KR20170131909A - An anti-microbial peptide, Oxyasin-3 isolated from Oxya chinensis sinuosa and its synthetic composition - Google Patents

Abstract

The present invention relates to a novel anti-microbial Oxyasin-3 peptide isolated from Oxya chinensis sinuosa and to a composition thereof. In the present invention, the chemically synthesized peptide, Oxyasin-3, has no cytotoxicity while exhibiting strong antibacterial activity against gram-positive bacteria, gram-negative bacteria or fungi, thereby being usefully used as antimicrobial agents, antifungal agents, antibiotics, food preservatives, cosmetic preservatives, pharmaceutical preservatives, and the like.

Description

An antimicrobial peptide oxyoxy-3 derived from a rice-grasshopper and its composition {An anti-microbial peptide, Oxyasin-3 isolated from Oxya chinensis sinuosa and its synthetic composition}

More particularly, the present invention relates to a novel oxyacyl-3 peptide and a composition thereof, which exhibit an excellent antibacterial or antifungal activity while having little cytotoxicity from a rice-grasshopper.

Infection with pathogenic microorganisms is one of the most common and fatal causes of human disease, unfortunately the antibiotic resistance of pathogenic microorganisms has been caused by the abuse of antibiotics. In fact, the rate at which pathogenic microorganisms are resistant to new antibiotics is much faster than the rate at which analogs of new antibiotics are developed. For example, pathogenic microbial species such as Enterococcus faecalis , Mycobacterium tuberculosis , and Pseudomonas aeruginosa , which can pose a life threat, I have developed resistance to all antibiotics. Tolerance to antibiotics is distinct from resistance to antibiotics, which was first discovered in the 1970s by Pneumococcus sp. And provided important clues to the mechanism of action of penicillin. Resistant species stop growing in the presence of the usual concentration of antibiotics but do not eventually die. This resistance occurs because antibiotics inhibit the cell wall synthetase when autolytic enzymes such as autolysin do not activate, which makes penicillin an endogenous hydrolytic enzyme. Activation kills bacteria and bacteria also inhibit their activity, resulting in the survival of antibiotics.

It is clinically very important that pathogenic microorganisms have resistance to antibiotics, because the inability to eradicate the resistant microorganisms is ineffective in antibiotic treatment in clinical infections. In addition, tolerance is considered to be a prerequisite for resistance to antibiotics, which is due to the survival of strains despite antibiotic therapy. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow in the presence of antibiotics. Since virtually all pathogenic microorganisms showing resistance are known to have resistance, development of new antibiotics capable of killing pathogenic microorganisms resistant to such antibiotic resistance is urgent.

As described above, it is necessary to develop a new antibiotic to prevent damage caused by pathogenic microorganisms showing resistance to antibiotics, and to develop a new antibiotic that acts independently of autolysin activity. In addition, there is a need to provide pharmaceutical compositions for effectively treating such new antibiotics with an infection of pathogenic microorganisms.

On the other hand, some of the substances that play an important role in maintaining the homeostasis of organisms are physiologically active substances derived from various organisms. Many researches have been carried out on a number of biologically active substances, among which antimicrobial peptides isolated from various organisms are known to act in various ways, ranging from bacteria, fungi and viruses. Antimicrobial peptides are also known to play an important role in host defense and the innate immune system. Korean Patent No. 10-0625875 (a novel peptide isolated from Aspergillus nidulans and a pharmaceutical composition containing it as an active ingredient) and Korean Patent No. 10-0964136 Antimicrobial and antifungal peptide genes and synthetic peptides having antimicrobial and antifungal activity) have been known. However, no description has been made on novel peptides isolated from rice hermit and novel uses thereof.

Korean Patent No. 10-0625875 (Novel peptide isolated from Aspergillus nidulans and a pharmaceutical composition containing the same as an active ingredient, Applicant: Chosun University, Registered Date: September 12, 2006) Korean Patent No. 10-0964136 entitled " Antibacterial or Antifungal Peptide Gene Isolated from Ulmus japonicus Larvae and Synthetic Peptide Having Antibacterial or Antifungal Activity, Applicant: Korea, Date of Registration: June 09, 2010)

It is an object of the present invention to provide an amino acid sequence of oxyhyacin-3 of the antimicrobial peptide isolated from a rice-grasshopper, which can be usefully used for the prevention or treatment of diseases induced by Gram-negative bacteria, Gram-positive bacteria and fungi, and compositions thereof.

The present invention relates to a peptide represented by the following SEQ ID NO: 1 synthesized from the gene of an antibacterial or antifungal peptide isolated from a rice plant ( Oxya chinensis sinuosa ).

SEQ ID NO: 1: MCICCPTKKKKKKK-NH ₂

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

The peptide is characterized by being harmless to red blood cells.

The present invention provides a pharmaceutical composition, antibiotic, food preservative, cosmetic preservative or pharmaceutical preservative for antifungal or antifungal preparations containing the above-mentioned peptide as an active ingredient.

In addition, the present invention can provide a composition for treating food poisoning or enteritis, containing the peptide as an active ingredient. The food poisoning or enteritis is caused by at least one of Staphylococcus aureus and Escherichia coli 0-157 ( Escherichia coli 0-157).

The antimicrobial peptide oxyjacin-3 and its composition derived from the rice-grasshopper of the present invention have little cytotoxicity and exhibit excellent antibacterial or antifungal activity, and thus can be provided as an antibiotic, a food preservative, a cosmetic preservative, a medicine preservative, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing the antimicrobial activity or antifungal activity of a novel oxyacyl-3 peptide of the present invention derived from a rice plant.

Hereinafter, the present invention will be described in detail.

The present invention relates to a peptide represented by SEQ ID NO: 1 synthesized from the gene of an antifungal or antifungal peptide isolated from a rice plant ( Oxya chinensis sinuosa ). Accordingly, the present invention may relate to an antibacterial or antifungal peptide directly isolated from a rice plant ( Oxya chinensis sinuosa ) having the same sequence as that of SEQ ID NO: 1. Accordingly, the present invention may relate to an antimicrobial or antifungal peptide directly isolated from a rice plant ( Oxya chinensis sinuosa ) having the same sequence as that of SEQ ID NO: 1, wherein the peptide may have a carboxyl end in the form of -NH ₂ or -OH have.

In the present invention, in order to identify a peptide gene derived from the rice plant ( Oxya chinensis sinuosa ), firstly, the rice grasshopper is rapidly frozen with liquid nitrogen to isolate the total RNA, and the gene information about the rice hatchlot transcripts is confirmed using the RNA seq analysis method Based on the sequence of the translated amino acid from each transcript, it is possible to select a unicin which is presumed to be a peptide showing antimicrobial or antifungal activity using the properties of the previously discovered antimicrobial peptide.

The amino acid sequence of the peptide consists of 14 amino acids with MCICCPTKKKKKKK-NH ₂ and is named Oxyasin-3.

The present invention also provides a pharmaceutical composition for antibiotic or antifungal preparation comprising the peptide as an active ingredient, an antibiotic, a food preservative, a cosmetic preservative or a pharmaceutical preservative.

More specifically, the oxysacin-3 peptide is useful as an antibacterial activity against bacteria such as Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157), Candida albicans And exhibits antibacterial or antifungal activity in Candida albicans in a radial diffusion assay (see FIG. 1).

In addition, in the test for confirming the erythrocyte cell destructive ability, melitin, a bee venom known as an antifungal or antimicrobial effect is highly cytotoxic, whereas the novel oxyacin-3 peptide of the present invention is found to be almost cytotoxic ( See Table 2). That is, the peptide is characterized in that it is less toxic to red blood cells of the rat.

As described above, the pharmaceutical composition containing the novel oxyacin-3 peptide of the present invention as an active ingredient exhibits antifungal and antibacterial effects as well as almost no cytotoxicity, and thus can be effectively used as a safe antimicrobial agent or antifungal agent.

The pharmaceutical composition containing the oxyacin-3 peptide of the present invention can also be administered parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. The peptide or the composition containing the peptide may be administered in various forms of parenteral administration. In the case of formulation, the peptide may be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As the base of the suppository, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

In addition, the novel peptide of the present invention can be used in combination with various carriers permitted as pharmaceutical agents, such as physiological saline or an organic solvent, and can be used in combination with carbohydrates such as glucose, sucrose or dextran, Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers can be used as pharmaceuticals.

The total effective amount of the novel peptide of the present invention or a peptide having more than 95% homology thereto in the above pharmaceutical composition can be measured in a single dose by a bolus form or by infusion for a relatively short period of time, And may be administered by a fractionated treatment protocol in which multiple doses are administered over a prolonged period of time. The concentration is determined by taking into consideration various factors such as the route of administration and the number of treatments of the pharmaceutical composition as well as the age and health condition of the patient. Thus, in view of this point, It will be possible to determine an appropriate effective dose depending on the particular use as a pharmaceutical composition containing the novel peptide of the present invention.

In addition, the present invention provides antibiotics, food preservatives, cosmetic preservatives or pharmaceutical preservatives containing the novel peptide as an active ingredient. In this case, food preservative, cosmetic preservative or pharmaceutical preservative is an additive used to prevent deterioration, decay, discoloration and chemical change of food or medicine. It includes antiseptic and antioxidant and inhibits the growth of microorganisms such as bacteria, fungi and yeast And also includes functional antibiotics such as inhibiting the growth of microorganisms or sterilizing the microorganisms in foods or medicines. Ideal conditions for these food preservatives, cosmetics or pharmaceutical preservatives should not be toxic and should be effective in trace amounts.

As shown in FIG. 1 and Table 2, the novel oxyacin-3 peptide of the present invention exhibits antibacterial or antifungal activity and has almost no toxicity, so that it can be usefully used as a preservative for foods, cosmetics or pharmaceuticals.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the invention is not limited by these examples.

Example 1: Selection of an antimicrobial peptide gene derived from a rice-grasshopper

In order to identify the peptide gene that exhibits antibacterial or antifungal activity from rice hunters, firstly, the whole rice was frozen by rapid freezing of the rice hatchlot with liquid nitrogen, and the gene information about the rice hunter transcripts was confirmed using the RNA seq analysis method. Based on the sequence of the translated amino acids (peptides) from the dead bodies and the properties of the existing antimicrobial peptides, the entire unijin was filtrated and the genes presumed as new antimicrobial peptides were selected. The selected amino acid sequence was composed of 14 amino acids with MCICCPTKKKKKKK-NH ₂ and was named Oxyasin-3 (Table 1).

Peptides Amino acid sequence Oxyasin-3 MCICCPTKKKKKKK-NH ₂

Example 2: Oxyacin-3 synthesis and isolation Purification

In Example 1, the oxyacin-3 peptide derived from the rice-grasshopper was synthesized, isolated and purified.

First, in order to synthesize an oxyacyl-3 peptide, the present inventors used Merrifield's liquid phase method (Merrifield, RB., J. Am. Chem. Soc., 85, 2149, 1963 ) Was used to prepare the peptide. 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. The elongation of the peptide chain by coupling of the 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 subjected to a reverse phase-HPLC column (Delta Pak, C ₁₈ 300 Å, 15 袖 m, 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 antimicrobial peptide having the correct amino acid sequence was synthesized because the calculated molecular weight and the calculated molecular weight were identical.

Example 3: Antimicrobial activity of oxyacin-3 antimicrobial peptide against pathogenic microorganisms

RDA (radial diffusion assay) was performed to investigate the antimicrobial activity against oxyacin-3 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.

Thereafter, 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) were added to the underlay gel and poured into a square plate. When the underlay gel was hardened, a hole with a diameter of 3 mm was poured into 5 μl Into each hole.

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 composed of citrate phosphate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w / v) type low electroendosmosis agarose and 0.03% × 10 ⁶ colony forming units / ㎖) were mixed and 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 each peptide was added to the hole.

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. 1, it was confirmed that the oxyacin-3 peptide had activity against Gram-negative bacteria and positive bacteria, which is increased in a concentration-dependent manner. This shows that it can be used as a therapeutic agent against existing harmful strains.

[Results of antifungal activity]

As shown in FIG. 1, it was confirmed that a clear zone (CLEAR ZONE) was formed as a result of the measurement of the antifungal activity of the oxyacin-3 peptide, and the activity was increased in a concentration-dependent manner as in the case of Gram-negative bacteria.

Example 4: Cytotoxicity measurement of redox blood cells of oxyacin-3 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. Then, 100 μl of the 5.0% erythrocyte cell solution was dispensed into 96-well plates, and then a solution in which the antimicrobial peptide was diluted stepwise 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)

Cytotoxicity of Oxyacyl-3 Antibody Peptides to Rat Erythrocytes Condition Percent red cell destructive ability ^a (占 퐂 / ml) 0 12.5 25 50 100 200 Oxyasin-3 0 0.5 0.6 0.5 1.9 0.7 Melitin 0 75.8 83.1 89.8 88.3 91.4 [Note] ^a : Cytotoxicity is determined by the ability of the antimicrobial peptide to destroy red blood cells

As shown in Table 2 above, it was confirmed that the oxyacin-3 peptide had almost no destructive ability of red blood cells when treated at a concentration up to 200 μg / ml. In contrast, melitin, a potent antimicrobial peptide used as a control, has high cytotoxicity at low concentrations. These results indicate that the antimicrobial peptides found in the present invention are not only cytotoxic and exhibit excellent antibacterial or antifungal activity, and thus can be used as safe antibiotics, food preservatives, cosmetic preservatives and medicines.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereto will be.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> An anti-microbial peptide, Oxyasin-3 isolated from Oxya chinensis          sinuosa and its synthetic composition <130> O-3 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 14 <212> PRT <213> Oxya chinensis sinuosa <400> 1 Met Cys Ile Cys Cys Pro Thr Lys Lys Lys Lys Lys Lys Lys Lys   1 5 10

Claims (10)

A peptide represented by SEQ ID NO: 1 synthesized from a gene of an antimicrobial or antifungal peptide isolated from a rice plant ( Oxya chinensis sinuosa ).
SEQ ID NO: 1: MCICCPTKKKKKKK-NH ₂
The method according to claim 1,
Wherein said peptide exhibits an antimicrobial activity against any one or more of Staphylococcus aureus and Escherichia coli 0-157 ( Escherichia coli 0-157).
The method according to claim 1,
Wherein said peptide exhibits antifungal activity against Candida albicans .
The method according to claim 1,
Wherein the peptide is harmless to the red blood cells.
A pharmaceutical composition for an antibacterial or antifungal agent comprising the peptide of any one of claims 1 to 4 as an active ingredient.
An antibiotic comprising the peptide of any one of claims 1 to 4 as an active ingredient.
A food preservative containing the peptide of any one of claims 1 to 4 as an active ingredient.
A cosmetic preservative containing the peptide of any one of claims 1 to 4 as an active ingredient.
A pharmaceutical preservative containing the peptide of any one of claims 1 to 4 as an active ingredient.
A composition for treating food poisoning or enteritis, which comprises the peptide of any one of claims 1 to 4 as an active ingredient.

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