-atopic composition according to claim 1, wherein rice prolamin is formulated in a form contained in rice extract.

A method for preventing or treating atopy, comprising administering rice framine in a therapeutically effective amount.

Use with rice prolamin for the preparation of oral anti-atopic composition.