KR101740551B1 - An anti-microbial peptide, Oxyasin-2 isolated from Oxya chinensis sinuosa and its synthetic composition - Google Patents

Abstract

The present invention relates to a novel oxyacyl-2 peptide and its composition isolated from a rice-grasshopper.
The chemically synthesized peptide (Oxyasin-2) according to the present invention showed a strong antibacterial action against Gram-positive bacteria, Gram-negative bacteria and fungi. The antimicrobial peptide according to the present invention can be used for the development of antibiotics, food preservatives, cosmetic preservatives, and medicines containing antifungal agents because of their low cytotoxicity and strong antimicrobial activity.

Description

An antimicrobial peptide derived from a rice-grasshopper, oxy-2, and an antimicrobial peptide, Oxyasin-2 isolated from Oxya chinensis sinuosa and its synthetic composition,

More particularly, the present invention relates to a novel oxyacyl-2 peptide and a composition thereof, which exhibit antibacterial and antifungal activity while exhibiting insufficient 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 may 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 when the antibiotic inhibits the cell wall synthetase because the autolytic enzyme activity of the bacteria such as autolysin does not occur. This fact suggests that penicillin is 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 also known to be resistant, development of new antibiotics capable of killing pathogenic microorganisms resistant to these antibiotic resistance is urgent.

As described above, it is necessary to develop a new antibiotic to prevent damages caused by pathogenic microorganisms showing resistance to antibiotics, and to develop new antibiotics that act 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 Priority 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, there have been no studies on novel peptides isolated from rice hermit and novel uses thereof.

It is an object of the present invention to provide an amino acid sequence of oxyhyacin-2, an antimicrobial peptide isolated from a rice-grasshopper, which can be usefully used for the prevention and treatment of Gram-negative bacteria, positive bacteria and human fungal diseases, and compositions thereof.

In order to achieve the above object, the present invention provides an antibacterial and antifungal peptide (SEQ ID NO: 1) isolated from a rice plant ( Oxya chinensis sinuosa ).

The peptide is characterized by having antibacterial and antifungal activity.

The antimicrobial activity is characterized by exhibiting at least one of Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157).

The antifungal activity is characterized in that it appears to Candida albicans .

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

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

The present invention can provide antibiotic, food preservative, cosmetic preservative, medicine, and the like because it has excellent cytotoxicity and excellent antibacterial and antifungal activity by being provided with the antimicrobial peptide oxyacin-2 and its composition derived from the rice grasshopper .

Brief Description of the Drawings Fig. 1 is a diagram showing antibacterial activity and antifungal activity of a novel oxyacyl-2 peptide of the present invention derived from a rice plant.

Hereinafter, the present invention will be described in detail.

The present invention provides antibacterial and antifungal peptides (SEQ ID NO: 1) isolated from rice plant ( Oxya chinensis sinuosa ).

In the present invention, in order to identify a gene derived from a rice hatchery ( Oxya chinensis sinuosa ), first, a rice hatcher is rapidly frozen with liquid nitrogen to isolate total RNA, and then gene information on rice hatchlot transcripts is analyzed using RNA seq analysis Based on the sequence of the translated amino acid from each transcript, the unicin, which is presumed to be a peptide showing antimicrobial and antifungal activity, is selected using the properties of the previously identified antimicrobial peptide.

The amino acid sequence of this peptide is MYWKLLIKMAHPKKK-NH ₂ , which consists of 15 amino acids and is named Oxyasin-2.

In addition, the present invention provides a pharmaceutical composition for antibiotic and antifungal preparation containing the active ingredient as the active ingredient, or antibiotic, food preservative, cosmetic preservative or pharmaceutical preservative.

Specifically, the present inventors used Staphylococcus aureus , E. coli 0-157 ( Escherichia coli 0-157) as antibacterial activity target bacteria to measure antibacterial and antifungal activity of the oxyacin-2 peptide, , And the RDA assay ( Candida albicans ) as a target of the antifungal activity was carried out by the RDA assay (radial diffusion assay), showing strong antibacterial or antifungal activity similar to melitin having strong antibacterial or antifungal activity 1).

In addition, the inventors of the present invention investigated the cytotoxicity of the novel oxyacyl-2 peptide of the present invention, and found that the novel oxyacyl-2 peptide of the present invention shows little cytotoxicity, Melitin, a bee venom used as a control, was found to be highly cytotoxic (see Table 2).

From the above results, the pharmaceutical composition containing the novel oxyacin-2 peptide of the present invention as an active ingredient exhibits excellent antifungal and antimicrobial effects, and is also useful as a safe antimicrobial and antifungal agent because of its low cytotoxicity Respectively.

Such a pharmaceutical composition can be administered parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation.

That is, the novel peptide of the present invention can be administered in various forms of parenteral administration. In the case of formulation, the peptide is prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, . Formulations for parenteral administration include sterilized 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 and the peptide having more than 95% homology thereto in the pharmaceutical composition of the present invention may be 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. Since the concentration of the effective dose of the patient is determined in consideration of various factors such as the route of administration and the number of treatments as well as the age and health condition of the patient, in view of this point, Lt; RTI ID = 0.0 > of the < / RTI > novel peptide as a pharmaceutical composition.

In addition, the present invention provides an antibiotic, a food preservative, a cosmetic preservative or a pharmaceutical preservative containing a novel peptide as an active ingredient.

In this case, food preservative, cosmetic preservative and pharmaceutical preservative are additives used to prevent deterioration, decay, discoloration and chemical change of foods or medicines, including bactericides and antioxidants, and proliferation of microorganisms such as bacteria, fungi and yeast As well as functional antibiotics such as inhibiting the growth of microorganisms or sterilizing the microorganisms in foods and medicines. Ideal conditions for these food preservatives, cosmetics, and medicine preservatives should be non-toxic and have a very small effect.

As shown in FIG. 1 and Table 2, the novel oxyacyl-2 peptide of the present invention exhibits strong antibacterial and antifungal activity and is insignificant in toxicity, thus confirming that it can be effectively used as a food preservative, a cosmetic, and a medicine preservative.

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

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 amino acid translated from the dead bodies, the uni - jean was filtrated based on the existing antimicrobial peptide properties. As a result, genes presumed as new antimicrobial peptides were selected. The selected amino acid sequence was MYWKLLIKMAHPKKK-NH ₂ , which was composed of 15 amino acids and named Oxyasin-2 (Table 1).

Peptides Amino acid sequence Oxyasin-2 MYWKLLIKMAHPKKK-NH ₂

Example 2: Oxylazin-2 synthesis and isolation Purification

In Example 1, the oxyacyl-2 peptide derived from the rice-grasshopper was synthesized and isolated and purified.

First, in order to synthesize an oxyacyl-2 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.

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 μ, 19.0 mm × 10 cm) using an acetonitrile gradient containing 0.1% TFA 30, 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-2 antimicrobial peptide against pathogenic microorganisms

RDA (radial diffusion assay) was performed to investigate the antimicrobial activity against oxyacin-2 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 composed 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 Add 4 × 10 ⁶ colony forming units / ml to the underlay gel. Pour into a square plate. When the underlay gel is hardened, take a 3 mm diameter hole and add 5 μl of peptide per concentration I put it in a hole.

10 ml of an overlay gel (6% TSB, 1% agarose) was poured and cultured at 37 ° C again to confirm the 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 / ml) were mixed and poured into a square plate. When the underlay gel was hardened, a hole having a diameter of 3 mm was punched out 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 formation of a clear zone. Respectively.

[Results of antibacterial activity]

As shown in FIG. 1, the oxyacyline-2 peptide was found to have activity against Gram-negative bacteria and positive bacteria, which was found to increase in a concentration-dependent manner. This indicates that there is a possibility of treating the existing harmful strains as a therapeutic agent. Compared with melittin, a potent antimicrobial peptide, Gram - negative bacteria showed high activity similar to melittin.

[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 antifungal activity of oxyacin-2 peptide and it was confirmed that the activity was increased in a concentration-dependent manner as in the case of Gram-negative bacteria. In addition, it showed antifungal activity similar to that of melittin, a potent antifungal agent used as a control.

Example 4: Cytotoxicity measurement of redox blood cells of oxyacin-2 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, pH 7.4 (PBS), and then diluted with phosphate-sodium chloride buffer 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, pH 7.4 (PBS) was added to all the tubes so that the total amount became 200 쨉 l, and the cells were incubated 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 obtained results are shown in Table 2 below.

[Equation 1]

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

Cytotoxicity of Oxyacyl-2 Antibody Peptides to Rat Erythrocytes
% Red erythrocyte destruction ability ^a (ug / ml) 0 12.5 25 50 100 200 Oxyasin-2 0 0 0 0.16 0 0 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, it was confirmed that the oxyacin-2 peptide had a low ability to destroy red blood cells when treated at a concentration of up to 200 ug / ml. In contrast, melitin, a potent antimicrobial peptide, was found to be highly cytotoxic even at low concentrations.

These results indicate that the antimicrobial peptides found in the present invention are not only cytotoxic and exhibit excellent antibacterial and antifungal activity, they can be used as safe antibiotics, food preservatives, cosmetic preservatives, and pharmaceuticals.

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. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> REPUBLIC OF KOREA (MANAGEMENT: RURAL DEVELOPMENT ADMINISTRATION) <120> An anti-microbial peptide, Oxyasin-2 isolated from Oxya chinensis          sinuosa and its synthetic composition <130> P2015-0233 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 15 <212> PRT <213> Unknown <220> <223> Oxyasin-2, Oxya chinensis sinuosa <400> 1 Met Tyr Trp Lys Leu Leu Ile Lys Met Ala His Pro Lys Lys Lys   1 5 10 15

Claims (10)

Peptides synthesized from amino acids deduced from genes of antimicrobial and antifungal peptides isolated from rice plant ( Oxya chinensis sinuosa ), represented by SEQ.
SEQ ID NO: 1: MYWKLLIKMAHPKKK-NH ₂
The method according to claim 1,
Wherein said peptide has antibacterial and antifungal activity.
3. The method of claim 2,
Wherein the antimicrobial activity is exhibited for at least one of Staphylococcus aureus , Escherichia coli 0-157 ( Escherichia coli 0-157).
3. The method of claim 2,
Wherein said antifungal activity is exhibited against Candida albicans .
The method according to claim 1,
Characterized in that the peptide is less toxic to red blood cells.
A pharmaceutical composition for an antibacterial and antifungal preparation, which comprises the peptide of any one of claims 1 to 5 as an active ingredient.
An antibiotic comprising the peptide of any one of claims 1 to 5 as an active ingredient.
A food preservative containing the peptide of any one of claims 1 to 5 as an active ingredient.
A cosmetic preservative containing the peptide of any one of claims 1 to 5 as an active ingredient.
A pharmaceutical preservative containing the peptide of any one of claims 1 to 5 as an active ingredient.

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