KR101809800B1 - A pharmaceutical composition comprising anti-allergic peptide isolated from abalone intestine - Google Patents

Abstract

The present invention relates to a peptide having an anti-allergic effect, and more particularly to a peptide having a functional peptide composed of Pro-Phe-Asn-Gln-Gly-Thr-Phe-Ala-Ser derived from the intestines of Haliotis discus hannai. The peptide having an anti-allergic effect exhibits an excellent effect of inhibiting an allergic reaction induced in activated human mast cells. Therefore, a composition containing the peptide as an active ingredient can be used for an allergic reaction-related disease .

Description

[0001] The present invention relates to a pharmaceutical composition comprising an anti-allergic peptide isolated from abalone,

The present invention relates to a peptide having an anti-allergic effect, and more particularly, to an anti-allergic peptide comprising Pro-Phe-Asn-Gln-Gly-Thr-Phe-Ala-Ser derived from Haliotis discus hannai And to a pharmaceutical composition for preventing and treating an anti-allergic reaction-related disease.

Abalone has been recognized for its excellence from ancient times, and the efficacy of Abalone in Korea has been described as "efficacy of abalone to lighten the body and brighten the eyes" . In the Joseon Dynasty, the scholastic poetry of the Joseon Dynasty introduced the abalone in the name of puffer in an asset speech book, and said, 'The lean meat can be eaten with the taste, and it is good to eat it, but the best method is to dry it and eat it. The chick can be cooked, it can be eaten in seaweed, and it is effective in treating boils. In spring and summer there is poison. When it touches this poison, the flesh becomes the boil of booster, and the lesion bursts. "In general, shellfish are excellent for restoring fatigued nerves, especially abalone, The shells of abalone are used in conjunctivitis and cataracts as well, which is called "Seonjyeongmyeong" in one room. It also relieves the symptoms of throat burning, chest tightness, strengthens the liver function, When it is weak, boiling abalone makes it cheerful, urine comes out well, and it also helps jaundice and cystitis.

Abalone has excellent nutritional value even in excreta and is used as a treatment for diabetes and hypertension. Abalone is abundant in proteins, rich in amino acids, and rich in minerals and vitamins such as phosphorus, iron, and iodine. Therefore, abalone has been treated as an aquatic product from ancient times, skin beauty, nourishment, and postpartum cooking, and has excellent efficacy in fragile constitution, many people eat for the purpose of medicinal as well as edible.

If a person lacks iodine and thyroid hormones, obesity due to basal metabolic slowdown and accumulation of fat in blood vessels may occur. In addition, thyroid hormones are involved in the physical, mental, and sexual growth of the growing children. A lack of iodine can lead to significant hypothyroidism, rough skin, and thinness of the hair. Therefore, it is safe to consume iodine in foods such as seaweeds. Abalone has high iodine content. It is known that when the head is sick or the ringing and the tongue and throat are dry, eating the abalone improves the strength of the liver because it is cleverly healed. It is said that when milk of maternity does not come out within seven days after the birth, it is said that it will have a great effect when it is fed orphan over abalone, because there are many minerals in abalone. In addition, abalone is a high-protein component, high absorption rate in the body is good food for pregnant women, obesity, liver cirrhosis. So it is used as medicine for the recovery of liver function and pulmonary tuberculosis. Abalone is rich in amino acids such as methionine and cysteine, and is good for regeneration and fatigue after suffering sickness. Research on abalone has been mainly carried out in the field of aquaculture and breeding. Recently, intestine has been studied for its functions such as enzymes and lectins. Studies on the physiological activity of abalone have been partially carried out on antioxidant and antihypertensive effects on the extracts of flesh tissue and intestine, State.

Allergy is a type of hypersensitivity reaction caused by immune imbalance. It is a reaction that occurs to a specific antigen called allergen, and IgE antibody specific to the antigen is induced to bind to mast cell or basophile . The mast cells or basophils activated by the IgE antibody secrete inflammatory cytokines and histamine to cause vasodilatation and inflammatory reaction to cause allergic atopy, asthma, skin allergy, and the like. In severe cases, it is an inflammatory reaction that occurs infiltration of inflammatory cells locally and belongs to type 1 hypersensitivity reaction. The incidence of such allergic diseases has been greatly increased, and the importance of mitigating and preventing diseases has been emphasized. However, in functional foods, there is almost no development of immune-active materials derived from marine natural products, except that spirulina is used as an immuno-active health food material as a whole.

In the prior art of the present invention, a digestive digestive digestive digestive gland and a pharmaceutical composition containing it as active ingredients are known in Korean Patent No. 10-2013-0029597, which has an effect of inhibiting MMP activity, (AIGID) as an active ingredient. The efficacy and function of abalone has been known for some time in folk remedies and some ancient literature. However, there is no evidence based on scientific facts so far, so scientific and empirical studies have shown that efficacy of abalone Verification and re-evaluation is an urgent task.

It is an object of the present invention to provide an allergic functional peptide consisting of the amino acid sequence of Pro-Phe-Asn-Gln-Gly-Thr-Phe-Ala-Ser derived from abalone. It is another object of the present invention to provide a pharmaceutical composition for preventing and treating an allergic disease containing the peptide.

This object of the present invention is achieved by a method for the treatment of haliotis discus hannai, Obtaining an AIGID fraction from the hydrolyzate obtained in said step; Identifying a peptide consisting of the amino acid sequence of Pro-Phe-Asn-Gln-Gly-Thr-Phe-Ala-Ser from the AIGID fraction; Evaluating the effect of inhibiting the allergic effect derived from human mast cell in the peptide identified in the above step; And a pharmaceutical composition for the prevention and treatment of an allergic disease in which the peptide is contained.

The peptide purified from the inverted embryo of the present invention has an effect of inhibiting the allergic reaction in human mast cells, and the pharmaceutical composition containing the peptide as an active ingredient has an excellent effect in the prevention and treatment of allergy-related diseases.

1 is a result of analyzing the molecular weight and amino acid sequence of the hydrolyzate derived from abalone according to the present invention.
FIG. 2 is a graph showing the results of toxicity determination of the overturned hydrolyzate of the present invention on human mast cells.
FIG. 3 is a graph showing the effect of the overturned hydrolyzate of the present invention on the inhibition of histamine secretion in human mast cells.
FIG. 4 is a graph showing the effect of the hydrolyzate of the overturned endogenous hydrolysates according to the present invention in the anti-IgE-induced in vivo antibody reaction in animal skin.
FIG. 5 is a graph showing the effect of the purified peptides according to the present invention on the inhibition of cytokine mRNA expression in human mast cells.
FIG. 6 shows the results of inhibiting the secretion of cytokines in human mast cells by the purified peptides according to the present invention.
FIG. 7 shows the results of inhibition of activation of MAPKs in human mast cells by purified peptides according to the present invention.
Figure 8 shows the results of inhibition of IκBα activation and NF-κB nuclear transfer in human mast cells according to the present invention.

The present invention provides a composition for the prevention and treatment of allergic diseases, comprising the peptide derived from Haliotis discus hannai as an active ingredient.

According to the present invention, the peptide purified by the above method has an anti-allergic effect and can inhibit the allergic reaction induced in human mast cells. Therefore, a composition comprising the peptide as an active ingredient can be useful for allergic diseases.

The composition of the present invention can be used as a pharmaceutical composition. In this case, in addition to the above-mentioned active ingredient, a formulation comprising a pharmaceutically acceptable carrier, an additive, an edible carrier or a food additive may be prepared. Examples of the carrier or the additive include diluents, sweeteners, stabilizers, perfumes, vitamins, and antioxidants.

For example, the diluent may be lactose, cereal starch and the like. Examples of the sweetener include sugar, liquid fructose, crystalline fructose, sucralose, sorbitol or aspartame, As the stabilizer, sodium carboxymethyl cellulose, beta-cyclodextrin or xanthan gum may be used. Natural flavors such as fruit flavors, herbal flavors, natural flavors such as natural flavors and flavonoids, sweeteners such as fructose, honey, sugar, and acid anhydrides such as citric acid and sodium citrate may be further added to improve the taste of the composition . The composition of the present invention may further contain, as an auxiliary component, an inorganic component such as vitamin B, C, E or beta-carotene, Ca, Mg, Zn, or a phospholipid component such as lecithin or an oligosaccharide, amino acid or taurine .

Meanwhile, the pharmaceutical composition of the present invention may be in the form of a tablet, a capsule, a dragee, a bead, a granule, a solution, a suspension, or a sachet It can be formulated into one.

The present invention will be described in more detail with reference to the following specific examples and experimental examples, but the following examples and experimental examples are intended to illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1. Preparation of AIGID using UF membrane bioreactor.

The digestive hydrolysates of abalone (AIGID) were separated from the abalone, washed several times with physiological saline, and lyophilized and powdered. 100 mL of distilled water was added to 20 g of each of the abalone and embedded powder samples and the pH was adjusted to 1.8 to 2.2 at the same pH as the digestion conditions. The pepsin was added at a substrate to enzyme ratio of 250: 1, Min to 2 hours under gastric digestion conditions. The hydrolyzate was then incubated with a mixture of bile salts (0.125 M taurocholate, 0.125 M glycodeoxycholic acid), 1 M CaCl2, 0.25 M bistritose (pH 6.5), 0.1% (w / v) After mixing with porcine pancreatic lipase, the cells were treated with trypsin and alpha-chymotrypsin under pH 8.0 at a substrate-to-enzyme ratio of 250: 1, a temperature of 35 to 40 ° C, and a reaction time of 2 to 3 hours under intestinal digestion conditions ≪ / RTI >

The secondary hydrolyzate was centrifuged at 10,000xg for 15 minutes at 4O < 0 > C, and the supernatant was lyophilized for 5 days to obtain AIGID powder, which was fractionated, concentrated and desalted in a UF membrane reactor system to obtain AIGID I (10 kDa or more) Three fractions were obtained: AIGID II (5-10 kDa) and AIGID III (5 kDa or less) (Fig. 1).

Experimental Example 1 Identification of cytotoxicity against hydrolysates derived from abalone.

The cytotoxicity of the three fractions (AIGID I (10 kDa or more), AIGID II (5-10 kDa) and AIGID III (5 kDa or less)) prepared from the overturned hydrolyzate obtained in Example 1 of the present invention was measured by MTT assay Respectively. The above MTT assay is an assay for measuring cytotoxicity or survival rate. It is tested using a microplate reader using the ability of mitochondria to reduce the yellow water-soluble substrate MTT tetrazolium to a bluish-purple water-insoluble MTT formazan by dehydrogenase Method.

To evaluate this, human mast cells were treated with the three fractions prepared from the hydrolyzate derived from the abalone obtained in Example 1 at a concentration of 100 to 500 μg / ml. Control group was compared with PMACI and AIGDP treated group and PMACI treated group only.

As shown in FIG. 2, there was no significant difference in the concentration of 100 to 300 μg / mL, but the survival rate of the cells treated with 500 μg / mL decreased by about 35%. As a result, the present inoculum-derived fractions showed no cytotoxic effect at a concentration of 100 to 300 μg / mL.

Experimental Example 2 Inhibitory Effect of Histamine Secretion on Hydrolysates Derived from Abalone Induced by the Invention.

Three fractions (AIGID I (10 kDa or more), AIGID II (5-10 kDa), and AIGID III (5 kDa or less)) prepared from the overturned hydrolyzate obtained in Example 1 according to the present invention inhibited the histamine secretion The effect was confirmed by histamine secretion level test. Histamine is an amino acid histamine decarboxylated, stored in mast cells, basophils, and the like, and is a substance that causes an allergic or anaphylactic reaction by external stimuli.

To assess this, PMACI was induced in human adipocytes and histamine secretion levels were examined in the cells. For the histamine secretion level test, AIGIDs derived from abalone organs on human adipocytes were treated at a concentration of 100 to 300 μg / mL as determined in Experimental Example 1, and PMACI was treated after 1 hour. As a control group, AIGID and PMACI treated group were compared with PMACI treated group.

As shown in FIG. 3, when PMACI was treated, histamine secretion was definitely increased and the AIGID I and III treated groups showed a significant decrease compared to the PMACI treated group. As a result, it was found that the antiallergic effect was excellent when AIGID I and III derived from the abalone embryo of the present invention were treated at a concentration of 300 μg / mL.

Experimental Example 3. Inhibitory Effect of IgE - Induced In vivo Antibody Antibody in Animal Skin against Hydrolysates Derived from Abalone.

Animal skin IgE (AIGID I) (10 kDa or more), AIGID II (5-10 kDa) and AIGID III (5 kDa or less)) obtained from the overturned hydrolyzate obtained in Example 1 according to the present invention Induced in vivo antigen antibody reaction inhibition was confirmed by PCA (passive cutaneous anaphylaxis) reaction. The PCA reaction is a method for examining the in vivo antigen antibody reaction. It is a method to see the reaction between a small amount of antibody and an antigen in vivo. After injecting a small amount of antibody into the skin of an animal, It is a way to observe that pigment collects a few minutes later.

To evaluate this, 50 mg / kg of AIGIDs was intraperitoneally administered with 100 ug of DNP-HSA to the PCA mouse model. One hour later, 500 ng of anti-DNP-IgE was administered to the DNP-HSA antigen in the skin. Using the spectrometer, The degree of reaction was measured. The control group was compared with those not treated with DNP-IgE and DNP-HSA alone.

As shown in FIG. 4, when AIGID III was administered, DNP-IgE and DNP-HSA alone were significantly inhibited compared with the control group. As a result, it was confirmed that AIGID III derived from Abalone intestine according to the present invention is superior to AIGID I or AIGID II in the treatment of allergy, and AIGID III was used for in vitro experiments.

Example 2. Molecular Weight and Amino Acid Sequence Analysis of Peptides Purified from Abalone Derived Hydrolyzate.

Peptides were obtained from the AIGID III fraction of three fractions (AIGID I (10 kDa or more), AIGID II (5-10 kDa) and AIGID III (5 kDa or less)) prepared from the overturned hydrolyzate obtained in Example 1 according to the present invention After purification, the molecular weight and amino acid sequence were analyzed by Q-TOF mass spectrometry.

For this purpose, AIGID III was purified by chromatographic method. The exact molecular weight and amino acid sequence of AIGDP were analyzed by repeating FPLC and RP-HPLC.

The molecular weight of the peptide (AIGDP) isolated from AIGDP was confirmed to be 1,175.2 Da, and the entire amino acid sequence was confirmed by PFNQGTFAS (Pro-Phe-Asn-Gln-Gly-Thr-Phe-Ala-Ser).

Experimental Example 4. Inhibition of PMACI-induced cytokine mRNA expression in human mast cells of purified peptide (AIGDP) purified from abalone-derived hydrolysates.

In order to confirm the suppressive effect of PMACI-induced cytokine (IL-1, IL-6, TNF-?) MRNA expression in human mast cell peptides comprising the amino acid sequence obtained in Example 2 according to the present invention RT-PCR. Cytokine is a glycoprotein that is used as a signaling substance to control the body's defense system and plays an important role in immunity, infection, hematopoietic function, tissue regeneration, development and growth of cells, Control and stimulate. Thus, cytokine is an important indicator for reducing inflammation induced by allergies. RT-PCR is a method of amplifying a target gene using a primer through a reverse transcription reaction.

To evaluate this, human adipocytes were pretreated with 100 to 300 μg / ml of AIGDP for 1 hour and stimulated with PMACI for 6 hours. RNA was then isolated from the cells using TRIzon reagent. CDNA was prepared from total RNA (1.0 ug) of cells using M-MLV reverse transcriptase, and cDNA was amplified using specific primers. The amplified cDNA was electrophoresed to confirm the protein produced by PCR reaction on agarose gel. Control group was compared with PMACI and AIGDP treated group and PMACI treated group only.

As shown in FIG. 5, when compared with the control group treated with PMACI alone, the expression levels of cytokines IL-1, IL-6 and TNF- induced by PMACI in human mast cells by purified peptides were significantly The amount of cytokine expression was inhibited in the treated group. In particular, the expression level of cytokine was significantly inhibited at a concentration of 300 μg / mL of AIGDP.

Experimental Example 5 Inhibition of secretion of PMACI-induced cytokines in human mast cells of purified peptide (AIGDP) from the overturned intracellular hydrolysates.

The peptide obtained in Example 2 according to the present invention was confirmed by cytokine assay to confirm the secretion inhibition of PMACI-induced cytokines (IL-1, IL-6, TNF-) in human mast cells. The cytokine assay is a general ELISA method using antigen-antibody binding. Cytokine is a glycoprotein that is used as a signaling substance to control the body's defense system and plays an important role in immunity, infection, hematopoietic function, tissue regeneration, development and growth of cells, Control and stimulate. Thus, cytokine is an important indicator for reducing inflammation induced by allergies.

To evaluate this, human adipocytes were pretreated with 100 to 300 μg / ml of AIGDP for 1 hour and then stimulated with PMACI for 8 hours. The expression level of cytokine was confirmed by ELISA kit reader at 450 nm wavelength using an ELISA kit reader. Control group was compared with PMACI and AIGDP treated group and PMACI treated group only.

As shown in FIG. 6, the expression levels of cytokines IL-1, IL-6, and TNF- induced by PMACI in human mast cells induced by purified peptides were compared with those of control group treated with PMACI alone, The amount of cytokine expression was inhibited in the treated group. In particular, at the concentration of 300 μg / mL of AIGDP, the amount of cytokine expression was greatly suppressed.

Experimental Example 6. Inhibition of PMACI-induced activation of MAPKs in human mast cells by purified peptide (AIGDP) from the overturned intracellular hydrolysates.

The western blot method was used to confirm the inhibitory effect of PMACI-induced MAPKs (p38, ERK, JNK) activation in peptides consisting of the amino acid sequence obtained in Example 2 according to the present invention in human mast cells. MAPK is a mitogen-activated protein kinase, an enzyme found as a serine-threonine kinase that is activated by stimulation of various cell growth factors or carcinogenic promoters. It is present in the intracellular signaling pathway that controls cell proliferation and differentiation, And the like. In particular, p38 and JNK are activated by various stress stimuli, inflammatory cytokines, and are involved in apoptosis, stress response, differentiation, etc., and ERK is involved in cell proliferation. Thus, MAPKs play an important role in immune responses such as cell activation, survival, differentiation and cytokine production. Therefore, the MAPK pathway is important for the pharmacological treatment of allergies. Western blot is a technique for detecting a specific protein from a mixture of proteins. It is a technique for detecting a specific protein by using an antibody by moving a protein separated by electrophoresis to a membrane.

To evaluate this, human adipocytes were pretreated with AIGDP for 1 hour and reacted with 1 uM of A23187 for 30 min to activate MAPKs with PMA. Then, the column was washed three times with PBS, and the same amount of protein was added to 10% SDS gel and subjected to electrophoresis. After transferring to nitrocellulose membrane, primary ab and secondary ab were reacted at room temperature for 1 hour each. After washing three times with TBST, proteins were identified. In order to confirm the activation level of MAPKs induced by PMACI, both humanized and non - humanized proteins were identified and compared. As a control group, PMACI and AIGDP treated group and PMACI treated group were used Respectively.

As shown in FIG. 7, activation of MAPKs induced by PMACI in human mast cells by purified peptides increased in all three types of JNK, P38 and ERK 1/2, but ERK 1/2 and p38 In contrast, JNK induced phosphorylation by PMACI. It was confirmed that the anti - allergic effect was excellent by inhibiting the activation of MAPKs by the peptides derived from abalone.

Experimental Example 7. Inhibition of Iκ-Bα activation and Nuclear transfer of NF-B purified from the overturned hydrolysates of the present invention.

The peptide consisting of the amino acid sequence obtained in Example 2 according to the present invention was subjected to western blot and electrophoretic mobility shift assay to confirm Iκ-Bα activation induced by PMACI and inhibition of nuclear transfer of NF-κB in human mast cells EMSA) was used. EMSA is an experiment to confirm the interaction between protein and DNA. It is an experiment to analyze the characteristics of transcription factors. This is an experimental technique for confirming whether a DNA binding to a strong binding DNA is bound to a DNA binding site of a protein by adding an antibody and shift occurs when a transcription factor in a nuclear extract binds to DNA and reads it into a fluorescent light previously labeled. . Many transcription factors are involved in the pathophysiology of allergies, and NF-κB is activated by various stimuli such as allergies. The NF-κB dimer is usually present in the cytoplasm in an inactivated state in association with inhibitors such as Iκ-Bα in most cells. It is dissociated from NF-κB by specific phosphorylation of Iκ-Bα by external stimuli and migrates to the nucleus, leading to extensive expression of inflammatory genes, cytokines, enzymes, cell adhesion molecules and acute phase proteins. AIGDP inhibits PMACI-induced NF-κB activation by Iκ-Bα phosphorylation and its degradation inhibition. In this way, NF-κB activation measurement is a technique to inhibit cytokine and reduce inflammation induction in allergy.

To evaluate this, human mast cells were pretreated with AIGDP (100 to 300 μg / mL), and PMACI was stimulated for 2 hours after 30 minutes. Levels of Iκ-Bα and p-Iκ-Bα were measured by western blot and levels of NF-κB were measured by EMSA. Control group was compared with PMACI and AIGDP treated group and PMACI treated group only. All experiments were repeated 3 times.

As a result, as shown in FIG. 8- (a), activation of NF-κB induced by PMACI stimulation in human mast cells by purified peptide was decreased by AIGDP in the nucleus, and Iκ- Bα was increased, whereas phosphorylation of Iκ-Bα was inhibited. As shown in FIG. 8- (b), NF-κB was increased by PMACI stimulation, but NF-κB was inhibited by AIGDP treatment. It is shown that inhibition of Iκ-Bα phosphorylation inhibits NF-κB activation and suppresses allergic effects through cytokine inhibition.

INDUSTRIAL APPLICABILITY As described above, the present invention can be used to inhibit allergic reactions in human mast cells. Thus, a composition comprising the peptides as an active ingredient can be useful for allergic diseases, This is a very useful invention.

Claims (5)

Hydrolyzing the haliotis discus hannai under digestion conditions; Obtaining an AIGID fraction of the hydrolyzate obtained in said step; Wherein the step of identifying a peptide comprising the amino acid sequence of Pro-Phe-Asn-Gln-Gly-Thr-Phe-Ala-Ser from the AIGID fraction obtained above is characterized.
A peptide derived from an abalone formed by the amino acid sequence of Pro-Phe-Asn-Gln-Gly-Thr-Phe-Ala-Ser produced by the method of claim 1.
A pharmaceutical composition for preventing and treating atopic diseases, asthma, and skin allergy diseases containing the peptides derived from abalone as defined in claim 2.
The pharmaceutical composition according to claim 3, wherein the composition further comprises at least one pharmaceutically acceptable carrier or additive selected from a diluent, a sweetener, a stabilizer, a flavoring agent, a vitamin, and an antioxidant.
The composition of claim 3 may be in the form of a tablet, a capsule, a dragee, a bead, a granule, a solution, a suspension, or a sachet ≪ / RTI >

Images