KR20180119175A - Composition comprising peptide Periplanetasin-4 isolated from Periplaneta americana for preventing or treating pseudomembranous colitis - Google Patents

Abstract

The present invention relates to a composition comprising peptide periplanetasin-4 isolated from periplaneta americana for preventing or treating pseudomembranous colitis, wherein the peptide periplanetasin-4 has an excellent effect of inhibiting inflammation caused by toxin secreted by Clostridium difficile, and can be easily used as an anti-inflammatory composition or a composition for treating the pseudomembranous colitis, and health functional food.

Description

[0001] The present invention relates to a composition for preventing or treating acute pseudomembranous colitis, which comprises a peptide of the cockroach derived periplanetasin-4, and a composition for preventing or treating acute pseudomembranous colitis,

The present invention relates to a composition for preventing or treating acute pseudomembranous colitis, which comprises the cockroach-derived peptide Periplanetasin-4.

In general, Clostridium difficile ( CD) bacteria cause enteritis by secretion of hyperoxic toxin (CD toxin) in the intestine, and it is known that the toxin derived from the bacteria strongly destroys microtubules in the cytoskeleton Lamont, Gastroenterology, 2007, 138: 875). Among these enteritis, acute pseudomembranous colitis is the causative agent of CD toxin, and it is one of the diseases increasing worldwide since 1999 (Lamont, Gastroenterology, 2007, 138: 875). Pseudomembranous colitis usually occurs during hospital admission and is fatal if it occurs in patients requiring chemotherapy or long-term antibiotic treatment. CD infection is more problematic when the total number of bacteria found in the intestines is reduced by the use of antibiotics rather than by intestinal inflammation itself. In general, the normal bacterial flora inhibits the growth of CD bacteria, which are pathogenic microorganisms. However, when these normal bacterial flies are reduced due to the administration of anticancer drugs or antibiotics, the pathogenic bacterium, CD bacteria, is proliferated and various enteritis including pemphigus colitis occurs (Lamont, Gastroenterology, 2007, 138: 875).

When Clostridium difficile (CD) is overproduced, toxin (CD toxin) is secreted and secreted toxin attacks and destroys epithelial cells in colon, causing acute enteritis. Colonic epithelial cells act as physical barriers to intestinal pathogenic microorganisms, and when the cytoskeleton is destroyed by CD toxin, these barrier functions are lost. Cholerotoxin, which causes similar enteritis symptoms, differs in that it causes simple diarrhea without inflammation in the intestine, whereas CD toxin causes inflammation and diarrhea in the colon simultaneously. Recent reports indicate that CD toxin generates an excess of reactive oxygen species in human colonic epithelial cells and induces activation of p38 MAPK. Such signal activation is known to cause massive fluid secretion, or diarrhea, in the colon (Lamont, Gastroenterology, 2007, 138: 875).

In addition to these intestinal hypermethylation, CD toxin strongly induces cell apoptosis in intestinal epithelial cells. CD toxin stimulation in human colon epithelial cells causes continuous expression of p21 protein. Increased p21 eventually stops the cell cycle (G2-M arrest), and this sequence of processes activates cellular suicide factors such as Bak, resulting in suicide of colon epithelial cells. The increased intestinal epithelial cell suicide process leads to the loss of barrier function, allowing penetration of intestinal materials into the body. In other words, various intestinal pathogenic substances are continuously exposed to the immune cells in the body, resulting in an inflammatory reaction and eventually develop into pseudomembranous colitis (Lamont, Gastroenterology, 2007, 138: 875).

Meanwhile, in spite of the multi-dimensional studies on CD toxin, there has been no development of a direct and efficient therapeutic agent for CD toxin-induced diseases until now. In addition, many accumulated research results do not lead to a fundamental treatment for epidemic treatment of the disease. Therefore, in the therapeutic aspect, more specific and more research and development for enhancing efficacy is urgently required.

Accordingly, the present inventors have studied the various physiological activities of insect-derived functional peptides, and confirmed that the cockroach-derived peptide Periplanetasin-4 has excellent therapeutic effect against acute pseudomembranous colitis, thus completing the present invention Respectively.

Korean Patent No. 10-1471975 (Title of the Invention: Peptides Having Antimicrobial Activity and Compositions Containing the Antibacterial Activity, Applicant: Chung-Ang University Industry & University Collaboration Foundation, Registered Date: December 05, 2014) Korean Patent No. 10-1021226 (Name of the invention: Composition for treating acute pseudomembranous colitis, Applicant: Industry-Academic Collaboration Foundation of Daejin University and Korea, Registered: March 03, 2011)

It is an object of the present invention to provide a composition for the prevention or treatment of acute pseudomembranous colitis which comprises the cockroach-derived peptide Periplanetasin-4.

The present invention relates to a peptide represented by SEQ ID NO: 1 synthesized in a gene of a peptide isolated from a cockroach ( Periplaneta americana ).

The present invention relates to a composition for preventing or treating acute pseudomembranous colitis, which comprises a peptide of Periplaneta americana having the amino acid sequence of SEQ ID NO: 1 below, Periplanetasin-4.

SEQ ID NO: 1: LRHKVYGYCVLGP-NH ₂

The acute pseudomembranous colitis is characterized by a toxin-induced disease secreted by Clostridium difficile .

The peptide may inhibit the inflammatory response, cytotoxic response, cellular suicide, p38 MAPK signal or p21 signal of human colon epithelial cells caused by toxin secreted by Clostridium difficile .

Accordingly, the present invention provides an anti-inflammatory composition containing the peptide periplonetasin-4, and the composition has an anti-inflammatory effect against toxin secreted by Clostridium difficile to be.

The present invention also provides a health functional food for preventing or ameliorating acute pseudomembranous colitis, which comprises the peptide Periplanetasin-4 derived from a cockroach ( Periplaneta americana ) having the amino acid sequence of SEQ ID NO: 1.

The peptide may exhibit an antimicrobial activity against at least one of Staphylococcus aureus and Escherichia coli 0-157. Alternatively, the peptide may exhibit antifungal activity against Candida albicans .

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for preventing or treating acute pseudomembranous colitis, which comprises the cockroach-derived peptide periplanetasin-4 having the amino acid sequence of SEQ ID NO: 1. In addition, the composition containing the peptide may be provided as a composition for anti-inflammation against toxin secreted by Clostridium difficile causing acute pseudomembranous colitis.

The method of providing the peptide of the present invention is as follows. Preferably, in order to identify a peptide gene derived from a cockroach ( Periplaneta americana ), the cockroach is first rapidly frozen with liquid nitrogen to isolate the total RNA, and the gene information on the cockroach transcripts is confirmed using the RNA seq analysis method. A method of screening for a putative unicin that has a therapeutic activity against the above-mentioned disease using an experiment showing activity against a previously discovered pseudomembranous colitis treating agent based on the sequence of amino acids translated from the transcripts of the present invention.

The present invention can provide a pharmaceutical composition for antiinflammation or a pharmaceutical composition for the prevention or treatment of acute pemphigus colitis, which comprises the cockroach-derived peptide periplanetasin-4 having the amino acid sequence of SEQ ID NO: 1. The cockroach-derived peptide periplanetasin-4 may be added in an amount of 0.001 to 30% by weight to the pharmaceutical composition of the present invention.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, body weight, the specific disease or condition to be treated, the severity of the disease or condition, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The present invention provides a health functional food for preventing or ameliorating acute pseudomembranous colitis, which comprises the cockroach-derived peptide periplonetasin-4 having the amino acid sequence of SEQ ID NO: 1. The health functional food may include periplanetasin-4 and a pharmaceutically acceptable food-aid additive. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the peptide of the present invention can be added include various kinds of drinks, meat, sausage, bread, Snacks, noodles, ice cream, dairy products, soups, ionic drinks, beverages, alcoholic beverages, gums, tea and vitamin complexes.

The present invention relates to for the prevention and treatment of acute pseudomembranous colitis containing cockroach-derived peptide Ferry New Plastic Neta -4 (Periplanetasin-4) the composition, the peptide Ferry New Plastic Neta -4 is Clostridium difficile seal (Clostridium difficile , and thus can be easily used as a composition for the treatment of anti-inflammatory compositions and compositions for treating acute pseudomembranous colitis, and as a health functional food.

Figure 1 shows a tissue staining photograph showing that the cockroach-derived peptide periplanetasin-4 (Peri-4) inhibits acute inflammatory responses caused by Clostridium difficile toxin (CD toxin, Tx) ) And ELISA (bottom).
2 is a graph showing MTT assay results indicating that the cockroach-derived peptide periplonetasin-4 (Peri-4) inhibits cytotoxic responses induced by CD toxin (Tx) in human colon epithelial cells.
FIG. 3 is a result of immunoblot analysis showing that the cockroach-derived peptide periplanetasin-4 (Peri-4) blocks cytotoxic cytotoxicity induced by CD toxin (Tx) in human colon epithelial cells.
FIG. 4 is a result of immunoblot analysis showing that the p38 MAPK and p21 signaling molecules strongly activated in the human colon epithelial cells treated with CD toxin (Tx) are inhibited by the cockroach-derived peptide periplanetasin-4 (Peri-4) treatment .
Fig. 5 is a diagram showing the antibacterial activity and antifungal activity of the novel periplanetasin-4 peptide of the present invention derived from a cockroach.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

≪ Example 1 > Screening of a cockroach-derived peptide gene &

In order to identify the peptide gene showing anti-inflammatory activity from the cockroach, the cockroach was rapidly frozen with liquid nitrogen to isolate the total RNA. The gene information on the cockroach transcripts was confirmed using the RNA seq analysis method, Based on the amino acid sequence translated from the colonies, the uninjine whole was filtrated through the identification of the therapeutic agent for pseudomembranous colitis. As a result, a gene presumed to have a therapeutic activity for the pseudomembranous colitis was selected. The selected amino acid sequence was composed of 13 amino acids with LRHKVYGYCVLGP-NH ₂ and named as Periplanetasin-4 (Table 1).

Peptides Amino acid sequence Periplanetasin-4 LRHKVYGYCVLGP-NH ₂

≪ Example 2: Synthesis and separation and purification of periplanetasin-

Peptide periplanetasin-4 derived from the cockroach identified in Example 1 was synthesized and isolated and purified.

First, to synthesize peptide periplanetasin-4, the present inventors used Merrifield's liquid phase method (Merrifield, RB, J. Am. Chem. Soc., 85, 2149 , ≪ / RTI > 1963).

Method of the peptide synthesis the Fmoc (9-fluorenylmethoxycarbonyl) amino acids of the N _α were synthesized and used as a protecting group (protecting group) of the amino group (amino group) to the conventional method (solid phase method).

Concretely, the peptide having carboxyl terminal in the form of -NH ₂ used Rink Amide MBHA-Resin as the starting material, and the peptide having carboxyl terminal in the form of -OH used Fmoc-amino acid-Wang Resin as a starting material.

Elongation of the peptide chain by coupling of Fmoc-amino acid was performed by DCC ( N -hydroxybenzo-triazole (HOBt) -dicyclo-hexylcar-bodiimide) method.

After the Fmoc-amino acid of the amino terminal of each peptide was coupled, the Fmoc group was removed with 20% piperidine / N-methylpyrrolidone (NMP) solution, washed several times with NMP and dichloromethane (DCM) It was dried with gas.

A solution of TFA (trifluoroacetic acid) -phenol-thioanisole-H ₂ O-triisopropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) Was added and the reaction was carried out for 3 hours. The peptides were separated and the peptide was precipitated with diethyl ether. The crude peptide thus obtained was purified by HPLC on a reverse phase HPLC column (Delta Pak, C ₁₈ 300 Å, 15 μm, 19.0 mm) using an acetonitrile gradient containing 0.1% TFA × 30 mm, Waters).

After the synthetic peptide was hydrolyzed with 6 N HCl at 110 ° C for 24 hours, the resulting residue was concentrated under reduced pressure, dissolved in 0.02 N HCl, and the amino acid composition was measured with an amino acid analyzer (Hitachi 8500 A).

The molecular weight was calculated based on the sequence of the synthesized peptides, and the exact molecular weight was measured using a MALDI mass spectrometer (MALDI).

As a result, it was confirmed that the peptide having the correct amino acid sequence was synthesized because the measured molecular weight and the calculated molecular weight were identical.

Example 3: Confirming the anti-inflammatory and mucosal barrier function-enhancing effects of periplanetasin-

The anti-inflammatory efficacy of periplanetasin-4 and the barrier-enhancing effect of mucosa were evaluated using an animal model of acute gastric mucositis.

To perform this experiment first, The toxin (CD toxin, Tx) secreted from Clostridium difficile bacteria was obtained. Briefly, bacterium VPI10463 (American Type Culture Collection, Rockville, Maryland, USA) was cultured in a meat broth (Difco company, product name: cooked meat medium) under anaerobic conditions. Then, the toxin was induced to be excessively secreted by using a different culture medium (Difco Co., Ltd., heart brain infusion culture medium), and then the supernatant was separated and concentrated 10 times or more to be used as a source of acute pseudomembranous colitis.

Next, an animal model of acute pseudomembranous colitis was established using 6-week-old CD1 mice purchased from Korean BioLink. In order to reduce the amount of stool in the digestive tract, feeding was stopped before the day before surgery and only water was supplied. A mouse with a body weight of 40 g was intraperitoneally injected with 0.1 ml of anesthetic (Ketara 2: rumen 1 ratio), and after performing an open surgery, the ileum was looped by tying 3 cm of the ileum near the cecum using a surgical thread. . In the obtained loop, 1 μl of concentrated CD toxin was injected or CD-toxin and cockroach peptide (Peri-4, 100 μg / ml) were injected together, and the abdominal wall was stitched again. . Control mice were injected with 100 μl of PBS in a prepared loop. The mouse abdomen was excised and only the ileal loop was removed. The tissue was stained using H & E staining to observe the ileal mucosal villi structure, which is shown in FIG. 1 (A).

Referring to FIG. 1 (A), it is confirmed that the normal mucosal structure observed in the control group is severely destroyed by CD toxin (Tx) injection. However, it can be seen that the mucosal damage due to CD toxin (Tx) is remarkably inhibited in the mouse colon injected with periplanetasin-4 (Peri-4).

Next, the amount of cytokine in the loop tissue was measured to determine whether the inflammatory response was reduced. For this purpose, the loop tissue was disrupted by ultrasonication, and the supernatant was extracted and used for the IL-6 enzyme-linked immunosorbent assay (ELISA). The results are shown in FIG. 1 (B).

Referring to FIG. 1 (B), CD toxin (Tx) stimulation strongly increases the amount of IL-6 in the loop tissue, but in tissues treated with periplanetasin-4 (Peri-4) do. Given that IL-6 is a potent inflammatory agent, it shows that periplanetin-4 inhibits the inflammatory response in the colon as well as suppresses the process of epithelial tissue destruction induced by CD toxin.

Example 4 Confirmation of Cytotoxicity Decreasing Effect of Periplanetasin-4 < RTI ID = 0.0 >

Since CD toxin is known to strongly induce cytotoxicity, it has been confirmed that the treatment with periplacetin-4 reduces CD toxin-induced cytotoxicity.

To this end, human colon epithelial cells (HT29 cells) were seeded at a density of 2 × 10 ⁴ cells in a 96-well plate, maintained in McCoy's 5A medium (Invitrogen), and treated with periplanetasin-4 (100 μg / After 1 hour, 1 μl / ml of CD toxin concentrated in Example 3 (about 1 nM of toxin concentration) was exposed for 48 hours.

Then, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) solution was treated and allowed to flow into the cells, followed by the activation of mitochondrial enzymes until a colored precipitate (formazan) Additional reaction was carried out for 2 hours. After completion of the reaction, the culture solution was completely removed and 100 μl of DMSO was added. The color change of the culture solution was measured at 570 nm using an ELISA reader. The results are shown in FIG.

Referring to FIG. 2, CD toxin (Tx) shows very high cytotoxicity in human colon epithelial cells, but cytotoxicity thereof is remarkably reduced when periplanetasin-4 (Peri-4) Is confirmed. Furthermore, in the cells treated with periplanetasin-4, no cytotoxicity was observed as in the control (untreated group, con), suggesting that periplanetasin-4 is a non-toxic substance. Thus, it can be seen that periplanetasin-4 can be used as a therapeutic agent for acute enteritis caused by CD toxin.

Example 5 Confirmation of Cellular Suicide Inhibitory Effect of Periplanetasin-

CD toxins are known to induce suicide signals in a variety of cells. Increased apoptosis in colon epithelial cells eventually leads to the loss of barrier function in the digestive tract. Thus, in Example 3, it was confirmed that the periplanetasin-4 treatment inhibited the cytotoxic-induced cell suicide process.

For this purpose, periplanetin-4 (Peri-4, 100 / / ml) was treated with human xanthine epithelial cells cultured and maintained in a 6-well plate at a density of 1 × 10 ⁶ cells and after 1 hour, 1 μl of CD toxin was treated and cultured for 48 hours. The cells were completely degraded using a cell disruption buffer and an ultrasonic wave, followed by immunoblot analysis. More specifically, the primary reaction was carried out using the caspase-3 antibody used as an indicator protein for apoptosis (16 hours), followed by additional reaction with HRP-conjugated secondary antibody, England Bio Labs Inc.) to visualize protein changes by sensitizing them to a film. The results are shown in FIG.

3, it was confirmed that CD toxin (Tx) stimulation on human colon epithelial cells caused very strong caspase-3 activity (lower band), but periplanetasin-4 (Peri-4) In conclusion, the expression of caspase-3 was significantly decreased in the control group, whereas in the cells treated with periplanetin-4 alone (Peri-4 alone), apoptosis did not proceed at all in the control group .

&Lt; Example 6: Confirmation of expression inhibition effect of p38 MAPK and p21 protein >

To investigate the mechanism of action of periplanetasin-4 to inhibit the toxicity of CD toxin, periplanetasin-4 (Peri-4, 10-100 占 퐂 / ml) was treated with human colon epithelial cells and after 1 hour, 3 &lt; / RTI &gt; of concentrated CD toxin was exposed for 3 hours. After the cells were completely degraded using ultrasound, immunoblot analysis was performed in the same manner as in Example 5. However, in this experiment, it was confirmed that the signaling molecules in the cell, which are known to mediate CD toxin-mediated cell suicide and toxicity, are inhibited by periplanetasin-4 treatment. For this, experiments were performed using p38MAPK and p21 protein-recognizing antibodies, which are well known as representative toxic mediators. The results are shown in Fig.

Referring to Figure 4, CD toxin (Tx) stimulation is found to increase the active phosphorylation of the p38MAPK protein, thereby also increasing the amount of p21 protein. However, when periplanetasin-4 (Peri-4) is treated with CD toxin, the activation of p38MAPK protein and the increase of p21 are all reduced. On the other hand, no other signal molecules in the human colon epithelial cells (phosphorylation of the transcription initiation factors Sp1 and Histone H3: phospho / acetylation: Ac) were observed, suggesting that periplanetasin- It is suggested that the target-signaling molecules that react in cells are p38MAPK and p21 proteins, and this process is a specific pathway.

Taken together, these results suggest that periplanetin-4 inhibits p38 MAPK and p21 signaling, thereby blocking cytotoxic cell suicide against colon epithelial cells.

<Example 7: Antimicrobial activity of periplanetasin-4 antimicrobial peptide against pathogenic microorganisms>

A radial diffusion assay (RDA) was performed to investigate the antimicrobial activity against the periplanetasin-4 peptide. Melittin, a powerful antimicrobial peptide isolated from bees, was used as a positive control in the present invention. In the present invention, the antimicrobial peptide was stored at -20 ° C and dissolved in sterilized distilled water.

[Antimicrobial activity measurement]

Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157), 3% (w / v) TSB (trypticase soy broth) was used to measure the antimicrobial activity of the antimicrobial peptide ) At 37 ° C and 200 rpm for 18 hours, and then cultured for 2 hours and 30 minutes at a concentration of 4 × 10 ⁶ CFU (colony forming units) / ml under the same conditions.

After that, a sterilized underlay gel consisting of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w / v) type I (low electroendosmosis) agarose and 0.03% TSB (4 × 10 ⁶ colony forming units / ml) was added to each well and poured into a square plate. When the underlay gel was hardened, a hole with a diameter of 3 mm was pierced to prepare 5 μl of peptide per concentration .

10 ml of an overlay gel (6% TSB, 1% agarose) was poured and cultured at 37 ° C again to confirm formation of a clear zone (CLEAR ZONE) The results are shown in Fig.

[Measurement of antifungal activity]

Candida albicans , a pathogenic fungus, was grown overnight at 30 ° C and 200 rpm in YPD medium (1% yeast extract, 2% peptone, 2% dextrose) And incubated for 2 hours to become logarithmic phase.

Bacteria cultured on sterilized underlay gel consisting of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w / v) type agarose and 0.03% ⁶ colony forming units / ㎖). After mixing, the mixture was poured into a square plate. When the underlay gel was hardened, a hole having a diameter of 3 mm was pored and 5 μl of the peptide was added per concentration.

10 mL of overlay gel (6% TSB, 1% agarose) was poured and cultured at 37 ° C again to confirm the formation of a clear zone (CLEAR ZONE) Respectively.

[Results of antibacterial activity]

As shown in Fig. 5, the periplanetasin-4 peptide was found to have activity against the pathogenic bacteria and positive bacteria, and it was found that this increased in a concentration-dependent manner. This indicates that there is a possibility of treating the existing harmful strains as a therapeutic agent. However, when compared with melittin, a potent antimicrobial peptide, all of the pathogenic bacteria have lower activity than melittin.

[Results of antifungal activity]

As shown in FIG. 5, it was confirmed that a clear zone (CLEAR ZONE) was formed as a result of measuring the antifungal activity of the periplanetasin-4 peptide, and it was confirmed that the activity was increased in a concentration-dependent manner as in the case of the negative strain. But exhibit lower antifungal activity than melittin, a potent antifungal agent used as a control.

<Example 8: Cytotoxicity measurement on the red blood cells of periplonetin-4 peptide>

The measurement of cytotoxicity against Rat red blood cells was carried out as follows.

Rat erythrocytes were washed three times with phosphate-sodium chloride buffer (PBS, pH 7.4) and then diluted with phosphate-sodium chloride buffer (PBS, pH 7.4) to prepare a 5.0% red blood cell solution. Thereafter, 100 μl of the 5.0% red blood cell solution was dispensed into 96-well plates, and then a stepwise diluted solution of the antimicrobial peptide was mixed. Then, phosphoric acid-sodium chloride buffer (PBS, pH 7.4) was added to all the tubes so that the total amount became 200 쨉 l, and cultured in a 37 째 C incubator for 30 minutes. After the culture was completed, the supernatant was transferred to a 96-well plate by centrifugation at 1000 rpm for 10 minutes in a centrifuge.

The destructive capacity of the red blood cells was measured by measuring the absorbance at a wavelength of 550 nm using an ELISA reader. 100% destructive capacity was calculated for 0.1% Triton X-100 as a control, and 0% destructive capacity for only red cell. At this time, the destructive ability of the peptide was calculated by the following equation (1), and the results are shown in Table 2 below.

[Equation 1]

% Destructive ability = (absorbance of the peptide-treated solution 550 nm-absorbance of the phosphate-sodium chloride buffer solution 550 nm) / (absorbance of the solution treated with Triton X-100 550 nm-absorbance of the phosphate-sodium chloride buffer solution 550 nm)

Measurement of cytotoxicity of antimicrobial peptides of periplanetasin-4 on rat erythrocytes Condition Percent red cell destructive ability ^a (占 퐂 / ml) 0 25 50 100 200 Periplanetasin-4 0 0 0 0 0 Melitin 0 106.9 104.9 106.41 104.8 [Note] a: Cytotoxicity is determined by the ability of the antimicrobial peptide to destroy red blood cells

As shown in Table 2, it is confirmed that the periplanetasin-4 peptide does not have the ability to destroy red blood cells. In contrast, melittin, a potent antimicrobial peptide, is highly cytotoxic even at low concentrations.

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

200 g of the peptide periplanetasin-4 of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

Formulation Example 1-2. Injection preparation

1 g of the peptide periplanetasin-4 of the present invention, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

The peptide periplanetasin-4 of the present invention was added to the cooking sauce in an amount of 1 wt% to prepare a cooking sauce for health promotion.

Formulation Example 2-2. Manufacture of dairy products

The peptide periplanetasin-4 of the present invention was added to milk in an amount of 0.1 wt%, and various dairy products such as butter and ice cream were prepared using the milk.

Preparation Example 2-3. Vegetable juice manufacturing

0.5 g of the peptide periplonetasil-4 of the present invention was added to 1,000 ml of tomato juice or carrot juice to prepare vegetable juice for health promotion.

Formulation Example 2-4. Manufacture of fruit juice

0.11 g of the peptide periplonetasil-4 of the present invention was added to 1,000 ml of apple juice or grape juice to prepare fruit juice for health promotion.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> Composition comprising peptide Periplanetasin-4 isolated from          Periplaneta americana for preventing or treating pseudomembranous          colitis <130> <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 13 <212> PRT <213> Unknown <220> <223> Periplaneta americana <400> 1 Leu Arg His Lys Val Tyr Gly Tyr Cys Val Leu Gly Pro   1 5 10

Claims (12)

(Periplanetasin-4) derived from a cockroach ( Periplaneta americana ) having the amino acid sequence of SEQ ID NO: 1 below.
SEQ ID NO: 1: LRHKVYGYCVLGP-NH ₂ The method according to claim 1,
Wherein said acute pseudomembranous colitis is a toxin-induced disease secreted by Clostridium difficile . The method according to claim 1,
Wherein said peptide inhibits the inflammatory response of human colon epithelial cells caused by toxin secreted by Clostridium difficile. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
Wherein said peptide inhibits the cytotoxic response of human colon epithelial cells caused by toxin secreted by Clostridium difficile. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
Wherein said peptide inhibits apoptosis of human colon epithelial cells induced by toxin secreted by Clostridium difficile . The method according to claim 1,
Wherein said peptide inhibits the p38 MAPK signal of human colon epithelial cells caused by toxin secreted by Clostridium difficile . The method according to claim 1,
Wherein the peptide inhibits the p21 signal of human colon epithelial cells caused by toxin secreted by Clostridium difficile . (Periplanetasin-4) derived from a cockroach ( Periplaneta americana ) having the amino acid sequence of SEQ ID NO: 1 below.
SEQ ID NO: 1: LRHKVYGYCVLGP-NH ₂ 9. The method of claim 8,
Wherein the composition has an anti-inflammatory effect against toxins secreted by Clostridium difficile . (Periplanetasin-4) derived from a cockroach ( Periplaneta americana ) having the amino acid sequence of SEQ ID NO: 1 below.
SEQ ID NO: 1: LRHKVYGYCVLGP-NH ₂ (Periplanetasin-4) derived from a cockroach ( Periplaneta americana ) having the amino acid sequence of SEQ ID NO: 1 below.
SEQ ID NO: 1: LRHKVYGYCVLGP-NH ₂ The peptide represented by SEQ ID NO: 1 synthesized in a gene of a peptide isolated from a cockroach ( Periplaneta americana ).
SEQ ID NO: 1: LRHKVYGYCVLGP-NH ₂

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